Structural Aspects of the Fibrinogen to Fibrin Conversion*

STRUCTURAL ASPECTS OF THE FIBRINOGEN TO FIBRIN CONVERSION* By

R . F . DOOLITTLE

Department of Chemistry. University of California. San Diego. La Jolla. California

I . Introduction . . . . . . . . . . . . . I1. Structure and Properties of Fibrinogen . . . . . . A. Physicochemical Data . . . . . . . . . . B . Electron Microscopy of Fibrinogen . . . . . . C. X-Ray Studies on Fibrinogen . . . . . . . D. Biochemical and Organochemical Characterization . . . 111. Structure and Properties of Fibrin . . . . . . . A . Definitions of Fibrin . . . . . . . . . . B. Some Physical Properties of Fibrin Gels . . . . . C . Studies on Fibrin Monomer . . . . . . . . D . X-Ray Studies on Fibrin . . . . . . . . . E . Electron Microscopy of Fibrin . . . . . . . F. Chemical Composition of Fibrin . . . . . . . G. Fibrin Split Products . . . . . . . . . . IV . The Conversion of Fibrinogen to Fibrin . . . . . . A . General Remarks on Fibrin Formation . . . . . B. Release of Fibrinopeptides . . . . . . . . C . Polymerization Steps . . . . . . . . . . D . Electric Birefringence Studies . . . . . . . . E . Thermodynamic Aspects of Fibrin Formation . . . . F. Functional Groups Involved in Polymerization . . . V . Covalent Cross-Linking of Fibrin . . . . . . . . A . Nature of the Cross-Links in Stabilized Fibrin . . . . B . Polypeptide Chains Involved in Fibrin Cross-Linking . . C . y-y Dimers and Polymerization Contact Sites . . . . D . Thc Significance of a-Chain Multimers . . . . . E . Relative Contributions of y- and a-Chains to Cross-Linking under Various Conditions . . . . . . . . . F . Unnatural Kinds of Covalently Reinforced Fibrin . . . VI . Other Aspects of the Fibrinogen-Fibrin Conversion . . . A . Variant Human Fibrinogens . . . . . . . . B . Human Fetal Fibrinogen . . . . . . . . . C . The Influence of Calcium Ions on Fibrin Polymerization . D . Cobalt-Fibrinogen . . . . . . . . . . E . Evolutionary Considerations . . . . . . . . F. Biosynthesis and Assembly . . . . . . . . VII . Reevaluation of Various Models . . . . . . . .

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.

. . . . . . . .

. . . . . . . .

. .

2 5 5

10 14 15

27 27

28

29 30 30 34 35

36 36 36

38 42 47 50 52 52 53

56 62 63 65 69 69 74 75

76 77 86 88

*This article was written while the author Ivas a visitor in the Department of Biochemistry, Oxford University, and was supported by a Career Development Award from the U . S. Public Health Service . 1

2

R. F. DOOLITTLE

Properties of the Ideal Fibrinogen Model . . . . . . . Conditions Attached to the Ideal Scheme of Fibrin Farmation . Reevaluation of Models from Electron Microscopy . . . . . Comments on Schematic Depictions Derived from Bioorganochcmical Observations . . . . . . . . . . . . . . E. Implementation of the Hall and Slaytcr Model . . . . . . F. Fibrin Formation with a Hall and Slayter Model . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

A. B. C. D.

.

1.

88 89

90 93 96 98 100

102

I. INTRODUCTION The central cvcnt in the coagulation of vertebrate blood is the transformation of a soluble plasma protein, fibrinogen., into an insoluble polymeric gel termed fibrin (Fig. l ) . The complex series of events which precedes this conversion is primarily directed toward the production of thrombin from prothrombin by limited proteolysis; the thrombin in turn catalyzes the release of a few small peptides (fibrinopeptides) from fibrinogen molecules, the resulting “fibrin monomers” polymerizing spontaneously to form the fibrin gel. fibrinogen

thrombin H20

(fibrin monomer) -+ fibrin gel

+

fibrinopep tides

Almost a generation has gone by since this rough outline of fibrin formation was developed following a period of intcnsc physicochemical characterization of fibrinogen and fibrin (Ferry, 1952). The initial contributions from organochcinical procedures-cnd-group analyses and peptide isolation (Bailey et aZ., 1951)-were in good accord with the physical picture, and it appeared as though it would be only a matter of time before the fundamental questions posed by Fcrry and Morrison (1947) would be answered in detail: “Elucidation of the conversion process involves two virtually independent problems; the nature of the chemical bonds which link the fibrinogen units together; and the geometrical arrangement of the fibrinogen units in the fibrin structure.” I n fact, very little progress has been made on either of these problems in the ensuing quarter century. The basic difficulty has been tied to a third unsolved problem-namely, the general shape and strueturc of the fibrinogen molecule itself. I n this area it has sometimes seemed that the more we learn, the less we know. In particular, there has been great difficulty reconciling certain organo- and biochemical observations on fibrinogen structure with widely accepted physicochemical data. One of the objectives of this review is to state as precisely as possible what these

FIBRINOGEN TO FIBRIN CONVERSION

3

FIG. 1. Scanning rlcctron micrograph of an crythrocytc rnmcslicd in fibrin. Abont X20,500. From Brrnstrin and Iiairincn (1971).

points of conflict arc. and how thcy affect various hypothcscs of fibrin formation. The disagreements fall mainly into three categories. First, there is no real accord on the general shape of the fibrinogen molecule, conceptions ranging all the way from long rigid rods and ellipsoids of revolution to flexible strings of globules and even symmetrical sponges. Much of the difficulty stems from the traditional hydrodynamic problem of distinguishing macromolecular asymmetry from hydration, as well as from having to make judgments about a hydrated protein on the basis of anhydrous

4

R. F. DOOLITTLE

specimens observed in the electron microscope. The second area of dispute has to do with the nature of the forces holding subunit portions of fibrinogen together. There have been a number of claims over the years that the molecule can be dissociated into half-molecules without the rupture of any covalent bonds. The overwhelming weight of recent evidence, however, indicates that all six of the constituent polypeptide chains are held together by disulfide bridges. In either case, there are implications for the other areas of controversy. The third unresolved issue involves the general location of the fibrinopeptides in the parent molecule. Elegant studies imploying electric birefringence positioned the fibrinopeptides A very near the ends of a long asymmetric molecule. Recent organochemical evidence, however, indicates that the two fibrinopeptides A are within twenty-four amino acid residues of each other, their respective polypeptide chains being held together by disulfide bonds. In order to appreciate the fine points of these seemingly irreconcilable dilemmas, it is necessary that we review a considerable amount of data on the structure of fihrinogen and fibrin amassed in several different fields. We must examine the conversion process itself, as well as the separate problem of fibrin stabilization. In the latter process the fibrin gel becomes covalently reinforced, and by locating the cross-linked regions of the chains involved one can make some surmises about the structures of the individual units and their arrangement in the gel. Also, the inaccessibility of cross-linking acceptor sites in fibrinogen-as opposed to fibrinhas led to the notion that these sites are masked by the fibrinopeptides (Lorand and Ong, 1966). Recent structural studies suggest, however, that the key acccptor sites are actually quite far removed from the fibrinopeptides, wherever they may be. Finally, a number of ancillary considerations, including studies on variant fibrinogens, possible fetal types and certain unusual clcrivatiws, also bear on the problem, as do evolutionary and biosynthetic aspects. After discussing the problem from all these viewpoints, we can make an attempt to find the most accommodating model. The extraordinarily large number of publications which has appeared in recent years on the general topics of fibrinogen and fibrin has necessitated an arbitrary-and sometimes perhaps too harsh-selection procedure in the citation of “significant observations.” Let the reader be cautioned that there are sufficient data strewn through the literature to support virtually any model or hypothesis explaining fibrin formation if other (conflicting) data are neglected. Although certain important studies from the “classical period” are cited in this review, a previous article in this series (Scheraga and Laskowski, 1957) should be consulted for detailed references to many ob-

5

FIBRINOGEN TO FIBRIN CONVERSION

servations discussed. Also, a number of more restrictive reviews have appeared during the last decade, including some on fibrinogen structure (Mihalyi, 1968, 1970), its conversion to fibrin (Blomback, 1967), its physiology (Lorand, 1970), its evolution (Doolittle, 1970), and the electron microscopy of fibrin (Bang, 1967), to name just a few.

11. STRUCTURE AND PROPER TIE^

OF

FIBRINOGEN

A . Physicochemical Data There is general agreement that the molecular weight of native human and bovine fibrinogens is 340,000 -I 20,000 (Table I ) . This value has been established by sedimentation and diffusion methods (Shulman, 1953 ; Caspary and Kekwick, 1957), as well as by light scattering (Kate et al., 1952). Physical characterization of the subunits separated after the cleavage of disulfide bonds is in agreement with this value (Henschen, 1964a; McKee et al., 1966) when taken together with quantitative endgroup data which clearly show that the native molecule is a dimer (Blomback and Yamashina, 1958). The molecular weights of fibrinogens from most other mammals examined arc in this range also, although occasionally higher values are found (Section VI,A,l). Other physicochemical parameters, such as the rotary diffusion coT.wLI.:I A cceptcd Values of Physicochemical Parameters of Human and Bovine Fibrinogena Molecular weight Sedimeritation coefficient Translational diffusion coefficientb Rotary diffusion coefficient Intrinsic viscosity Partial specific volume Frictional ratio Molecular volume (calculated, unhydrated) Extinction coefficientc

Af S2X.W

D*,," ezo. 1"

It I i,

f/b

El1% om 280

IEP

Isoelectric pointd Percent or-helix8

-

~

~~~

340,000 f 20,000 7.9 s 2 . 0 X 10-7 em? sec-l 40,000 see-' 0 25 dl/gm 0 . 71-0.72 2.34 3 . 9 x 102Aa 15-16 5.5 33

~

Although most of these vakies have been determined rigorously only for human and bovine fibrinogeris (Scheraga and Laskowski, 1957), the physical properties of most vertebrate fibrinogens follow the same general pattern. A recent report, using a laser-dependent quasielastic scattering approach sliggwts that the translational diffusion Coefficient might be as low as 1.5 X lo-' em2 sec-' (Birnboim and Lederer, 1972). At neutral pH. Seegers el al. (1945). Mihalyi (1965).

6

R. F. DOOLITTLE

efficient, intrinsic viscosity, ctc. (Table I ) , are not in dispute in themselves, but the various abstractions to which they have been lent are certainly subject to question. The fundamental problem lies with the attempts of physical chemists to deduce the shape of the fibrinogen molecule on the basis of these parameters alone, using approaches that have in fact been reasonably successful with other proteins. I n all these cases, however, certain assumptions are made with regard to other molecular properties, placing restrictions on these models which may sometimes be totally invalid. Without doubt the biggest difficulty has been distinguishing true dissymmetry from hydration effects, the relative contributions of which are reciprocally related for many hydrodynamic properties (Fig. 2 ) . For smaller, more compact proteins, acceptable compromises havc been made-usually assuming 0.1-0.3 g of water per gram of protein-without distorting the principal aim of finding out something about the moleculc being studied. Supposing, however, that an uncharacteristic protein has a unique architecture that binds an unusual amount of water. What shape would the physicochemical parameters prescribe then? I n fact, this is a critical point precisely because a model for fibrinogen has been proposed that postulates just such properties (Section VII,C,3). Even though physical chemists have always injected notes of caution with regard to their “equivalent models,” too often the biochemist seems 10

OBLATE

5

PROLATE

8-

u

P

-b/O

I

u/b-

FIQ.2. Relationship between degree of hydration and axial ratio for frictional ratio (f/fo) = 2.34 and viscosity increment ( Y ) = 35 ( v = Iql/V X 100). Adapted from Oncley (1941).

7

FIBRINOGEN TO FIBRIN CONVERSION

willing to accept their portrayals literally. Hydrodynamically, fibrinogen has most often been described as its equivalent prolate ellipsoid of revolution (rigid and impenetrable) . The dimensions of the ellipsoid are usually based on a ‘lreasonable value” for its axial ratio (most often 10-20), assuming a “reasonable” amount of hydration. The upper limit of the latter is usually taken to be 0.3g of water per gram of protein. Accordingly, most classical models put the length of the equivalent ellipsoid of revolution a t 600-7OOA (Table 11),and its width a t 35-60A. Although protein chemists were, and still are, fixed on compact, more or less impenetrable, proteins as being thermodynamically most reasonable in an aqueous environment, they were aware that perfect model geometries--e.g., spheres and ellipsoids-were abstractions that could only approximate reality. Accordingly, some early attempts were made to refine these models and to provide a wider range of shapes, especially for larger proteins. Shulman (1953), following an earlier lead of Kuhn et al. (1951), showed how the hydrodynamic data derived from measurements of fibrinogen could be accommodated equally well by a model of linked nodules (Fig. 3). Shulman (1953) also noted that, in the case of equivalent ellipsoids of revolution, the axial ratios derived from viscosity measurements on fibrinogen were not in very good agreement with those obtained from sedimentation and diffusion data unless a very high degree of hydration was postulated (Fig. 2 ) . Similarly, attempts to estimate the shape of fibrinogen using the p-function approach of Scheraga and Mandelkern (1953) also indicated a large degree of hydration. This method gives T A n m 11 Lengths of “Eqiiiualmt Ellipsoids” Ddrrmincd for Fibrinogen b?j Variom Mrthodsa Length

(A)

Axial ratio

Sedimentation-diffusion (via f/fo)

860

Light scattering (dissymmetry) Flow birefringence (via 0) Viscosity (via &function)

660 670 500

29* (No hydration)

Method

1fV 18d

5 (Hydration not limited)

Reference Shulman (1953) Katz et al. (1952) Hocking et al. (1952) Edsall (1954)

a As indicated in Fig. 3, the lengths of rigid chains of spheres satisfying the same data will be significantly different from the equivalent prolate ellipsoids of revolution described here. * See also Fig. 2, however. Assumed minor axis = 40 A, Assumed for purposes of calculation.

8

R. F. DOOLITTLE

k 5 0 0 8

4

FIG.3. Some schematic depictions of fibrinogen which can accommodate hydro-

dynamic data: (1) ellipsoid and string of beads consistent with intrinsic viscosity and sedimentation data; (2) ellipsoid and string of beads from sedimentation and diffusion data; (3) compromise of ellipsoid and string of beads for sedimentation and diffusion data; (4) equivalent hydrodynamic ellipsoid for a fully hydrated molecule. Redrawn from Shulman (1953).

information about the total effective volume of a protein, as well as its axial ratio, by combining sedimentation (s), intrinsic viscosity (171) and molecular weight ( M ) data according to the relationship

where N is Avogadro's number, V is the partial specific volume, p the solvent density, and yo the viscosity of the solvent. Edsall (1954) employed the outside limits for these parameters and found that the p function of fibrinogen must lie between 2.05 and 2.28 X 1C6. Since 2.12 X los is the theoretical value attributable to a sphere, and any value lower than that should be impossible, the axial ratio according to this method must lie between 1 and 5 (Fig. 4 ) . Edsall noted that this would be consistent only with a "greatly swollen ellipsoid, decidedly less elongated than anyone had previously proposed." At the time he tended to dismiss the conclusion as being simply too hard to believe, the weight of evidence from other sources being that proteins must have more compact structures (Edsall, 1954). Somewhat later Yang (1961) reconsidered these observations, and, although he noted that the p-function of Scheraga and Mandelkern ( 1953) has several limitations, the notion that

9

FIBRINOGEN TO FIBRIN CONVERSION

1

2

4

16

8

32

64

128

alb

FIG.4. Relationship of axial ratio ( a / b ) to p-function of Scheraga and Mandelkern (1953). Calculations employing the outside limits of hydrodynamic parameters of fibrinogen indicate that the /3-function of fibrinogen must lie between 2.06 and 2.28 X lo". From Edsall (1954).

fibrinogen might be a highly swollen protein with an effective volume more than five times that of the dehydrated protein was cast as a real possibility, suitable alternative explanations of the data still being wanting. On the other hand, the fact that fibrinogen exhibits such a marked flow birefringence argues for an extremely elongated molecule (Edsall et al., 1947). This argument has been countered by noting that extensive deformation of proteins may occur when they are exposed to high shearing forces; the same forces would also be operable in viscosity measurements and could lead to extended structures (Yang, 1961). There are a number of other interpretive aspects that can be misleading about hydrodynamic data, as, for example, would arise from flexible linkages between domains. Furthermore, most of the data can be interpreted from the point of view of flattened discs just as well as for elongated ellipsoids, the fundamental choice of prolate over oblate for fibrinogen usually being made a priori on the basis of its characteristic flow birefringence (Edsall et al., 1947). Without drawing any rigid conclusions about the actual shape of fibrinogen a t this time, we can make some preliminary judgments about what kinds of structure the data can accommodate. At the extremes, if fibrinogen is not in the least hydrated, then it corresponds to a n equivalent ellipsoid of revolution with an axial ratio of about 30. On the other hand, if its idealized shape is approximately spherical, then it must be hydrated to the extent of about 8 g of water per gram of protein (Fig. 2). Between

10

R. F. DOOLITTLE

these unlikely limits, the data are quite consistent with a nodular structure with an axial ratio of 5-10 ( a = 350-500A; b = 50-80A) and a larger than usual amount of accompanying water. With these broad approximations in mind, we can turn our attention to another controversial area, the electron microscopy of fibrinogen.

B. Electron Microscopy of Fibrinogen Although early attempts to visualize the structure of fibrinogen by electron microscopy were not nearly as successful as studies performed on fibrin (Section III,E), their influence on the thinking of biochemists working on fibrinogen has been considerable. Generally the electron microscopist sprays a dilute solution of fibrinogen in a volatile buffer on to a suitably backed grid and evaporates the water and buffer. As a general rule, protein volumes measured directly from electron micrographs approximate the molecular volumes calculated from partial specific volumes and molecular weights, indicating a tendency to collapse into a minimal volume upon drying (Haschemeyer, 1970). This is obviously a q a t t e r of great concern when dealing with a protein like fibrinogen where the hydrodynamic data suggest an unusual degree of hydration. The existence of a nodular fibrinogen structure was reported by Siege1 et al. (1953), who observed a molecule measuring 60-80 X 500 A comprised of four linked globules, consistent with one of the possible shapes depicted by Shulman (1953) on the basis of hydrodynamic data. The first electron microscope study of fibrinogen to meet with wide acceptance, however, was that of Hall and Slayter (1959). Their shadowcast micrographs showing three linearly attached globules have received considerable exposure and have served as a prototype model for workers in almost all areas of fibrinogen chemistry. Other workers have obtained quite different results, however, and Hall and Slayter’s conclusions-which will be referred to throughout this article-must be examined critically. Hall and Slayter (1959) sprayed fibrinogen solutions on to grids and then fixed them by spraying on a dilute formaldehyde solution; the specimens were shadowcast with platinum. Their micrographs showed quite clearly a preponderance of triads about 480-500A long, although there were some dyads and some single globules, as well as a few higher combinations (Fig. 5, top). The triads had globular spheres a t the ends which were about 65A in diameter, whereas the one in the center was somewhat smaller, averaging about 50A in diameter. The spheres were joined together by a thin “thread” which could not be resolved in their micrographs, but which they construed to have a diameter of 8-15A. Calculation revealed that the volume of the three-globule particle was very close to the molecular volume computed by Shulman (1953) on the basis of his hydrodynamic characterization.

FIBRINOGEN TO FIBRIN CONVERSION

11

FIG.5. Two sti3iingly diffrrrnt. elcctron microgr:ipI~of bosinc fibrinogen. Top: Shadowcast specimen similar to tliosr published by Hall and Slaytrr (1959) (courtesy of H. S. Slayter). Bottom: Negatively stained preparation of Kiippel (1966).

Subsequently, Bang (1964), while not challenging thc existencc of a three-globule-cluster, came u p with a slightly differcnt structure on the basis of his micrographs, which were also obtained by shadowcasting. His fibrinogen molecules appeared t o have subunits which were much more

12

R. F. DOOLITTLE

elongated and closer together. H e estimated an axial ratio of 6-7, in contrast to thc value of 9 which one obtains from the Hall and Slayter model. The length of the Bang molecule was put at 375 2 40A and its width close to 60A. Later Kay and Cuddigan (1967), basing thcir conclusions on negatively stained fibrin preparations, proposed a model of fibrinogen which was a “linear array of nodular elements,” the average width of which was 3 W 5 A . They estimated the length of the native fibrinogen molecule to be 690 A. At about the same time, Koppel (1966) published electron micrographs of negatively stained fibrinogen preparations which unexpectedly showed that fibrinogen might have a cagelike geometry approximating that of a pentagonal dodecahedron (Fig. 5, bottom). Koppel (1967) subscqucntly put forth pcrsuasive argumcnts about how his model could also accoininodatc the hydrodynamic data, espccially since its cagclikc structure could explain the anomalously high hydration values suggested by previous investigations. We will rcturn to tlicsc widely divergent fibrinogen models when wc considcr the clcctron microscopy of fibrin (Section II1,E). In the mcnntime, it is difficult for the non-electron microscopist to know how highly to regard these different dcpietions, capccially since many articles imply that other models h a w been modificd by their inventors sincc their original publication. This is often said about the Hall and Slayter model in particular, but it should be pointed out that as recently as 1969, after a decade of debate, E. M. Slayter (1969) republished the original pictures of Hall and H . S. Slaytcr (1959), making it quite clear in the text that there has been no backing away from the original three-ball model. Electron microscopy of proteins can givc rise to artifacts, however, and slight changes of conditions can make drastic differences in the final product. For example, Stewart (1971) reports that while working in Hall’s laboratory she observed the production of fibrinogen triads on a routine basis, but when she moved to another laboratory and varied her technique she never again saw triads. She suggested that fibrinogen might be a “coiled spring” which unravels t o different degrees under different conditions. It is intercsting to note that Edsall e t al. (1947) also considered the possibility of a disc-shaped model of fibrinogen with the properties of a coiled spring. In the meantime, we are left with the same uneasy vagueness about fibrinogen structure as determined by electron microscopy as we were bequeathed by the physical chemists. Supposing fibrinogen is a highly hydrated structure, how can conventional electron microscopy in m c u o ever give rise to a true picture? Recently Tooney and Cohcn (1971, 1972) reported thc electron microscopy of microcrystals obtained from a filrinogen I m p r a t i o n which had been partially degraded by trcatnicnt with a bacterial enzyme preparation.

13

FIBRINOGEN TO FIBRIN CONVERSION

The removal of a relatively small portion of the molecule (apparently from the carboxy-terminal section of the a-chain) was evidently sufficient modification that limited crystallization a t low ionic strength was possible. The pictures exhibit a high degrec of order, and a packing unit of dimensions 90 X 450 A has been detected (Fig. 6 ) . It should be noted that a cylinder of these dimensions has a volume of 2.9 X 106A3, and, depending on the disposition of the peptide chains in that volume, can obviously accommodate either a compact structure or the highly swollen molecule suggested by some of the hydrodynamic data. Optical superposition of these plates yields n characteristic fibrin pattern, emphasizing how much like the nativc structure these preparations are. Utilization of three-dimensional image rccoiistruction techniques (DcRosier and

b

a

6. 7'0p: Electron micrograplrs of modified fibrinogrn niicrocrystals showing X220,000. , ! h t t o n i : 0111ic.:d tiiffraction 1):iltc~ms obtained from From Tooncq- and Colicn (1972). clcctron microgml,lra altoviii abo\. FIG.

450 ,i repeat unit.

(1.

14

R. F. DOOLITTLE

Klug, 1968) may ultimately result in the refinement necessary to yield an approximate shape for these molecules. Until then, the biochemist is depending more and more on less direct methods.

C . X-Ray $tudies on Fibrinogen Fibrinogen has never been crystallized in a form suitable for detailed X-ray diffraction studies, but the protein can be gelled in a pseudocrystalline fashion by a variety of methods, and these procedures have provided some important observations. Bailey et aZ. (1943) showed th a t fibrinogen films exhibit a-patterns similar to the keratin-myosin family, giving strong meridional reflections with a spacing of 5.1 A, a characteristic that has since been attributed to the existence of “coiled coils” (Crick and Kendrew, 1957). Strycr et aZ. (1963) used a low-angle X-ray approach t o demonstrate that packed fibrinogen gels give the same 226 A repeat unit as that observed in fibrin; the significance of this observation will be developed further during our discussions of fibrin and its formation. Recently Lederer (1972) performed a small-angle X-ray study on a dilute solution of bovine fibrinogen and compared the experimental data with computer predictions for various models derived from physico-

-

28 ( rad)

FIQ. 7. Small-angle X-ray scattering curve observed with bovine fibrinogen compared with computer-predicted plots for various models (dashed line : cxperimental scattering curve after extrapolation to c = 0). From Lcderer (1972).

15

FIBRINOGEN TO FIBRIN CONVERSION

chemical studies and electron microscopy. His conclusion was that the data best fit the interesting model proposed by Koppel (1967), although even in that case there were significant discrepancies (Fig. 7 ) .

D . Biochemical and Organochemical Characterization 1. Amino Acid and Carbohydrate Composition

The amino acid compositions of the four mammalian fibrinogens which have been analyzed are similar and undistinguished, all the usual amino acids being present in reasonable amounts (Fig. 8 ) . Analyses performed on automatic analyzers (Mihalyi et al., 1964; Henschen and Blomback, 1964; Cartwright and Kekwick, 1971) have not proved significantly different from those reported a generation ago (Tristram, 1953). A few unusual amino acids do occur in fibrinogen, including a tyrosine0-sulfate which is present in most fibrinopeptides B (Bcttelheim, 1954).

n W

>

0: W

cn

m

. 0 0

0

I

2

3

4 O/o

5

6

7

8

9

10

II

12

EXPECTED

FIG.8. Average frequency of occuncnce of amino acids in mammalian fibrinogen (four species : human, pig, sheep, ox) compared with theoretical distribution (solid line) based on random distribution of triplets with observed maminalian base composition. A = fibrinogen; 0= frequency in 53 other vertebrate proteins which have been completely sequenced and which have been depicted in this fashion by King (1971). The fibrinogen amino acid frequencies were calculated from the compositions reported by Cartwright and Keliwiclc (1971);sec also Table X. Tlic iatios of upartic acid/asparaginr and glutamic ncid/glutamine linve been set arbitrarily to 1.5. For a discusaion of the widely observed “aryinine moindy,” sc‘c King (1971).

16

R. F. DOOLITTLE

This unusual derivative-found elsewhere in only a few polypeptide hormones and vasoactive factors-has been identified in a wide variety of fibrinopeptides B, including a fish (Doolittle, 1965b), the frog (Gladner, 1968) and a large number of mammals. It is not present in higher primates and the rat, however (Blomback and Blomback, 1968; Wooding and Doolittle, 1972). A phosphoserine residue occurs in the fibrinopeptide A of several higher primates, including man (Blomback e t ul., 1962; Doolittle et at., 1971b), and also the dog (Osbahr et al., 1964). Small amounts of phosphorus may also occur in parts of the fibrinogen molecule other than the fibrinopeptides (Blomback et al., 1963; Krajewski and Cierniewski, 1972). Fibrinogen has no free sulfhydryl groups, all its cysteine being involved in disulfide bridges. Henschen's data on bovine and human (Henschen, 1964b) indicate that there should be 28-29 disulfide bridges per 340,000 molecular weight, but the data of Cartwright and Kekwick (1971) suggest that the number might be as high as 32-34. On the other hand, some investigators put the number as low as 21-22 (Latallo et al., 1971). Mammalian fibrinogens contain 4-576 carbohydrate, mostly consisting of neutral hexoses, glucosamine, and sialic acid (Blombilck, 1958a). Its location in the molecule is described more fully in following sections. 2. End-Group Determinations

Amino-terminal analysis has played an enormously important role in our understanding the conversion of fibrinogen to fibrin, proving without doubt that limited proteolysis is the triggering mechanism for fibrin formation (Bailey et at., 1951). Beyond that, it was the quantitative determination of amino terminals of a variety of fibrinogens and fibrins which showed that the native fibrinogen molecule is a dimer, each particle of 340,000 molecular weight having three pairs of nonidentical polypeptide chains (Blomback and Yamashina, 1958). Two of the three different chains were found to undergo a change in amino terminal as a result of thrombin-catalyzed fibrin formation, corresponding to the release of the fibrinopeptides A and B. By convention, the chains that lose the fibrinopeptides A are called the a-chains and the ones that lose the fibrinopeptides B the P-chains.l The chain whose amino-terminal residue The International Committee on Nomenclature of Blood Clotting Factors has designated the fibrinogen chain with the fibrinopeptide A still attnrhed An, and the chain with the fibtinopeptide B a t its amino terminus the Bp. The corresponding chains from fibrin, after the removal of the fibrinopeptides, are called the a-chain and /3-chain. I n this article the Aa and B/3 chains are simply called the fibrinogrn achain and P-chain.

17

FIBRINOGEN TO FIBRIN CONVERSION

TAIILI': I11 Amino- and Curboxy-2'crminal GTOUPS of Six Murnmaliun Fibrinogens" a-Chain Mammal

ox

Sheep Pig Human Dog Horse

Amino-

Carboxy-

Glu Ala Ala Ala Thr Thr

Pro Pro Pro Val Pro Val

&Chain Amino-

&lu

GlY Ala ElU His Leu

7-Chain

Carboxy-

Amino-

Carboxy-

Val Val Val Val Val Val

TYr TYr TYr *Yr TYr TYr

Val Val IIe Val Ile Val

Amino-terminal data from Ijayhoff (1969); carboxy-terminal data from Okude and Iwanaga (1971).

remains unchanged is termed the 7-chain. The formula a2pzy2apparently applies t o all vertebrate fibrinogens (see also Section VI,E,l) . The amino terminals of fibrinogen a-chains vary in the more than fifty species that have been examincd, although there is a decided preference for alanine or threonine (Dayhoff, 1969). Similarly, although a wide variety of residues exists a t the amino terminus of the p-chains from various species, a disproportionatc number of pyrrolidone carboxylic acid residues2 ( m l u ) has bcen found (BlombBck and Doolittlc, 1963a,b; Mross and Doolittle, 1967; Wooding and Doolittle, 1972). Most mammalian y-chains have amino-terminal tyrosinc (BlombBck and Yamashima, 1958), although several cxccptions have now been found (O'Neil and Doolittlc, 1973). Thc onc lower vertebrate which has bccn studied has serine a t the amino terminus of its y-chains (Doolittle et al., 1963; Doolittle, 1965a). The carboxy terminals of fibrinogens from six different mammals have been determined (Okude and Iwanaga, 1971). I n four of these species, the a-chain terminates with proline (ox, sheep, pig, and dog) ; in the other two it is valine (human and horse). All the p-chains examined have carboxy-terminal valine. I n the casc of the y-chains, pig and dog end with isoleucine, whereas tlie remaining four havc carboxy-terminal valine (Table 111). 3. Structural Studies on Fibrinopeptides

The amino acid sequences of fibrinopeptides from a large number of species have been determined. Most of these have been accomplished

' Konstandard abbreviations u s d in this nrticlc include SDS (sodium dodecj 1 aulfate), PAS (periodic ncid-Scliiff), and m l u (pyrrolidone carboxylic acid).

18

R. F. DOOLITTLEI

in Blomback’s laboratory in Stockholm (see, e.g., Blomback and Blomback, 1968) or in the author’s laboratory (Doolittle, 1970), and are tabulated in Dayhoff’s Atlas (Dayhoff, 1969). These structures are exceptionally variable and have been of great interest in the area of molecular phylogeny and evolution (Doolittlc and Blomback, 1964). On the other hand, some gcneralizations about structurcfunction relationships can be made on the basis of these data also. I n spite of the great variability-which implies a nonspccific function that can be satisfied by a large number of amino acid combinations-certain features of these peptides have been conserved during the course of evolution. For example, the junctions split by thombin are always arginyl-glycine bonds, in lower vertebrates as well as mammals (Doolittle, 1965a). Furthermore, all the fibrinopeptides examined bear a substantial negative charge (ranging from -2 to - 6 in mammals), a property consistent with early notions that the fibrinopeptides prevent polymerization by the mutual electrostatic repulsion of individual fibrinogen molecules. This tendency toward electronegativity is also reflected in the occurrence of the sulfated tyrosines, the phosphorylated scrines, and the pyrrolidone carboxylic acidterminal residues. The lengths of the fibrinopeptides range from 13 to 21 residues in mammals, although they may be as long as 4&45 residues in lower vertebrates (Doolittle, 1965b; Gladner, 1968). A number of substantial deletions, terminal and internal, has occurred during the evolution of these structures without apparent consequence to the parent fibrinogen molecule. I n two closely related buffalo, for example, the only differences in the amino acid sequences of their fibrinopeptides A is an internal deletion of four residues (Mross and Doolittle, 1967). The fibrinopeptides B tend to be somewhat more changeable than the A peptides, including several large-scale deletions (Wooding and Doolittle, 1972), suggesting that their role is even less demanding than that of the A pcptidc. I n spite of this great tolerance for change, some amino acids have never been found in any of the fibrinopeptides examined. In this regard, cysteine, methionine, and tryptophan appear to have been selected against. I n the case of cysteine, there could be an obvious disadvantage if a free sulfhydryl group existed in these presumably exposed locations, since bridge formation leading to premature polymerization would always be a threat. Similarly, tryptophan and methionine might be thcrmodynamically undesirable in these polar charge clusters. The liberated fibrinopeptides exhibit the characteristic spectrum of a random coil when examined by circular dichroism methods, although this may have no bearing on their natural configuration before release from the parent fibrinogen molecule.

FIBRINOGEN TO FIBRIN CONVERSION

19

4. Separation and Characterization of Subunits I n 1962 two laboratories independently reported the separation of the three nonidentical chains by chromatography after sulfitolysis of fibrinogen (Henschen, 1962; Clegg and Bailey, 1962). Henschen (19Ma) was subsequently able to prepare thc individual chains in sufficient quantity for a rigorous chemical characterization. Using similar procedures, McKee et al. (1966) determined the molecular weights of the individual chains from human fibrinogen and found them to add up to approximately 170,000, the accepted valuc for a half-molccule of fibrinogen. The individual values, which were dctermined by sedimentation equilibrium in the prcscncc of 6 M guanidine, were @-chain = 63,500, ,@-chain= 56,000, and 7-chain = 47,000 (there was some confusion about nomenclature in that report, the designations C, B, and A bcing used for the chains we now call p, and 7). The amino acid compositions of thc three chains, while unremarkable, are significantly different from each other. Hence, the a-chains from a variety of species all have considerably less tyrosine than the p- and y-chains, but arc richer in arginine and scrinc (Cartwright and Kekwick, 1971). 7-Chains have significantly less prolinc than the other two chains, and p-chains are richer with regard to methioninc and cysteinc. All threc chains have been found to contain carbohydrate (Henschen, 1964c; Mills and Triantaphyllopoulas, 1969), although on SDS gel electrophoresis only ,@-chains and 7-chains are PAS-positive (Pizzo et al., 1972; Gaffney, 1972). I n the case of the 7-chain, one carbohydrate attachment site has been located at asparagine-52 (Iwanaga et al., 1968). Various groups have reported different degrees of heterogeneity in their fibrinogen preparations. Gerbeck et al. (1969) have reported thc existence of two different 7-chains in bovine fibrinogen, and Henschen and Edman (1972) have rcported licterogcneity among human y-chains. Muraiio et al. (1971), on the other hand, find extensive heterogeneity among human @-chains, but not in 7-chains. Whether or not these rcports of hetcrogcncity rcflcct genetic polymorphisms, incomplete carbohydrate attachment :mcl/or rcmoval, or incidental protcolysis in vivo or during preparation, or all of these, is not yet clear. (Y,

5. Halj-Molecule Claims Once it had been established that the native vertebrate fibrinogen molecule was a dimer composed of threc pairs of nonidentical chains, the central problem was to find out how thcse wcre arranged. I n particular, the big question was, how can the six polypcptide chains bc packed into the tlircc balls making up the Hall and Slaytcr model? It was clear

20

R . F. DOOLITTLE

Thnm IV Sonic Rrports on tho Dissociation of Fihrinogn without Prior Ruptzrrr of Dim@dr Bonds Conditions

Methods of analysis

Low fibrinogen concentration

Ultracentrifuge, diffusion measurements Light scattering, viscosity Ultracentrifuge

Low ionic strengih Pretreatment, with EDTA Maintenance at 37°C

Ultracentrifuge, light scattering

Reference Caspary and Kekwick (1957) Sowinski rt al. (1050) Blombiirk rt al. (1966) Capet-Antonini and Guinand (1970)

that much of the integrity of the fibrinogen molecule depended on disulfide bonds, but it was by no means certain that all six of the chains were interconnected by such bridges, Over the years a number of reports (Table IV) have claimed that fibrinogen can be dissociated into half-molecules or their equivalent (presumably a-p-y) without the rupture of disulfide bonds, including observations in the ultracentrifuge a t low fibrinogen concentrations (Caspary and Kekwick, 1957), conditions of low ionic strength (Sowinski e t al., 1959), pretreatment of the fibrinogen with ethylene diamine tetraacetate (EDTA) (Blomback et al., 1966; Capet-Antonini, 1970) and maintenance of the fibrinogen a t 37°C (CapetAntonini and Guinand, 1970). All these results are a t variance with a variety of other experiments, which indicate that fibrinogen molecules cannot be dissociated without first breaking disulfide bonds. For example, Johnson and Mihalyi (1965a) showed that the molecular weight of fibrinogen is unchanged when measured in the presence of 5 M guanidine, a solvent that could reasonably be expected to dissociate a noncovalently bonded dimer. Similarly, when unreduced fibrinogen is run on SDS-polyacrylamide gel electrophoresis after treatment with concentrated SDS-urea solutions, the native molecular weight of 340,000 is observed. Furthermore, other workers (Endres and Scheraga, 1971) have been unable to confirm some of the earlier claims about temperatureinduced dissociation, although it should be noted that they were working a t significantly higher fibrinogen concentrations. Finally, there is a growing mass of evidence accumulating from structural and degradativc studies that all six chains are indeed bound together by disulfide bonds. There is a tendency to ignore the “half-molecule data” as mcrcly curiosities. The data that have appeared, however, clearly indicate that so?nething happened to the fibrinogen molecules under the experimental conditions rcportcd. The ultracentrifuge results could he rationaliacd as bcing duc to somc deformation of the native structure, keeping in mind

21

FIBRINOGEN TO FIBRIN CONVERSION

that the theoretical deformation would have to make the structure less compact or more hydrated in order to make it sediment more slowly. On the other hand, some of the observations (Table IV) were made using light scattering and viscosity as experimental tools, and these data also indicated dissociation into subunits. These observations are cited here on two counts. First, one of the areas of greatest conflict in reconciling physical and biochemical observations hinges on the existence of disulfide bonds between the two halves of the molecule. Second, if fibrinogen was not dissociating in these experiments, then its anomalous behavior might be helpful in deciphering other puzzling structural aspects. A t t e m p t s to Produce Half-Molecules after Mild Reduction. If, in fact, the dimeric halves of the fibrinogen molecules are joined by disulfide bonds, then it might be possible to produce half-molecules after mild reduction. The success of workers in the immunoglobulin field in producing half-molecules of rabbit y-globulin a t very low levels of reducing agent (Palmer and Nisonoff, 1964) prompted a number of similar experiments on fibrinogen. Budzynski and Stahl (1969) observed that a small number of disulfide bonds in fibrinogen was reduced faster than the remainder. Moreover, full clottability was maintained, as were those physical parameters measured by viscometry, electrophoresis, and gel filtration under nondissociation conditions. The assignment of these easily reduced bonds to particular chains has been accomplished by using [“C] iodoacetamide as an alkylating agent after reduction a t varying concentrations of dithiothreitol (Fig. 9 ), followed by SDS-polyacrylamide electrophoresis to locate the radioactivity

32 CI

i

24

x

16

8

0

0 0

0.2

0.4

0.6

0.8

1.0

DTT (moles/liter)

FIG.9. Inrorporntion of [“Cliodonretamidr (IA) into native bovinc fibrinogrn

as n function of dithiothreitol (DTT) concentration.

22

R . F. DOOLITTLE rnm FROM ORIGIN

800

FRACTION NUMBER

Fro. 10. Distribution of radioactivity in sodium dodecyl sulfate-polyacrylamide gels after electrophoresis under fully reducing conditions. B ( - . - .1, c ( - - - ) , D (-1, and E refer to points in Fig. 9 corresponding to different amounts ( . - . a )

of ["Cliodoacetamide incorporated. a, /3, y indicate positions of fibrinogrn chains.

in the individual chains (Fig. 10). The first two disulfide bonds to be broken both appear to be in the a-chains, two moles of alkylating agent being taken up in those chains before any radioactivity appears elsewhere. The third bond to be split is apparently between two y-chain segments. As in the case of the Budzynski and Stahl (1969) experiments, no loss of clottability was occasioned by these mildly reduced preparations. Nor were half-molecules observed on the SDS gels under these conditions. I n fact, when the dithiothreitol concentration was raised, small amounts of the individual a-, p-, and y-chains began to appear more or less simultaneously, suggesting a complicated interchain structure. 6. The Disulfide Knot

Cyanogen bromide digestion of whole human fibrinogen, which contains about thirty methionine residues per 170,000 molecular weight, results in a very large number of virtually irresolvable fragments. B. Blomback et aE. (1968) arbitrarily divided a Sephadex G-100 gel filtration efiuent of such a digest into a number of fractions and examined each for the release of the fibrinopeptides A and B after the addition of thrombin. Coincidentally, both pcptides were released in the same fraction, and when this material was purified further, it was also found to contain the aminoterminal tyrosine of the y-chain. I n a single cyanogen bromide fragment, then, all three amino-terminal segments were found bound together. The

FIBRINOGEN TO FIBRIW CONVERSION

23

fragment also contained about half of the cysteine in fibrinogen, even though accounting for only 15% of thc mass. The piece, which is refcrred to as “the disulfide knot” (DSKj , has bceii thoroughly characterizcd. Originally the molecular weight of the fragment was put at 26,500 as iiicasurcd by sedimcntatioii equilibrium (B. Blombiick et al., 1968). The amino acids thought to be present at the time added up to a molecular weight of 21,700, but this number was subsequently revised upward when the /?-chain piece turned out to be twice as large as originally thought (115 residues instead of 57). The DSK also contains carbohydrate. A major shift in thinking was effected, however, when subsequent molecular weight determinations yielded a molecular weight of 56,000 (Blomback, 1971a,bj. This reinvestigation was prompted, a t least in part, by the isolation of disulfide-linked pairs of a-chain and 7-chain peptides, respectively. The conclusion which has now been reached is that all six amino-terminal regions of the polypeptide chains comprising a molecule of fibrinogen are bound together in the disulfide knot (Fig. 11). This unexpected development is a t odds with certain physical data that position the fibrinopeptides A at opposite ends of the molecule (Section IV,C) , and it is certainly not consistent with any notion of a noncovalently bonded dimer. It is in good agreement, however, with other fragmentation studies discussed in the following section. The question arises whether an artifact due to disulfide exchange could have occurred. I n fact, great carc appears to have been taken to prevent that possibility, including treating preparations of fibrinogen with iodoacctamide during purification as well as keeping the pH low throughout the experiment

FIG. 11. Schematic depiction of disulfide bond arrangemcnt in disulfide knot (DSK) isolated from cyanogen bromide digest, of human fibrinogen. Redrawn from

Blombiick (1971b).

24

R . F. DOOLITTLE

(Blomback, 1971a). On the other hand, it is disconcerting that higher polymers of the DSK, apparently disulfide linked, have also been observed (Blomback, 1972).

7. Enzymatic Degradation of Fibrinogen Proteolytic fragmentation has been artfully applied to the characterization of a number of large proteins, including the immunoglobulins, myosin and collagen. In each of these cases some regions of the molecules were more accessible and/or vulnerable than the remaining, presumably more compacted, domains. Judicious selection of conditions has allowed major portions of each of these proteins to be characterized in their own right, independent of the parent molecule. Furthermore, it was possible to draw conclusions about the overall arrangement of these fragments which were subsequently borne out by other approaches. Accordingly, this same strategy was adopted by Mihalyi and his coworkers, using trypsin and chymotrypsin in early attempts to produce a limited fragmentation of fibrinogen (Mihalyi and Godfrey, 1963). These initial studies, which were interpreted mainly in terms of the Hall and Slayter three-ball fibrinogen model (Johnson and Mihalyi, 1965b), were soon overshadowed by a host of studies employing plasmin as the degradative agent, primarily because of the clinical implications of fibrinogensplit-products (Niewiarowski and Kowalski, 1958; Triantaphyllopoulas, 1958). Plasmin has a more restrictive specificity than trypsin, displaying a definite preference for lysine side chains (Weinstein and Doolittle, 1972), and its action on fibrinogen results in a reproducible set of fragments which have been characterized extensively (Marder et al., 1969). Briefly, the first intermediate to appear during the plasmin digestion of fibrinogen is termed fragment “X”; it has a molecular weight of about 240,000 and is fully clottable. Fragment “X” is then broken down into two major pieces, “Y” and “D”, the molecular weights of which are about 155,000 and 85,000, respectively; these two pieces are not clottable but do inhibit fibrin formation when mixed with undigested fibrinogen. Fragment “Y” is subsequently fragmented further into another molecule of “D” and a second product termed “El’, the molecular weight of which is about 50,000. Marder, noting the asymmetric mode of several of the cleavages, has shown how the intermediate and final products of this digestion correlate very well with the Hall and Slayter model of fibrinogen (Fig. 12). Fragment “E” has subsequently been shown to be virtually identical with the disulfide knot (DSK) released by cyanogen bromide fragmentation of fibrinogen (Marder, 1971), an observation very much in keeping with the discovery that the DSK contains all six aminoterminal sections of the parent molecule, which in turn fits well with

FIBRINOGEN TO FIBRIN CONVWION

1 J ”D”

“E”

25

“X”

”Y”

“0“

FIG.12. Schematic depiction of the degradation of fibrinogen by plasmin showing how compatible the pattern of split products is with Hall and Slayter’s model. Molecular weights: fibrinogen = 330,000; “X” = 240,000; “Y” = 155,000; “D” = 85,000; “E” = 50,000. Adapted from the description of Marder (1970).

Marder’s earlier proposal that fragment “E” is the central sphere in the Hall and Slayter model. Recently a plethora of studies has appeared which utilized the convenience of SDS-polyacrylamide gel electrophoresis for following the plasmin degradation of fibrinogen. These reports have clearly established that a-chains are the first to be significantly degraded by plasmin, the conversion of the native molecule to fragment “X” being primarily due to major losses from the a-chain carboxy terminal (Gaffney and Dobos, 1971). I n addition to being the first chains attacked by plasmin, a-chains are also readily attacked by certain snake venom enzymes. A r ~ i n for , ~ example, reduces the molecular weight of a-chains from 65,000 to 40,000 when inducing the transformation of fibrinogen to fibrin (Mattock and Esnouf, 1971). Mills and Karpatkin (1971) have also reported that a-chains are particularly susceptible to proteolytic attack and have The most commonly used snake venom enzyme preparations used in this regard are Reptilase, from the venom of Bothrops javaraca (also marketed under the trade name of Bemostase), and Arvin, an extract from the venom of Agkistrodon r h o d o s t o m . In the latter case the active principle is also called Ancrod.

26

R. 1.“. DOOLITTLE

even suggested that thrombin can degrade a-chains over and beyond the removal of the fibrinopeptide A. It should be recalled that the bacterial enzyme prcparation used by Tooney and Cohen (1972) to produce fibrinogen microcrystals also degraded the a-chain (Section II,B) . All these studies suggest that the a-chain has an open and more exposed structure than the p- and y-chains. Other SDS-gel electrophoresis studies have confirmed and refined the sequence of events depicted by Mardcr (1970) (Fig. 12). Pizzo et al. (1972), Mills (1972), and Furlan and Beck (1972) all report that fragment “E” has properties corresponding to the DSK. One significant difference between fragment “E” and the DSK is that a portion of the ,&chain amino-terminal region-including the fibrinopeptide B-is chewed off by plasmin early in the digestion (Mills, 1972; Furlan and Beck, 1972). There is some disagreement, however, as to the chain composition of fragment “D”, Furlan and Beck (1972) finding it to be composed of pieces of ,8- and 7-chain only, whereas the others report contributions from all three chain-types. SDS-gel studies employing carbohydrate stains have not been in good agreement with earlier studies. Henschen (1964~)found all thrce chains to be PAS positive upon examination by papcr electrophoresis, and Mills and Triantaphyllopoulas (1969) identified carbohydrate in all three chains after separation by chromatographic techniques. I n contrast, two studies using SDS-gels (Pizzo et al., 1972; Gaffney, 1972) have found only the p- and 7-chains to be PAS positive. Mills and Triantaphyllopoulas (1969) found that the y-chain carbohydrate ends up with fragment “E” after plasmin digestion of fibrinogen, an observation confirmed by the SDS-gel studics. On thc other hand, they concluded that the carbohydrate found in fragment “D” originated in the a-chain, whereas the SDS-gel experiments attribute it to P-chains. 8. Large-Scale Amino Acid Sequence Studies on Fibrinogen

The amino acid sequences of those portions of human a-, p-, and y-chains isolated as the disulfide knot have been completely determined (Iwanaga et al., 1968, 1969; Blombtlck, 1971b; Blombiick et al., 1972). I n the case of the a-chain this amounts to a 54-residue sequence from thc amino terminus, including the previously studied fibrinopeptide A. The 8-chain fragment is 115 residues long, including the fibrinopeptide B, and the y-chain piece consists of 78 amino acids. These scqucnces are discussed further in Section VI,E,3. I n addition to the studies on the DSK, a 28-residue section a t the carboxy terminus of human and bovine y-chains has been sequenced (Chen and Doolittle, 1971; Sharp et al., 1972). At this writing more than 80% of the primary structure of fi-

FIBRINOGEN TO FIBRIN CONVERSION

27

brinogen remains to be detcrmincd, although studies are currently under way in a number of laboratories. Summing to this point, the physical chemistry of fibrinogen indicates that it is either a highly elongated and/or very highly hydrated molecule with an approximate molecular weight of 340,000. Electron microscopy has provided a wide selcction of photographs and models derived therefrom, most of which can accommodate the bulk of the physicochemical data if suitable assumptions are made. The most popular of these models has been that proposed by Hall and Slayter (1959) consisting of three linked globules. A small-angle X-ray study appears to be more in agreement with a symmetrical, highly hydrated molecule, but electron micrographs of fibrinogen-derived microcrystals indicate a fundamental molecular unit of dimensions 90 x 450 A. Biochemical and organochemical fragmentation of the fibrinogen molecule has resulted in the characterization of three pairs of nonidentical chains, all of which appear to be joined near their amino terminals by disulfidc bonds. Proteolytic fragmentation with plasmin gives rise to a set of fragments which is in good agreement with a thrcc-ball model of fibrinogen. About 300 of the 1500 amino acids in a half-molecule of (human) fibrinogen have been positioned in a linear sense (sequenced), most of these being near the amino-terminal ends of the three different chains.

111. STRUCTURE AND PROPERTIES OF FIBRIN A. Definitions of Fibrin Basically, fibrin is the product that results upon the polymerization of fibrinogen. The transformation can take place in a variety of ways, however, and the physical and chemical properties of the final product are not equivalent in all cases. For example, fibrin can be prepared in which only the fibrinopeptides A have been removed, either by digestion with certain snake venom enzymes3 (Blomback et al., 1957) or by the use of distantly related (heterologous) thrombins (Doolittle e t al., 1962; Doolittle, 1965s). Fibrinlike gels can also be prepared in which no fibrinopeptide material a t all has been removed, either by the addition of stoichiometric amounts of protamine sulfate (Stewart and Niewiarowski, 1969), which presumably neutralizes the fibrinopeptides and/or other negative charge clusters, or even by simply pelleting pure fibrinogen solutions in the ultracentrifuge (Stryer et al., 1963). Fibrin gels with the fibrinopeptides still attached can also be prepared by direct covalent cross-linking by certain transamidases (Farrell and Laki, 1970) or by the introduction of covalent bonds by direct chemical procedures (Section V,F). Furthermore, there are significant differences in the properties of

28

R. F. DOOLIlTLE

thrombin-induced fibrin depending on the environmental conditions during the polymerization, and there are naturally certain differences if the fibrin is subsequently stabilized by the action of activated factor XIII (Section V ) . I n this section, however, we will be concerned primarily with fibrin produced by the action of thrombin on fibrinogen in which, unless otherwise stated, it is presumed that both pairs of fibrinopeptides have been removed and no covalent bonds have been introduced.

B. Some Physical Properties of Fibrin Gels Protein gels can be categorized into two broad classes (Ferry, 1948), fully oriented, on the one hand, or the network type on the other. The first of these is typified by concentrated solutions of tobacco mosaic virus, in which case the elongated particles become oriented in an infinite hexagonal array in order to minimize mutual coulombic repulsion. I n the network kind of gel, which is the basis of gelatin as well as fibrin, fibers pervade the system in three dimensions, forming a large open water trap (Fig. 13). The distinction is an important one because network gels have considerably less contributory solid than do fully oriented gels. It must be regarded as remarkable that in the case of fibrin, the system may contain as little as 0.05% protein and yet behave as a rigid solid (Ferry, 1948). It must be emphasized that the polymerization of fibrinogen is primarily directed a t fiber generation and not a fully oriented packing. The constitution of these fibers, and especially their breadthwhich can reach macroscopic proportions-is very sensitive to environmental conditions during the polymerization process, including the concentration of fibrinogen, ionic strength, pH, concentration of thrombin and the presence or absence of a variety of small molecules.

(a)

FIG.13. Comparison of molcculnr arrays in

work type gcl (right).

(b)

n fully orirntrtl grl

Redrawn from Fcrry (1948).

(Irft) nntl

:I

not-

FIBRINOGEN TO FIBRIN CONVERSION

29

Classically fibrin gcls h a w been classified as “coarse” (highly opaque) or “fine” (transparent), although all gradations between these two extremes can exist (Ferry and hlorrison, 1947). Generally speaking, opaque clots arc thought to have coarser fibers and are formed under conditions of modcratcly low pH (6-7) and low ionic strength (0.1-0.2), whereas fine clots are prepared a t higher p H (8-9) and higher ionic strength (0.3-0.5). Many other factors can alter this situation, including the fibrinogen concentration. I n this regard, most of the fibrin preparations discussed in these sections were prepared starting with 0.20.5% fibrinogen solutions. I t might just be mentioned that the upper concentration limit for fibrin formation is simply a matter of the solubility of fibrinogen, which under ordinary circumstances is approximately 4 g per 100 ml (4%). Naturally there are some obvious diff erences between fibrin gcls formed in vivo and those prepared from purified fibrinogen solutions. Under natural conditions the propagating fibers tend to wrap around the formed elements of the blood (Fig. I ) , including red and white blood cells and platelets. Platelets contain a contractile protein which is the basis of natural clot retraction, resulting in the squeezing out of trapped fluids. This phenomenon should not be confused with the syneresis of fibrin gels prepared from fibrinogen solutions, which can also be mechanically compacted, by pressing out the clot with a glass rod, for example. I n the latter case the fibers tend to collapse upon each other under the influence of local-attractive forces, this process also leading to the elimination of trapped solvent.

C. Studies o n Fibrin Monomer Ordinary fibrin prepared by the addition of catalytic amounts of thrombin to solutions of purified fibrinogen can be reversibly dispersed by traditional unfolding solvents, including strong urea or guanidine solutions. These gels are also rendered soluble by dilute acids or base or various combinations of low pH and high salt. Accordingly, a large number of physicochemical studies have been conducted on dispersed fibrin preparations, including flow birefringence (Donnelly et al., 1955), light-scattering determinations (Steiner and Laki, 1951), viscosity measurements (Mihalyi, 1950a), electric birefringence experiments (Haschemeyer, 1963), isoelectric point determinations (Mihalyi, 1950b), and a variety of molecular weight measurements (Ehrlich et al., 1952). All these observations have been in accord with the general notion that the monomeric unit in fibrin (fibrin monomer) has virtually the same size and shape as the starting fibrinogen molecule, cxcept for those properties that reflect the removal of the electroncgative fibrinopeptides. I n this

30

R. F. DOOLITTLE

latter regard, the isoelectric point of fibrin is slightly higher than that of fibrinogen (Mihalyi, 1950b), and the fibrin monomer has a measurable transverse dipole moment, indicating that fibrinopeptides are not located symmetrically with regard to the longitudinal axis (Haschemeyer, 1963).

D. X-Ray Studies on Fibrin Thirty years ago Astbury’s group (Bailey et al., 1943) discovered that

fibrin and fibrinogen gels prepared by ammonium sulfate precipitation gave essentially identical patterns when studied by wide-angle X-rays. Bailey et al. (1943) concluded that individual fibrinogen units maintained their size and shape during the transformation into the gel: “In other words, fibrin is no other than an insoluble modification of fibrinogen without any fundamental change in molecular plan.” As we have seen, the physical chemistry of fibrin monomer is in total agreement with the conclusions of that early X-ray analysis. It is the detailed arrangement of those units which interests us, however, and another X-ray study, performed twenty years later, has provided a clue to those details. Stryer et al. (1963), using a low-angle approach, demonstrated that fibrinogen gels (prepared by pelleting in the ultracentrifuge) and fibrin both exhibited repeat distance of 226A. Assuming that the individual molecular lengths were of the order of twice that distance (as they would be according to the Hall and Slayter model of fibrinogen), the authors proposed that the repeat distances corresponded to a staggered overlapping of the packed molecules. The same suggestion had been made previously by Ferry (1952) on the basis of certain other physical measurements, and we will discuss these in more detail in later sections.

E . Electron Microscopy of Fibrin Even the earliest electron microscope studies on fibrin provided dramatic pictures of smoothly branching fibers exhibiting very uniform widths over great distances (Fig. 14). The breadth of these fibers, as mentioned earlier, was dependent on the conditions of formation, but once a critical width had been achieved-perhaps as little as three or four molecules thick-a characteristic banding pattern appeared with a spacing measured a t 220-250 A. A number of different interpretat.ions of this repeat unit has been offered by various investigators, each group offering a rendition which is most compatible with its own model of fibrinogen (Fig. 15). Koppel (1967) , for example, believes that the 230 A banding is a direct reflection of the diameter of his polyhedrally shaped fibrinogen molecule (Fig. 15D), whereas a t the other extreme, Kay and Cuddigan (1967) propose that the 230 A spacing is due to a two-thirds molecular overlapping of fibrinogen molecules which are themselves about 690 A

FIBRINOGEN TO FIBRIN CONVERSION

4

W

pm

Fro. 14. An early electron micrograph of fibrin. From Emvn and Porter (1947)

31

32

R. F. DOOLITTLE A

D

FIG. 15. Depictions of fibrinogen and fibrin as derived from various electron microscopy studies: (A) Kay and Cuddigan (1967) ; (B) Hall and Slayter (1959) ; (C) Bang (1964) ; (D) Koppel (1967).

long (Fig. 15A). Bang (1964) also interpreted the banding in terms of overlapping molecules, but his scheme features a one-third overlap of fibrinogens which are about 375A long. As pointed out in the preceding section, the Hall and Slayter model would be consistent with an overlapping equal to half a molecular length yielding a repeat unit of approximately 230 A. Interestingly enough, Hall and Slayter themselves did not interpret the fibrin banding in that fashion. Rather, they proposed t h a t during the polymerization process therc is a massive compression of the fibrinogen molcculc leading to a packing unit about 230A in length (Fig. 15B). Their basis for this conclusion had to do with the fine structure of the banding pattern, which was not a simple alternation of light and dark bands of the same dimensions. In fact, the pattern they observed consisted of a dark wide band, a light band, a narrow dark band, and then a light band and a dark wide band. To them it seemed quite natural t h a t this pattern was a direct reflection of the packing of terminal spheres, on the onc hand, and central spheres, which they had found to be SmallCr, on the other. It was subsequently found that fibrin exhibits slightly different banding

FIBRINOGEN TO FIBRIN CONVERSION

33

patterns dcpcnding on conditions of staining. Typc I patterns, which arc the kind achieved by Hall 2nd SInytcr, arc commonly observed in positively stained or shatlowcast prcparations, whereas a second kind of banding, Type 11, is usually o l ~ s e ~ cind negatively stained specimens (Kargcs and Kuhn, 1970). Both types yield t’nc same overall spacing pattern, and Kargcs and Kuhn (1970) have explained the differences as being duc to an unevcn distribution of polar groups in the fibrin units in thc casc of positive staining, and differences in sample density in the case of negative staining (Fig. 16). They have concluded that the‘ most polar regions of the fibrin molecular units arc also the least dense, the implication being that the protein has a much less compact structure in these areas (Karges and Kuhn, 1970). I n any event, the demonstration of the two banding patterns on thc same fiber under different conditions does suggest that it is highly improbable that the Type I pattern correlates directly with the three balls of Hall and Slayter model, and it is perhaps fair to say that almost all investigators today agree that the fibrin banding is not duc to any drastic compression of the starting molecules, the vast majority attributing it to some degree of molecular overlap. Recently Stewart and Niewiarowski (1969) compared thrombininduced fibrin with fibrinogen polymerized by the addition of protamine sulfate. The average periodicity of “thrombin-fibrin” was found to be 239 A in shadowed and 228 A in negatively stained preparations. I n the case of protaminc-sulfate fibrinogen films, the corresponding periodicities

I+

240i

POLAR REGION

f

POSl TlVE STAl N I NG

A b A

1 1 1

TYPE

NEGATIVE

II

STAINING

240Aj

\DENSEST REGION

FIG.16. Dingraninintic inteiprctation of diffcrrnt strintion pnttcrns obscrwd in clcctron microgr:Lplls of fibrin aftt,r positi\ e and nraativr staining. The proposnl I S that the most polar rcgions of ihr incli\-idunl units air tlic least compact (dcnsc). Adapted from Iiarges and Iiiihn (1970).

34

R. F. DOOLITTLE

were 225 and 223 A. It might be mentioned in passing that in this latter case branching networks occurred only occasionally, most of the fibers being broad with blunted spurs.

F. Chemical Composition of Fibrin Everything we know with any certainty about the chemical composition of fibrin is consistent with its being identical with fibrinogen except for the absence of the fibrinopeptides. The amino-terminal end groups naturally reflect the fibrinopeptide release, four moles of glycine appearing for approximately every 340,000 molecular weight in virtually all species that have been examined (Blomback and Yamashina, 1958). The carboxy terminals of fibrin have been studied less intensively but appear to be constant within the limits we have set down regarding thrombin-induced fibrin. The vulnerability of the carboxy-terminal portion of the a-chain (Section II,D,7), while not in any way considered a fundamental aspect of fibrin formation, may give rise to some differences in the fibrin carboxy-terminal picture in certain situations. The disulfide bdnd situation appears to be the same in fibrin and fibrinogen (Henschen, 196413), although the unlikely possibility of a rearrangement of bridge partners has not been rigorously ruled out. Preparation of the individual a-,p-, and y-chains follows much the same lines as employed for fibrinogen (Henschen, 1 9 6 4 ~ ) . There have been assorted reports over the years that carbohydrate is released during the transformation of fibrinogen to fibrin (Scheraga and Laskowski, 1957). Blomback (1958a) did find a slightly lower hexose content in fibrin compared with fibrinogen, but the difference was within the experimental error of measurement. Laki and Mester (1962) reported that the clottability of fibrinogen decreases proportionately with the extent of destruction of carbohydrate by periodate oxidation, but one would be hard-pressed to conclude a specific role for carbohydrate on the basis of that correlation alone. Later, Chandrasekhar and Laki (1964) reported that carbohydrate was released during the covalent stabilization of fibrin, an amino sugar acting as leaving group upon introduction of the amino group involved in cross-linking. I n support of their proposal, they also reported that sialidase-treated fibrinogen cannot be rendered urea insoluble, a characteristic of covalently reinforced fibrin (Chandrasekhar et al., 1964). Subsequent reports have not supported the claims that carbohydratc is released, however. Hormann and Gollwitzcr (1966) found no significant differeiiccs in the carbohydratc content of bovine fibrin and fibrinogen, even though taking great pains to compare neutral hexoses, amino sugars and sialic acid. Raisys et nl. (1966) prepared rabbit fibrinogen

FIBRINOGEN TO FIBRIN CONVERSION

35

labeled with [l4CC]glucosamine and looked for the release of radioactivity during clotting. None mas released, either during the formation of ordinary fibrin or when it was covalently reinforced by activated factor XIII. The principal acceptor site for fibrin cross-linking has also been isolated from fibrinogen and found to be glutamine residue in a peptide with no carbohydrate (Chen and Doolittle, 1971). One is forced to conclude that carbohydrate is not ordinarily released during any phase of fibrin formation, whether or not it is stabilized by the introduction of covalent bonds.

G . Fibrin Split Products Digestion of fibrin with plasmin gives rise to a set of fragments that corresponds well with the series obtained by plasmin digestion of fibrinogen, the fragments “X”, ‘(Y”,“D”, and “E” having equivalent properties (Dudek et al., 1970). Apparently no new target regions are exposed by the polymerization process, nor are any of the originally accessible sites shielded. The same new amino terminals are exposed during plasmin digestion of fibrin and fibrinogen (Mills e t al., 1964). The liquefaction of fibrin-commonly referred to as the lysis time-occurs when a critical number of fibrin monomers has been degraded to “Y” and “D” fragments (Fig. 12). Apparently this situation can develop when as few as four to eight bonds per 340,000 molecular weight have been cleaved, although many more bonds are broken subsequently before the plateaucorresponding to the final, more or less stable, pieces-is reached (Weinstein and Doolittle, 1972). Once again, the data are completely in keeping with the long-standing proposal that the only difference between a fibrinogen molecule and a monomeric unit of fibrin is the absence of the fibrinopeptides from the latter. There is no convincing evidence for a large-scale conformational rearrangement of the protein during its transformation into fibrin. Unlike the physical chemical comparisons of fibrin monomer and fibrinogen, where the preparations are examined in unfolding solvents, the enzymatic probing comparisons are performed on native structures, and the results are still the same. It would be premature to conclude that the only change in three-dimensional structure accompanying the conversion of fibrinogen to fibrin is fibrinopeptide removal. Certainly there must be some shift in the accommodation of the groups involved in holding together the fibrin monomers, for example, ,z transition from intramolecular to intermolecular associations and/or a transfer from interactions with solvent (water and ions) to interactions with groups on neighboring molecules. What we can conclude, howcvcr, is that the differences between the two structures-one existing as a soluble moIecule and the other as a

36

R. F. DOOLITTLE

member of an array in a rigid gel-are absence of the fibrinopeptides.

relatively subtle, except for the

IV. THECONVERSION OF FIBRINOGEN TO FIBRIN A . General Remarks on Fibrin Formation I n the preceding section a wide variety of observations were reviewed

which strongly indicate that the individual units of the final product (fibrin) are not radically different from the starting material (fibrinogen), except for the removal of the fibrinopeptides. This conclusion leaves us far short of appreciating the underlying basis for the conversion, however, and we must now attempt a detailed consideration of the reaction itself. The development of the three-dimensional fiber network in fibrin probably proceeds in two distinct modes: first, a certain amount of linear chain development depending on specific “end-to-end” interactions, and second, a less specific lateral aggregation of these intermediate polymers (Ferry, 1952). The relative contributions of the two associative processes depends on environmental factors, and i t has been proposed that fine clots have a greater contribution of “end-to-end” polymerization and less lateral involvement than coarse clots (Ferry and Morrison, 1947). Experimental support for this notion was obtained from light-scattering studies in which the 1ength:mass ratios of fibrin during the initial phases of polymerization were found to vary with cxperimental ronditions in the same way as the clot opacity (Steiner and Laki, 1951). Situations were also found in which gel formation could be prevented by arresting the polymerization process a t the stage of large molecular weight, soluble aggregates which are referred to generally as intermediate polymers. Before either of these polymerization stages can occur, fibrinogen has to be (‘activated,” and the overall process came to be described in three steps: fibrinogen

2

F,, -+ fibrin

nF

(1 )

(2)

(3)

tv-here T is thrombin; P, the fibrinopcptidcs; F, fibrin monomer; and F,,, the intermediate polymer. Since thrombin is involved only in the release of the fibrinopeptidcs, we will consider stcp 1 in tlic next scctioii s c p rately from the spontaneous polymerization processes that occur in steps (2) and ( 3 ) .

B . Release of Fibrimpeptides The discovcry of the fibrinopcptidcs A and H W R Y the drainatic culmination of a broad series of experiincnts ctcsignect to explore the actioii

FIBRINOGEN TO FIBRIN CONVDRSION

37

of thrombin on fibrinogen, and although the events have been well reviewed in many other places (Scheraga and Laskowski, 1957), a few of the high points ought to be cited here. First, Mihalyi (1950b) demonstrated that the isoelectric point of fibrin monomer was slightly more basic than fibrinogen, suggesting that acidic material had been split off during the conversion. Then it was found that the amino terminals of fibrin were different from fibrinogen (Bailey e t al., 1951), the newly exposed glycyl end groups being (correctly) regarded as [‘scars” where peptide material had been removed. Lorand (1951, 1952) demonstrated that fibrin clot liquors contained about 3% nonprotein nitrogen and called this material “fibrino-l,gtide.” Bettelheim and Bailey (1952) then showed that there were actually two different acidic peptides, now generally referred to as the fibrinopeptides A and B. It was not realized that there were actually two pairs of each of these peptides per molecule -reflecting the dimeric nature of native fibrinogen-until the completion of a more quantitative end-group study (Blomback and Yamashina, 1958). Almost from the start it was clear that fibrinopeptides A were released by thrombin a t a faster rate than the fibrinopeptides B (Bettelheim, 1956), suggesting that the two different peptides did not necessarily play equivalent roles in mammalian fibrin formation. The slower rate of release of the B peptide has been observed in the cases of bovine (Blomback and Vesterrnark, 1 9 3 3 , rabbit (Shainoff and Page, 1960), and horse and human (Teger-Nilsson, 1967). A convenient method for assessing the relative contributions of the two different charge clusters became available with the discovery that certain snake venom enzymes (Reptilase) -which had long been known to possess clotting activity-only removed the fibrinopeptidc A during the process (Blornbiick et al., 1957) .3 As a result, it was possible t o compare fibrin lacking only its fibrinopeptides A with thrombin-induced fibrin with both A and B removed. I n one of the first experiments along these lines, Laurent and Blomback (1958) conducted. a light scattering study on these two kinds of fibrin which led them to suggest that the removal of the fibrinopcptide A allowed mainly “end-to-end” polymerization, whereas the release of the fibrinopeptidc B was essential for lateral aggregation. Although this hypotlicsi~is w r y attractive in terins of Ferry’s earlier suggestions about two distinct associative processes (Ferry, 1952), the Laurent and Blombiick data can be interpreted in a number of different ways. The light-scattering determinations were actually performed on fibrin preparations that wcrc partially dispersed in urea solutions, and :ilthough thc Rcptilaec-fihrin niatcrial did in fact cxliibit a lower mass: length ratio than the diq)crsctl throinbin-fibriii~this may only have re-

38

R. F. DOOLITTLE

100

5 0 Yo

0

I00

200

n

Time (min)

FIG.17. Rates of release of bovine fibrinopeptides A and B compared with incorporation of protein into mechanically separable fibrin gel (F). Note that when 80%

of tlie protein has been transformed into the gel, lrss than 40% of the fibrinopcptides B have been removed. From Blomback and Vestermarlr (1958).

flected an easier dispersal of interwoven polymers because of the extra repulsive charges contributed by the fibrinopeptides B. Another possibility is that the removal of the fibrinopeptide B is involved in fiber branching (as opposed to lateral association) , another phenomenon which would lead to higher mass :length ratios in thrombin-induced fibrin (Laurent, 1972). One reason for questioning the notion that fibrinopeptide B removal is necessary for lateral aggregation is that under ordinary (thrombincatalyzed) conditions of fibrin formation, only a small portion of the fibrinopeptides B is removed when the bulk of the parent protein molecules have been incorporated into the gel (Fig. 17). This would imply that the extent of lateral involvement is not determined until after the initial formation of a space network. Second, there was no indication in the experiments of Laurent and Blombiick (1958) that Reptilase-fibrin corresponded to the “fine clots” of Ferry and Morrison (1947) with regard to transparency. The suggestion that fibrinopeptide B removal encourages lateral aggregation is a reasonable on‘e, but final judgment ought to be reserved until more experimental basis is provided.

C . Po 1yinerization Steps 1. Formation of Intermediate Polymers

Under certain conditions, the gclation of thrombin-induced fibrin can be prevented, the process being interrupted a t the stage of intermediate polymers (Fig. 18). High concentrations of certain low molecular weight compounds, including 1,6-hexane-di-o1 (hexamethylenc glycol) and urea,

FIURINOGEN TO FIBRIN CONVERSION

30

FIG, 18. Electron micrograph of interinediatc polytncrs formcd during initial stages of fibrin formation as described by Hall and Slayter (1959) (courtcsy of H. S. Slayter).

are particularly effective in stabilizing the intermediate polymers under defined conditions of pH and ionic strength, presumably by preventing lateral aggregation of the so-callcd LLcnd-to-ciid”chains (Shulman et al., 1951). These stabilized polymers have been studied by a variety of techniques, including sedimentation and viscosity, flow birefringence, light scattering and electron microscopy, and were found to be composed of 15-20 monomeric units associated in a rodlike form twice the breadth of a fibrinogen molecule. This led Ferry (1952) to propose that the intermediate polymers were formed as a result of a staggcred overlap process leading to a bimolecular rod (Fig. 19, bottom). This notion led to the invention of some simple electrostatic models for explaining polymerization after the removal of the fibrinopeptides (Fig. 19). For example, it was possible that both fibrinopeptides A and B (it must be recalled that it was not yet understood that fibrinogen was dimeric) were a t one end of the molecule (Fig. 19, top). In this case thrombin attack could expose a new terminal positive charge center which would interact with a pre-

40

R. F. DOOLITTLE A

A

-I--

--

C

FIG.19. Schematic depiction of events leading to formation of intcimcdiate polymers formed from staggered overlaps of individual monomeric units. In sequence A the fibrinopeptides would be disposed a t one end of the fibrinogen molccule, whereas in sequence B they would be clustered in the middle of the molecule. In either case, a staggered overlap could occnr leading to the intermediate polymer depicted in C. (Redrawn from Ferry et nl., 1954.) I t has been pointed out (Oosana and Kasai, 1962) that the polymeric growth of fibiin by a half-staggered o w r h p process yields the equivalent of a helix with two residues per turn.

viously existing negative patch at the center of the molecule. Alternatively, the fibrinopeptides could be situated a t the center of the molecule, their removal leading t o a positive central patch for interaction with terminal negative clusters (Fig. 19, middle). Ferry and his coworkers favored this latter scheme, especially since electric birefringencc studies did not indicate the production of any signiscant longitudinal dipole moments (Tinoco, 1955). 2. Stage 3: Lateral Aggregation

Not much more can be said about the lateral aggregation of intermediate polymers which constitutes the final stage of gel formation,

FIBRIKOGEN TO FIBRIN CONVERSION

41

DlMERlZATlON

(COLLAPSED NETWORK)

FIG.20. Pictorial depiction of nssocititivc steps occurring during fibrin formntion.

except that i t is a process sensitive to pH, ionic strength, etc., as evidenced, for example, by the differences between fine and coarse gels, and may have something t o do with the removal of the fibrinopeptides B (Section IV,B). It should be remarked, however, that the “lateral” aspect does not imply a connection with the side-by-side interaction which Ferry has postulated giving rise to the so-called “end-to-end” chains discussed in the preceding section. Nor should it bc confused with the collapse of fibers which occurs during syneresis, which is probably very nonspecific and disordered (Fig. 20) . It might bc mentioned, however, that coarse gels arc much morc readily syncresed than fine gels (Ferry, 1948). Certainly onc of the main objectives in studying fibrin polymerization is to tliStinguisli thc diffcrcnt associative steps with regard to specific contact sites and functional groups involvcd. In this regard, less direct methods can be utilized in studying the two modes of polymerization also. For example, iii a later section (V,EI we will attempt to correlate different cross-linking arrangemcnts with the different stages of polymerization.

42

R. F. DOOLITTLE

3. Reaggregation of Dispersed Fibrin

Fibrin which has been dispersed in an unfolding solvent, such as strong urea or guanidine solutions and weak acids, can be readily reaggregated by return to an ordinary solution environment (Lorand, 1950; Ehrlich et al., 1952). The solvent change can be effected either by dialysis or simple dilution, and i t is therefore possible to study the polymerization steps without any concern about the rate of thrombin interaction with fibrinogen or the relative rates of release of the fibrinopeptides. Advantage has been taken of this “reversibility,” which can be effected repeatedly, to study not only the influence of solution variables and sundry small molecules, but also the inhibitory effects of fibrin and fibrinogen degradation products (Section II,D,7). The approach has also been useful in studying genetically defective fibrinogens (Section V1,A). Most of these investigations are simple kinetic studies of the rate of polymer formation, which is normally a first-order process (Sturtevant et al., 1955). Electron microscopy of reaggregated fibrin has also been informative, abortive fiber growth and unusual patterns being observed under certain conditions (Bang, 1967). Also, the characteristic banding pattern has been found to fluctuate in intensity with simple variables like pH, ionic strength, and temperature (Belitser et al., 1971). The most widely used reaggregation method employs fibrin dispersed in 1 M sodium bromide a t pH 5.1-5.3, a procedure that gives back a fibrin product with an essentially native structure.

D. Electric Birefringence Studies Not long after it was discovered that fibrin formation depended on the release of some acidic peptides, Tinoco (1955) attempted to study the phenomenon by electric birefringence. H e reasoned that the loss of 10-13 negatively charged groups (as determined by the shift in isoelectric point and preliminary characterization of the fibrinopeptides) should result in a substantial change in the electrostatic properties of the parent molecule, and if both the peptides were at one end, a very large dipole moment should arise. In fact, Tinoco found that fibrin monomers have only a very small dipole moment, and the tentative conclusion was that the fibrinopeptides must be located near the center of the fibrinogen molecule, as depicted by the second of the model proposals in Fig. 19. Tinoco (1955) did note the possibility, however, that the peptides A and B might be located a t opposite ends of the molecule, the compensatory charge losses canceling any dipole effects. It must be emphasized that the idea of fibrinogen being a symmetrical dimer had not yet arisen. Later, when it became clear that two pairs of fibrinopeptides were being

FIBRINOGEN TO FIBRIN CONVERSION

43

released from fibrinogen by thrombin, electric birefringence studies were taken up by Tinoco’s student, Haschemeyer (Haschemeyer and Tinoco, 1962 ; Haschemeyer, 1963). These experiments are so crucial-and presently so baffling-that i t is worthwhile reviewing them and their conclusions in detail. Haschemeyer (1963) concentrated on the early event in fibrin formation, reasoning that the release of a single fibrinopeptide from a dimeric fibrinogen molecule ought to give rise to a transient polar intermediate which would disappear when the companion peptide from the other half of the molecule was released. Earlier studies had already established that the starting fibrinogen molecule had a high degree of electrical symmetry (Tinoco, 1955; Haschemeyer and Tinoco, 1962). I n her initial experiments, Haschemeyer (1963) incubated fibrinogen with small amounts of thrombin for varying times, stopping the reactions by lowering the pH to about 4, under which conditions polymers should have been reduced to the monomer form. The acidified solutions were found to contain a highly polar molecule with the same rotary diffusion coefficient as fibrinogen and which had a dipole moment equal to 4300 Debye units. Knowing that the bovine fibrinopeptide A has an electrostatic charge of -4, it was a simple matter to calculate the distance of the charge cluster from the rotational center of the molecule according to the relationship 1.1 =

ver

where p is the dipole moment observed, v the number of charges, e the charge in electrostatic units, and r the distance from the molecule~scenter (in angstroms). The distance turned out to be 220A, or very near the ends of a dime,ric fibrinogen molecule thought to be about 500A long. Furthermore, the same intermediate was found when a snake venom enzyme3 (Hemostase = Reptilase), presumed to release only the fibrinopeptide A, was used instead of thrombin, confirming that the dipole was the result of losing a fibrinopeptidc A, and not a B. I n either kind of fibrin-thrombin-induced or that formed from the action of the snake venom enzyme-the dipole disappeared upon continued digestion, although a permanent transverse dipole moment of considerably smaller magnitude remained in both cases. This latter observation was taken to mean that the two fibrinopeptidcs A were on the same side of the parent molecule with regard to the central axis. At the same time, the fact that the two kinds of fibrin (thrombin-induced and Hemostase-induced) were indistinguishable in this regard meant that the fibrinopeptides B were located symmetrically relative to the central axis. In addition to providing critical data about the structure of fibrinogen, these electric birefringence experiments also shed light on certain aspects

44

R. F. DOOLITTLE

TABLIC V

Conclusions Drawn from the Results of Electric Birefringence Studima 1. The fibrinopeptides A are situated near opposite ends of an elongated fibrinogen molecule, 220 from the center of rotation (calculated from a dipole moment of 4300 I> for the transient polar intermediate). 2. The fibrinopeptides A are located on the same side of the parent molecule relative to the transverse axis, as indicated by the permanent trarisveme dipole moment observed in fibrin. 3. The fibrinopeptides B are situated equatorially, as evidenced by the fact that the transverse dipole moments of thrombin-induced fibrin and snake venom enzymeinduced fibrin are the same. 4. The transient dipole produced by the association of a molecule of fibrin monomer and one of the original polar intermediate has a rotational diffusion coefficient equivalent to a rionoverlapping, end-to-end dimer. 5. The transverse dipole moment of the iiitlermediate polar dimer indicates a parallel orientation for the two units.

Adapted from Haschemeyer (1963).

of the polymerization process itself. In many of the preparations a fibrin dimer was identified which had a rotary diffusion coefficient of an endto-end dimer (Table V ) . This is a very important point because the value was in excellent agreement wit,h a true end-to-end dimer but very T.tBLI*: VI Calculated IZoLary Digusion Coefin'mts for Some Hypothetical Fibrin D ~ T F ~ C T S ~

Ratio e monomer:O dimer For monomer a/b = 5

For monomer a / b = 10

5.7 8.0

6.3 s.O

5.1

5.0

4.1

3.s

v?== 3 . 0

2.8

T i J

1.4

Type of dimeriaation

-1 End-to-end Corner-to-corner 1/3 Overlap

2 / 3 Overlap

rn

1-1

I

Side-by-side Observed

G I

1

1.6 6 . 5b

Calculated from Perrin's equation (1934), e = [(3kT)/(16*?a3)](2 h i ( 2 a l b ) - 11. for fibrin monomer arid 6600 sec-I for the dimer (Haschemeyer, 1!)63).

* The observed rotary diffusion coefficients were e = 43,000 sec-'

FIBRINOGEN TO FIBRIN CONVERSION

45

far from the predicted value for the overlapping type of dimer postulated by Ferry, Katz, and Tinoco (1954) (Table VI) . Although the dimer was absent from fibrinogen solutions, it is surprising that i t was stable a t low pH, and i t may represent a small amount of covalently bound dimer (Section VI) . A transient polar intermediate corresponding to the dimer was also observed in some preparations, indicating that polymerization begins after the release of a single peptide, the second peptide presumably being removed by thrombin acting on the growing chain. Finally, i t was observed that the fibrin dimer preserved the transverse dipole moment found in fibrin monomer, showing that thc alignment of thc two units was parallel (Table V). The electric birefringence conclusions were widely accepted by investigators working on the chemical structure of fibrinogen during the period when it was convenient to cast their own findings schematically with thc fibrinopeptides A at opposite ends of a three-ball model. The revelation that all six amino-terminal segments arc bound togethcr by a series of disulfide bonds makes i t very difficult, if not impossible, to accommodatc those same data. In this regard, there seems to bc some confusion about what kinds of structure could possibly reconcile both sets of observations, and there has been some discussion about molecules where all six amino groups might be a t one end. Such a molecule could indeed be expected to yield a transient polar intermediate upon the relcase of one of the fibrinopeptides (Fig. 21), but it would soon be replaced with a molecule of even greater dipole moment as other fibrinopeptides were released. Since fibrin has no permanent longitudinal dipole moment, such a model cannot resolvc the impasse. [A full discussion of the nature of dipole moments in proteins relative to their geometric and electrostatic centers is given by Ptlysels (1953).] It is difficult to fault the electric birefringence studies, since they were obviously carried out with great care and attention to detail. In a dcsperatc cffort to find why thc results are in conflict with the doubledisulfide knot picture, howevcr, lct mc list a number of possibilities cornpiled with tlic benefit of ten years’ hindsight. 1. The polar intermediates were not found when the studics were conducted a t iieutral pH, and tlie suggestion that tlie fihrinopeptides might have rcmaincd associated under those conditions (Laskowski et al., 1960) sccms weak. More likely, there may have been technical limitations since the glycinc buffer which had to be employed a t that pH was probably not as effective as thc low-conductivity system used in the expcriments is attested to by the tcchnical afterthoughts expressed by Holcomb and Tinoco (1963). 2. The value talien for y, the anisotropic factor, althougli reasonable,

46

---/I

R. F. DOOLITTLE 220A

4

+++++++

+t+f+++ &;\I

NO DIPOLE

I -\T ,,

+++++++

1

NO DIPOLE

LARGE DIPOLE

- 1. - 3i N

T + $1:

+ I +

NO DIPOLE

T

+ $

LARGE DIPOLE

VERY LARGE DIPOLE

FIG.21. Simple illustration of dipoles induced by initial removal of fibrinopep-

tides from two different (hypothetical) fibrinogen molecules. I n the top sequence, which depicts what Haschemeyer (1963) reported, an intcrmediate of high dipole moment is initially produced by the action of thrombin (T), but upon removal of the other fibrinopeptide A, charge symmetry is restored. I n the other scquencc (bottom), a fibrinogen molecule is depicted with both fibrinopeptides A clustered at one end. In this case also, release of a single fibrinopeptide results in the formation of a polar intermediate, but the dipole moment of the final product is twice as great as that of the intermediate.

might be substantially off. The fraction of polar molecules would then have been underestimated and the value of r calculated proportionately

too large. 3. There is the possibility that the transient dipole, which represented only a very small fraction of the molecules, was due to cleavage of a moiety other than the fibrinopeptide A. The extreme vulnerability of a-chains to enzymatic degradation, including the possible action of thrombin (Mills and Karpatkin, 1971), snake venom enzymes (Mattock and Esnouf, 1971), and plasmin, has been discussed in earlier sections. It must be emphasized that these criticisms are directed only a t finding a possible way out of the present dilemma, since it is clear that the conclusions of the electric birefringence studies and the proposed disulfide bridge arrangements cannot both be correct. As long as this impasse remains, it would seem worthwhile to reexamine the electric birefringence approach with certain variations which can now be applied on the basis of information from other quarters. For example, the validity of the basic premise-that it is the loss of a fibrinopeptide A which gives rise to the transient dipole-can easily be assessed by performing measure-

FIBRINOGEN TO FIBRIN CONVERSION

47

ments on appropriate fibrin preparations from several different species. We now know that the pig, siamang, bovine, and camel fibrinopeptides A have -2, -3, -4, and - 5 net charges, respectively. The dipole moments for the transient intermediates should fall on a straight line extrapolating to zero if the loss of the A peptide is responsible. If all these fibrins give similar results, on the other hand, then the early products of plasmin digestion can be examined to see whether similar dipoles are produced. I n the meantime, we can only note that the results of one of the most elegant experiments ever performed on the fibrinogen-fibrin system do not agree with structural data derived from organo- and biochemical investigations of the molecule.

E . Thermodynamic Aspects of Fibrin Formation A completely satisfactory thermodynamic treatment of fibrin formation has not yet been achieved, and the area is presently ripe for reinvestigation. I n this section some of the early thermodynamic attempts are reviewed, primarily because they have been so influentiel in shaping general opinion as to what kinds of bonding develop during the conversion. Ferry (1952) tried to explain the polymerization process-and the formation of intermediate polymers in particular-by analogy with Debye’s theory of soap micelle formation (Debye, 1949). I n both these cases, it was reasoned, aggregation occurs only after the balancing of two opposing tendencies, the coulombic repulsion due to the approach of charged monomers on the one hand, and the energy gain associated with the accommodation of short-range attractive forces on the other. Ferry considered dipole interactions, hydrogen bonds, and the associations of nonpolar side chains as the short-range attractive forces most likely to be involved (Ferry, 1952). As in the case of soap micelle formation, the different dependencies of the opposing forces on concentration led to a critical concentration, below which the system should exist as monomers, and above which as intermediate polymers of a more or less definite size. By determining this critical concentration for intermediate polymers, Ferry was able to make a rough estimate of the free energy change for the reaction of fibrin monomers associating into intermediate polymers, a value of about -8 kcal/mole fibrin monomer being suggested. Later, when it was found that the size range of intermediate polymers did not vary significantly over a wide pH range-where a substantial change in coulombic repulsive forces would have been expected-Ferry retreated from this perhaps overly simple scheme (Ferry, 1954; Tinoco and Ferry, 1954), although the good features of this model ought not to be forgotten. About that time also, Mihalyi (1954) performed a set of differential pH titrations on fibrinogen and fibrin, and these data formed the basis of

48

R. F. DOOLITTLE

a number of subsequent theoretical interpretations of the bonds formed during fibrin formation. It was well known that fibrin gels could not form below pH 5.7 or above pH 10.5. The low p H limitation had previously been attributed t o a critical histidine residue being protonated and interfering with critical electrostatic alignments in a microscopic sense (Shulman and Ferry, 1950; Ferry, 1952). Other workers, combining the pH titration data with calorimetric determinations, came to the conclusion that the key bonds forming during the polymerization process were tyrosine-histidine hydrogen bonds, thereby accounting for gel limitations a t both high and low pH (Sturtevant et al., 1955). Originally i t was proposed that 19 tyrosine-histidine connections were possible, although the point was reemphasized later that only about half of these would be employed in gel formation a t neutral p H (Scheraga, 1963). The overall process of thrombin-catalyzed fibrin formation is a highly exothermic process, anywhere from 30-50 kcal/mole fibrinogen being released as heat depending on the conditions (Laki and Kitzinger, 1956). I n fact, the bulk of the heat release seems to be associated with the polymerization steps, as evidenced by the fact that approximately the same amount of heat is released during aggregation of fibrin monomers (Sturtevant e t al., 1955) as is lost during the thrombin-catalized gelation. Sturtevant et al. (1955) took advantage of the fact that dispersed fibrin preparations maintained a t p H 5.3-5.7 exist primarily in the monomeric form, but between pH 5.7 and 6.1, intermediate polymers predominate, and above pH 6.1 gel networks begin to develop. By judiciously adjusting the pH, they were thus able to measure the enthalpy change for thc conversion of monomers to intermediate polymers as well as the overall process leading to complete gelation, obtaining a value of 19 kcal/mole fibrin monomer for the first stage and approximately 45 kcal/mole for the combined polymerization steps leading to gelation. These relatively high enthalpy changes, as mentioned above, were interpreted as being due to extensive hydrogen bond formation. Over the years there has been a disproportionate amount of emphasis placed on the idea that all the steps in fibrin formation are “reversible,” and, in the author’s opinion, this has served to confuse the situation more than to clarify it. Working backward, certainly step 3 is “reversible” in the sense that fibrin gels can be repeatedly dispersed and reaggregated by alternating dispersing and nondispersing solvents, but this hardly implies that the polymerization process is freely reversible in the traditional sense of the term. With regard to step ( 2 ) , i t has indeed been demonstrated that simple dilution of intermediate polymer solutions can lead to their dissociation, but even those situations, which were denoted with a double arrow in Section IV,A, are somewhat artificial in that the

FIBRINOGEN TO FIBRIN CONVElRSION

49

solutions contain large amounts of antipolymerants. Finally, the relatively small enthalpy change associated with fibrinopeptide removal (although never measured directly) strengthened the rather misleading notion that this too was a “reversible” situation (Scheraga and Laskowski, 1957). The possibility that the fibrinopeptides might remain associated with the parent molecules after peptide bond cleavage by thrombin spawned the idea that under some circumstances the reaction might actually be driven backward. It was presumed that the noncovalent attachment of the fibrinopeptides to fibrin monomer depended on hydrogen bonds between the parent molecule and the ubiquitous carboxyl groups found in fibrinopeptides. Under ordinary conditions, release of the fibrinopeptides presumably depended on the reaction being pulled over by steps (2) and ( 3 ) , exposing the hydrogen bond donors which could now bond to carboxyl acceptors on neighboring molecules (Laskowski et al., 1960). As this hypothesis was developed, there was a shift away from the previous idea of tyrosine-histidine hydrogen bonds to a preference for tyrosine-carboxyl bonding. Later, as notions about hydrogen bonding in proteins became more refined, there was a rather formal renunciation of this conjecture about hydrogen bonds, a new theory proposing covalent attachment as the basis of polymerization being offered in its place (Endres et al., 1965). Unfortunately, this theory was predicated on misleading reports regarding the involvement of fibrin a-amino groups (Lorand et al., 1962) and is totally untenable. It might also be mentioned that the extreme variability of the fibrinopeptides, and especially of their side chain carboxyls, argues against any specific attachments with the parent molecule. I n spite of this vacillating history, hydrogen bonding is still likely to be involved as one of the main directive forces accounting for polymerization and one of the principal contributants of the large negative enthalpy change. The AH,,,.,, of a single hydrogen bond between two amino acid side chains which are completely exposed to environmental water is thought to be about -1.5 kcal/mole (Nkmethy e t al., 1963), suggesting that quite a large number of such bonds would be needed to account for the heat released during fibrin formation. I n addition, the heat absorbed by the entropy-driven association of nonpolar sidechains has to be allowed for (Nkmethy and Scheraga, 1962), making the equivalent number of hydrogen bonds required even greater. Not all of these would need to be intermolecular, some possibly arising intramolecularly as a result of conformational shifts. Because of the large negative enthalpy change, we can tentatively presume that fibrin polymerization is fundamentally different from the combination of bovine serum albumin with antibodies (made against

50

R. F. DOOLITTLE

scruni albumin), or the polymerization of insulin, associative proccsscs that are primarily entropy-driven and do not give off significant amounts of heat (Kauzmann, 1959). It is also different from the polymerization of tobacco mosaic virus coat protein (Stauffer et al., 1970), which is actually an endothermic reaction. Neither does it have any of the obvious characteristics of cooperative reactions of the type suggested for actin (Kasai et al., 1962), flagellin (Asakura et al., 1964), or glucagon polymerization (Gratzer and Beaven, 1969). On the other hand, it has all the features of the combination of ribonuclease S with the S peptide a highly exothermic association that depends on the formation of a large number of hydrogen bonds-both inter- and intramolecular-as well as on lesser contributions from hydrophobic and electrostatic interactions (Hearn et al., 1971). In summary, we still have no firm thermodynamic basis for postulating what kind or how many bonding situations exist between polymerized fibrin units. The fact that both the kinetics of clot formation and the final character of the clot are influenced by pH and ionic strength factors suggests that there has got to be some electrostatic influence. The large amount of heat released suggests a role for hydrogen bonding, and it would be expected that the close intermolecular associations would also have to involve a hydrophobic contribution. What the exact nature of these interactions is will not be fully appreciated until the actual contact segments arc identificd and their structures determined.

F . F~rnctionalGroups Involved in Polymerization A large number of experiments have been conducted t o find out which functional groups are involved in the binding situations holding together the fibrin clot. As in all such studies, one can seldom be sure that it is not some indirect configurational change or secondary aspect that inactivates a process, rather than proving that a particular group is spccifically and directly involved in a particular action. Sometimes, however, negative results can rule out the participation of a particular group, information that is often equally valuable. 1. Histidine

Fibrin polymerization does not occur a t any pH below 5.7, even though there is no change in the hydrodynamic properties of the molecule under these conditions. The good correlation of polymerization inhibition with the titration of the imidazole side chain is the basis for histidine involvement, although a t least two interpretations are possible. The protonation of certain key histidines could distort critical dipolar alignments, as sug-

FIBRINOGEN TO FIBRIN CONVERSION

51

gcsted by Ferry (1952),or it could prevent histidine side chains from serving as hydrogen bond acceptors (Sturtevant et nl., 1955). 2. Tyrosine

Trcntnicnt of fibrinogen with protcinasc-free mushroom tyrosinasc renders it unclottable (Sixer and Wagley, 1951). On the other hand, tyrosines in fibrin proved equally accessible to tyrosinase attack, and it would be presumptuous to conclude direct tyrosine involvement on the basis of these experiments alone. Fibrin formation is inhibited at high pH, however, and the close correlation with the pK of phenolic groups has led to the suggestion that tyrosines are involved as hydrogen bond donors in fibrin formation (Sturtevant e t al., 1955). Iodination of fibrinogen a t low levels ( < 1 atom/moIe) does not interfere with clotting (McFarlane, 1963), but extensive iodination leads to unclottable material (Laki and Mihalyi, 1949). Treatment of fibrinogen with tetranitromethane a t low levels results in spontaneous precipitation and extensive cross-linking of a-chains, and mild derivatization with acetylimidaxole also results in an unclottable product (Doolittle and Haskins, 1971). Mild dansylation of bovine fibrinogen yields primarily O-DNS-tyrosine after hydrolysis, the ratio of O-DNS-tyrosine to r-DNS-lysine being about 3 to 4:l (Mihalyi and Albert, 1971). No loss in clottability or drastic change in physical properties is observed when the degree of substitution is below six moles per mole. More extensive derivatixation results in discernible structural changes, an increasing substitution of lysinc side chains, and the formation of faster sedimcnting material (Mihalyi and Albert, 1971.) 3. Lysine

Fibrinogen acetylated to the point where approximately 35% of its lysine E-amino groups arc substituted is no longer clottable upon the addition of thrombin, even though fibrinopeptide release occurs (Caspary, 1956). Succinylation yields similar results, although in this case concentrated solutions will gel spontaneously-with or without fibrinopeptide removal-evidently as a result of long-range coulombic repulsive forces between the excessively negative particles (Doolittle, 1972). On the other hand, amidination of the nmino- groups in bovine fibrinogen t o an extent of 60-70rjO does not interfere with gel formation (Fuller and Doolittle, 1966), suggesting that the influence of most amino-group reagents is mainly attributable to changes in charge distribution, rather than interference with the participation of specific amino groups, since amidination leaves the charge distribution of a derivntized protein more or less undisturbed (Hunter and Ludwig, 1962).

52

R . F. DOOLITTLE

Q. Other Functional Groups The participation of carboxylic acid groups in fibrin polymerization has been suggested by Laskowski et al. (1960). The amide groups of glutamine have been suggested on equally hypothetical grounds (Doolittle et al., 1972), as have the amide groups of the peptide backbone (Bailey and Bettclheim, 1955). The participation of serinc and thrconinc hydroxyl groups might conceivably play a role in hydrogen bonding, but there is no experimental basis for such a proposal. Finally, in one instance, short-range interactions between the nonpolar side chains of two leucine residues in neighboring molecules have been proposed on the basis of space-filling models of a purported contact site (Doolittle et al., 1972).

V. COVALENT CROSS-LINKING OF FIBRIN A . Nature of the Cross Links in Stabilized Fibrin

It has been recognized for more than fifty years t h a t fibrin formed in the presence of calcium ions has different properties from that formed in their absence, but it was Robbins (1944), who demonstrated that it was the unsuspected presence of a calcium-dependent serum factor which was making the difference. Clots formed in the presence of this factor and calcium ions were found to be insoluble in dilute acid and base (Robbins, 1944) and strong urea solutions (Laki and Lorand, 1948) , suggesting t h a t the forces holding the monomeric units together were covalent in nature. The factor, which is present in plasma in an inactive form (Loewy and Edsall, 1954) and is activated by thrombin (Buluk et al., 1961; Lorand and Konishi, 1964), has been extensively purified and characterized (Loewy et al., 1961; Takagi and Konishi, 1972), and is now officially referred to as factor XIII. Other names used in the past include fibrinsta1)ilizing factor, Laki-Lorand factor, fibrinasc, fihrinoligasc, and crosslinking enzyme. The proenzymc is a contaminant of almost all fibrinogen preparations unless special care is taken to remove or destroy it, although it remains inactive in the absence of calcium ions. Activated factor XI11 reinforces fibrin by introducing covalent bonds between side-chains of neighboring monomers. At first it was thought that the newly exposed glycinc amino terminals of fibrin were involved in the cross-linking process, especially since certain glycinc derivatives could inhibit the stabilization (Lorand et al., 1962). This hypothesis was found to be mistaken, however, evidence being presented that thc donor groups wcre actually t h e p-amino groups of lysine sidcchaiiis (Lorand et aE., 1966; Fuller and Doolittle, 1966; Doolittle and Fuller,

FIBRINOGEN TO FIBRIN CONVERSION

Xlll

53

CH2

/ CHZ

thrombin

\ /

CHZ

FIG.22. Formation of f-(y-al~itnniyl)l!.sinc rross-links by ronctensation of glutamine and lysine side chains.

1967). T h c acceptor group had bccn idcntificd as glutamine (Mata?iE and Loewy, 1966), and the direct demonstration of L- (7-glutamyl) lysine cross-bridges (Fig. 22) aftcr total enzymatic digestion of cross-linked fibrin unequivocally established the type of bond involved in stabilization (MataEiE and Loewy, 1968; Pisano et al., 1968; Lorand et al., 1968).

B. Polypeptide Chains Involved in Fibrin Cross-Linking Interest in fibrin cross-linking extends beyond the stabilization process itself, inasmuch as information about the geometry and structure of crosslinked regions should offer insights into how fibrin units are packed together during the initial polymerization process, presuming there is no significant rcarrangement during the cross-linking reaction. I n this regard, there is no detectable difference between cross-linked and noncrosslinked fibrin when viewed in the elcctron microscope (Kay and Cuddigan, 1967). Accordingly, considerable effort has been spent isolating crosslinked fragments after digesting cross-linked fibrin in various ways. Chen arid Doolittle (1969) demonstrated that there were two different kinds of cross-linking arrangement, one being comprised of dimerically linked y chains, and a second they thought involved both a- and 7-chains. pChains were rcportcd not to be involvcd in the cross-linking process. Although the formation of 7-7 dimers was quickly confirmed (Takagi and Iwanaga, 19701, other workers (RlcKee et al., 1970) showed quite convincingly that the second kind of cross-linking situation involves achains exclusively, these chains being slowly welded together to form extensive multimeric arrays (Fig. 23). As in tlie case of 1)lasmin digestion of fibrinogen (Section II,D,7),the advent of SDS-gel electrophoresis has proved an elegant and simple tool

54

R. I”. DOOLITTLE

Y-Y

55

FIBRINOGEN TO FInRIN CONVERSION 1000

90

I

2-

gk

gz

-

-

ao70-

COMPLETELY SOLUBLE FIBRIN

60-

50-

1000

I00

10

DILUTION FOLD OF NORMAL PLASMA

1 X

1/27

FIG.21. Rrlntionsliip bctivcrn tlic solubility of fibrin in 2% acetic acid and the contcnt, of y-y dimers and high niolrcular wiglit polymers. From Scbwartz et al. (1971).

for conducting a variety of cspcriincnts on fibrin cross-linking. For cxample, Sclimnrtz e t a l . (1971) were ahle to show that it is the formation of 7-7 dimers which confers the resistance of cross-linked fibrin to dispersing solvents (Fig. 24). On the other hand, detailed structural studies still depend on the isolation of substantial amounts of material, and traditional chromatographic methods led to the isolation, characterization, and localization of the complete y-y cross-linking unit (Chen and Doolittle, 1970, 1971). These efforts were aided considerably by the employment of a radioactive substitute donor, [l4CC]glycine ethyl ester (Lorand and Jacobsen, 1964), originally with the aim of isolating the cross-linking acceptor site only (Fig. 2 5 ) . In fact, tryptic digestion of isolated y-chains containing the substitute donor yielded two radioactive fragments, one of which turned out to be a donor-acceptor unit composed of the other. I n other words, the acceptor peptide, quite conveniently, was also the donor peptide. Under natural circumstances two of these peptide segments from adjacent molecules are aligned in an antiparallel fashion and can become reciprocally cross-linked by two E - (y-glutamyl) lysine cross-bridges, the lysine side chain of onc segment being condensed with a glutamine side chain FIG.23. Sodium dodecyl sulfatr (SDS) gel electrophoresis of polypeptide chains produced by reduction of bovine fibrin forming undcr cross-linking conditions and poisoned at various time intervnls by the addition of a urea-SDS-mercaptoetlianol mixture. (1) zero time; ( 2 ) 1.5 minutes; ( 3 ) 5 minutes; (4) 10 minutes; ( 5 ) 25 minutes ; ( 6 ) 40 minutes. Note disnppcarance of y-chains concomitant with appcarance of y-y dimers. a-Chain dimcrs are strongest in (3) ; a-chain trimers are strong in (6). Patterned on the experiments of McKee et al. (1970).

56

R. F. DOOLITTLE

!I

FRACTION

FIG.25. Carbox) nictliS 1 celliilose clirornatogi iipliy of sulfitolgzcd bovine fibrin which had been clotted under cross-linliing conditions in tlic prcsencc of ["Clglycinc ethyl estcr (which acts as a substitute donor in the cross-linking reaction) dcinoiistrating preferential incorporation into y-cali:unb. x-x = .i,,,,; 0- - - 0 = q i i n ~ . ) From Chen and Doolittle (1969).

of the other and vice versa. I n the unnatural situation involving the substitute donor, not only was a radioactive acceptor peptide produced, but also a linked system in which one of the two possible cross-bridges was formed naturally, but a radioactive substitute donor was incorporated into the reciprocal acceptor site (Chen and Doolittlc, 1970). The isolation of doubly and singly linked natural units, as well as singly-linked and non-cross-linked radioactive peptides, led to thc characterization of this region of the y-chains from both human and bovine fibrin; it turned out to be the carboxy-terminal segment of these chains (Fig. 26). The tryptic peptidcs from these two species differ in only two of twenty residues, indicating that this is a highly conserved region. The fact that one of the differences was a histidine-glutamine interchange made it possible to demonstrate the existence of a hybrid cross-linked unit, formed by the clotting of a mixture of human and bovine fibrinogens, thereby establishing without a doubt that y-y dimer formation is between neighboring molecules and is not the result of intramolecular bonding betwccn y-chains of the same inolcculc (Doolittlc et nl., 1971a).

C.

7-7

Dimers and Polymerization Contact Sites

The question arises, are cross-linking sites also contact sites for the spontaneous polymerization of fibrin monomers? There is a large differ-

Hicincrn

5 10 15 20 . . . Ler~-Thr-Ile-G1~-Cl~i-~ly-Glii-Clii-Hi~-Hi~-Leu-~ly-C;ly-Ala-Lys-~lii-Ala-C;ly-A -COOH

1

T

NOOC-Val -A4spC~ly-Ala-Glii-Lys-Ala-G1~--Gly-Len-Hia-His -Gln-Cln-Gly-Glii-Gly-Ile-Thr-Leu. 20 15 10 5

.. E 0

Ijovinc

5

10

20

15

. . . Leu-Ala-Ile-G1~-Glu-C;ly-G1n-Gln-Hi~-C;11~-Len-Gl~-Gly-Ala-Lys-Gln-i\la-Gly-Aap-~~l-COOH

T

i.

HOOC-Val-Asp-G1~-i\la-Glri-Lys-Ala-Gly-Gly-Leu-Glri-His -Gln-Gln-Gly-Glu-Gly-Ile-illa-Leu. . . 20

15

10

5

FIG.26. Amino acid sequences of c:ubosy-terminal segments of y-chains shorring locations of rrciprocal ci,oss-linlis (arrows) between antip:udlel neighboring chains. From Clwn and Doolittlc (1971).

58

R. F. DOOLITTLE

I

v

~

FIBRIN

I /

0

FIBRINOGEN

60

I20

I80

TIME (min)

FIG.27. Relative rates of incorporation of dansyl-cadaverine (DC) into filrinogcxn (A) by factor SIII*. From Lorand el al. (1966).

(0) and fibrin

ence in the accessibility of cross-linking acceptor sites to small molecular weight substitute donors in fibrinogen compared with fibrin (Fig. 27), suggesting that the sites are shielded by the presence of the fibrinopeptidcs (Lorand and Ong, 1966). Furthermore, Reptilase3 is just as effective as thrombin in exposing these sites to the action of activated factor XI11 (Lorand and Ong, 1966), and indeed, y-y dimerization takes place in fibrin formed under these circumstances (Mattock and Esnouf, 1971), indicating that only fibrinopeptides A need to be removed. Also, during the progressive plasmin digestion of fibrinogen, clottability is lost a t thc same point as the ability to form y-y dimers (Gaffney and Dobos, 1971). What we have, then, is a close correlation between those actions that allow polymerization and thosc that permit 7-7 dimcr formation. It seems reasonable a priori that thc segments should be in contact before the introduction of covalent boiids, as opposed to the enzyme having to impose a drastic rcorgaiiization before catalyzing the condensation reactions. I n fact, it may bc that the polymerization process per sc--as opposed to the release of fibrinopeptides-affords the necessary rcadjustment that results in the exposure of the cross-linking sites. If it is presumed that the y-chain tcrniinal scgment is involved in some aspect of the initial polymerization, then one can ask what fc-atures of this region suit i t for the task. I n an effort to provide some insight into that question, space-filling models of the y-y donor-acceptor unit have been constructed, with a full awareness of the risks inherent in making models on the basis of amino acid sequence data only. The helical-wheel approach of Schiffer and Edmundson (1967) indicated that the glutamine acceptor site and the lysine donor site, which are eight residues apart in the sequence (Fig. 26), both would project from the same side of a n a-

FIBRINOGEN TO FIBRIN CONVERSION

59

helix, an ideal arrangement for reciprocal bonding. Furthermore, the space between the two bridges would be occupied by only one other set of side chains, a reciprocal pair of leucines that would provide a desirable hydrophobic interaction. Accordingly, two a-helical eicosapeptides corresponding to the (bovine) y-chain carboxy-terminal sequence were constructed with space-filling models. When the two hcliccs were oriented in nntipnrallcl fashion (Fig. 28), they fittcd togcthcr rcmarknbly well, whether or not the reciprocal interchain peptide bonds wcre formed (Fig. 29). There are other general three-dimensional schemes that could be formulated, of course, including the formation of backbone hydrogen bonds leading to a p-structure association. It is of some interest that when fibrin is compressed and subjected to X-ray analysis there is an indication of a shift from an a-pattern to a /3-pattern (Bailey et al., 1943). Furthermore, the suggestion has been made that the reciprocally cross-linked segments are analogous to a large cyclic peptide ( n = 16), structures that frcquently employ reciprocal backbone hydrogen bonding for stabilization (hfosher and Blout, 1971). Polymerization inhibition experiments with synthetic peptides may ultimately provide a basis for choosing among thesc or other hypothetical arrangements, and thc models ought not to be takcn too seriously a t this stagc. Kegardlcss of whether the interaction between y-chain carboxy-terniinnl

I A L A /~L E U G LY

ALA ,GLy HIs

I

LEU 1

+KN ,GLY

GLU

I

1

GLN

1 VAL’

G LY

AdP

FIG.2s. Scliemntic dcpictioii of lwo nntiliai allcl y-cliniii acgiiieiits in a-Iidir:tI conformations. From Doolittlc e l al. (1972).

60

R. F. DOOLITTLE

FIG.29. Space-filling models of a pair of y-chain carboxy-terminal eirobnpt.1)-

ti&

in a-liclical configurations : ( A ) c~s~~osiirc sliowing cross-links foriiicd lwt w

~ i i

FIBRINOGEN TO FIBRIN CONVERSION

61

lysinrs-15 and glutamines-7; (B) models seen from oppositc side showing possible interaction between glutamines-8 and glutnmines-16.

62

R. F. DOOLITTLE

C

I

I

I I

I

I

I

1

I I

I

I I

FIG.30. Schematic representation showing two different mays reciprocal y-y dimers could be involved in the formation of intermediate polymers involving staggered overlaps.

segments involves helical stretches, ,&structure, or some other arrangement, however, the fact remains that the two strands are joined a t this point in such a way as to imply that neighboring molecules-each of which is a symmetrical dimer-are aligned in the same way, a conclusion also reached by Haschemeyer (1963) in her electric birefringence studies (Table V). Reciprocally bound antiparallel 7-y dimers could fit into Ferry’s scheme of initial polymerization in two ways, either as a function of the initial step involving lateral attachment with a staggered overlap, or in the subsequent end-to-end abutment that ensues upon addition of the next unit (Fig. 30). I n the first case, the carboxy terminals of the y-chains would have to be back near the center of the molecule; in the second, they would be located a t the extremities. Similarly, in one or the other of these instances the y-chain carboxy-terminals would be near or distant from the fibrinopeptides, depending on the locations of these amino-terminal portions. I n either case it is important to appreciate that virtually all y-chains become cross-linked in this fashion during gel formation under physiological conditions, implying that all fibrin monomeric units are oriented in precisely the same fashion a t this stage of polymerization.

D. The Significance of a-Ckain Multimers The structures of the a-chain cross-linking sites have not yet been determined, but some features of this process as it is presently understood already allow some judgments to be made about possible chain arrangements. The fact that a-chains form multimeric arrays (McKee e t al., 1970), as opposed to reciprocal dimers, must mean either that more than

l”Il3ltINOGEPi T O FIBRIN CONVERSION

G3

FIG.31. Diagraininatic explanation of how n staggered ovcrlnp can account for y-y dimers, on the one hand, and a-chain inultimcrs on the other, presuming that rach of tlicse chains has only one donor and one acccptor sitc cacli. The solid lincs depict contact surfaces between y-cliains; the brokcn lincs rcprescnt cu-chain contact snrfaccs. p-China, which arc not inrolvcd in factor SIII‘b-catalyzcd crobslinking, are not shown. Tlic dynd asis of syininctry is sliown in tlic cciiter of cacli unit.

one bonding sitc is involved and/or that the alignment in fibrin is staggered in such a way that the donor of one chain joins the acceptor of a chain different from the one with which its own acceptor interacts. I n this latter case, the donor and acceptor sites on a given chain would have to be quite distant in a spatial sense (Fig. 31). There is some evidence that there may be a t least two different acceptor sites in a-chains, since labeling with small molecular weight substitute donors results in two different labeled peptides (Chen, 1970; Doolittle et al., 1972). The introduction of a substitute donor, especially under the relatively sluggish conditions that prevail during the labeling of a-chains compared with ychain labeling, is not unequivocal evidence that a site actually participates in cross-linking, however, and a final judgment on this point will have to await tlic isolation of the cross-linkcd peptides themselves.

E . Relative Contributions of y- and &-Chains to Cross-Linking under Various Conditions The proposal has been made that y - y dimer formation is a direct reflection of the initial polymerization process leading to the formation of intermediate polymers, whcreas c-chain cross-linking, which is a much slower process, is more likely the conscqucnce of subsequent lateral association (Doolittle et al., 1972). Ferry and his co-workers (Kate et al., 1953) had long ago shown that the presence of calcium ions did not significantly alter the size and shape of intermediate polymers, but i t did prevent their dissociation upon dilution. Gollwitzer et al. (1970) observed factor XIII-cross-linked fibrin in the electron microscope after treating the preparations with various dispersing solvents, including guanidine solutions, dilute acids, etc. Although the cross-linked fibrin was no longer dispersible in thcsc SoIvcnts, its appearance in the electron

64

R. F. DOOLITTLE

0

100

200 TIME

300

400

(MINUTES)

FIQ. 32. Relative rates of disappearance of y-chains (hollow symbols) and a-chains (solid symbols) during clotting of fibrinogen at various ionic strengths. Clotting times increased as a function of ionic strength. A = 0.15; 0 = 0.28; 0 = 0.40; 0 = 0.53. Arrows indicate clotting times at 4, 18, 35, and 70 minutrs, respectively.

microscope did change, the material swelling up as though it were trying to break its shackles. Close inspection revealed that the cross-linked fibrin had divided into numerous subfibrils ordered lengthwise, the widths of the strands being about two molecules thick. The authors concluded that the formation of covalent cross-links is limited to the subfibril stage and does not occur between strands (Gollwitzer et al., 1970). On the other hand, Khodorova e t al. (1972) found cross-linking to occur much more readily in coarse clots than in fine clots, the implication being that cross-linking is encouraged by greater lateral involvement. The relative contribution of y-chains and a-chains t o cross-linking in coarse and fine gels was determined in a series of gels formed a t varying ionic strengths (Fig. 32). The gel-point times varied predictably with ionic strength, a n effect attributable both to slower fibrinopeptide release (Blomback, 1958b) and to the more rcluctaiit aggregation of filjrin monomers (Latallo et al., 1962a). Although the rate of y-y dimer formation was also dependent on ionic strength, the diminution in rate was less than expected on the basis of the delayed gel formation (Fig. 33), and at high ionic strengths 7 - y dimers were clearly evident before the onset of gelation. On the other hand, a-chain involvement is minimal a t high ionic strength (Fig. 32), strongly suggesting that a-chain multimers arc

65

FIBRINOGEN TO FIBRIN CONVERSION 2.0

-

1.6

-

1.2

-

0.8

-

0.4

-

-

In w

+

z=

5

I

w

z

s W

0 ' 0

I 01

I

02

I

03

I

I

04

05

I 06

IONIC STRENGTH

FIG.33. Influence of ionic strengtli on clotting time (0) xnd relative iniolvemcnt, of y-chains in cross-linking as determined by the disnppearnncc of individual y-chains on SDS gels ( X ) .

associated with some aspect of the lateral aggregation leading to coarse fibers. These observations indicate that whatever orientation is generally necessary for the formation of y-y dimers, it is accomplished during the association of intermediate polymers. They also suggest that the geometric constraints inherent in the phenomenon of @-chainmultimers may not be a factor in determining the initial packing possibilities, but may rather be a consequence of multiple site cross-linking during the later stages of gel development.

F . Unnatural Kinds of Covalently Reinforced Fibrin 1. Enz ymntic Methods

Fibrin that cannot be dispersed in unfolding solvents can be formed in a variety of unnatural ways, including both enzymatic and clicmical procedures. The packing of individual niolecular units is not necessarily identical in all these cases. Although it has not been stressed in preccding sections, i t should be evident that activated factor XI11 is a transamidase (transglutaminase) enzyme (Loewy et al., 1966) similar in its action to an enzyme first isolated from guinea pig liver hy Waelsch and his collcagues (Waelscli nnd hZycek, 1962). Thcsc enzymcn, which 1iam since been found in a

66

R. F. DOOLITTLE

variety of tissues (Chung and Folk, 1972), are capable of incorporating various amino compounds into suitable acceptor sites on proteins according to the general reaction R-NH,

+ protein-CO-NHp

-+

+ NHa

protein-CO-NH-It

Glutamine side chains are apparently the exclusive acceptor sites. Invariably these enzymes are calcium dependent and sulfhydryl activated. Farrell and Laki (197a) found that bovine fibrinogen could be gelled directly by preparations of the guinea pig liver transamidase, without the removal of fibrinopeptides. It was of interest then, to find whether the chain cross-linking involvement was similar to that which develops during the cross-linking of ordinary fibrin by activated factor XIII. I n fact, the pattern of cross-linking proved to be very different, a-chains being cross-linked a t a very fast rate, followed then by y-chains and finally even P-chains (Fig. 34). Evidently the liver transamidase is a very nonspecific enzyme which has gelled the fibrinogen solutions by cross-linking a variety of exposed lysine side-chains with a number of glutamine acceptors on neighboring molecules, leading to a random, patchGork system of intermolecular bonding. SignificantIy, y-y dimers did not appear during the gelation of bovine

0

2

4 TIME

6

8

10

(MINUTES1

FIG.34. Disappearance of individual (noncross-linked) chains during gelation of bovine fibrinogen by guinea pig liver transamidase. Individual concentrations were determined by quantitative scanning of sodium dodecyl sulfate gels shown in Fig. 35. After the completion of these experiments, several other groups reported simihr results (Chung and Folk, 1972; Gray and Lorand, 1972).

FIBRINOGEN TO FIBRIN CONVERSION

67

fibrinogen induced by the liver transamidase (Fig. 35). At first i t was supposed that this was because y-y dimerization was absolutely dependent on the release of fibrinopeptides. When bovine fibrinogen was first clotted with thrombin, however, and the fibrin dispersed and reaggregated in the presence of the liver transamidase, once again y-y dimers failed to appear, the principal involvcmcmt of a-chains hcing clear (Figs. 36 and 37). Control preparations in which activated factor XI11 was present exhibited normal 7-7 dimer formation after reaggregation, suggesting that there is some special aspect of factor XI11 that allows i t to function in that specific operation. 2. Chemical Methods

Fibrinogen solutions are readily gelled by a variety of chemical crosslinking reagents. Mihalyi and Lorand (1948) long ago demonstrated that formaldehyde was effective in this regard, for example. A much milder treatment involves intermolecular peptide bond formation brought about by thc action of small amounts of water-soluble carbodiimides in the cold. These reagents activate carboxyl groups and in the presence of suitable nucleophiles-in this case presumably amino groups on neighboring molecules-lead to amide linkages. Examination of these gels reveals that cross-link formation is limited exclusively t o a-chains (Fig. 38). Fibrin gels-as opposed to fibrinogen solutions-can also be rendered indispersible by the action of small amounts of these water-soluble carbodiimides (Fig. 39). As in other unnatural cross-linking situations, only a-chains become covalently bonded, as evidenced by examination on SDS-gels as well as by a variety of chromatographic procedures. Total enzymatic hydrolysis of this artificially cross-linked fibrin, using procedures that should cleave only peptide bonds involving a-linkages (Pisano e t ,uZ., 1969), revealed 2-4 moles of r-(7-glutamy1)lysine per mole of fibrin monomer. Although this is about the same amount as is isolated from fibrin cross-linked by activated factor XIII, it must he emphasized that the enzyme utilizes glutamine as an acceptor, whereas the carbodiimides work on free carboxyls, so the residues connected in the two situations cannot be the same. The main conclusion to be drawn from these experiments is that achains are able to come into close intermolecular proximity in both fibrinogen solutions and in fibrin gels, as evidenced by the fact that a variety of treatments is effective in splicing them together. 7-Chains, on the other hand, are much less accessible to these agents, and thc formation of y-7 dimers is n very specific event. p-Chains are the least available for intcrmolccular bonding, but even they can be reacted if

68

Q!

Y

R. F. DOOLITTLE

FIBRINOGEN TO FIBRIN CONVERSION

69

conditions are vigorous enough, as demonstrated by the sustained action of a liver transamidase (Figs. 34 and 35).

VI. OTHERASPECTSOF

THE

FIBRINOGEN-FIBRIN CONVERSION

A . Variant Human Fibrinogens The first suggestion of an abnormal human fibrinogen was made by Imperato and Dettori (1958), who studied a child with a mild bleeding

condition which could not be accounted for by deficiencies in other clotting factors. Unfortunately, it was not possible to obtain a complete physical and chemical characterization of this fibrinogen. Since that time, however, a t least eleven other variant human fibrinogens have been reported, all of which arc slow to form clots upon the addition of thrombin (Table VII). The reluctance to form gels varies considerably, as does the clinical severity, suggesting a wide variety of molecular defects. Of these, one has definitely been shown to involve a delayed fibrinopeptide release (Bethesda), most of the others being slow to clot because of defective polymerization (Table VII) . I n only one case has an amino acid replacement been identified in a variant human fibrinogen (M. Blomback et al., 1968), one of the residues in the disulfide knot of fibrinogen Detroit having undergone a n arginine to serine substitution. The location of the replacement is just two residues away from the amino-terminal glycine of the fibrin a-chain, a site predicted as a polymerization spot by Bailey and Bettelheim (1955). Although the possibility of othcr structural changes in this defective molccule cannot yet be ruled out (Rlammen et al., 1969), i t appears likely that this single amino acid replacement is responsible for the sluggish aggregation of fibrin monomers. This might be because this peptide segment actually represents a contact site for polymerization, or i t might be that the loss of the positively charged arginine side chain has changed the distrihution of chargc sufficiently that electrostatic alignment is hampered. The change might even have cffccted a Iarge-scale conformational shift which affects contact sites elsewhere in the molecule. Most of the variant human fibrinogens have becn examined by imFIG.35. Sodium dodecyl sulfatc (SDS) clcctrophorcsis gels of bovinr fibrinogen exposed t o guinea pig liver transamidase for various tinics before poisoning n i tli urc.n-SDS-inerc:il~toctlianol mixtnre : (I) zrro time ; (2) 0.25 minute ; (3) 0.5 ininute; (4) 1.0 minnte; ( 5 ) 2 0 minutes; (6) 4.0 minutes; (7) 10 minutes; (8) control (no enzyme). Gelation occurred a t 3.5 minutes. In each cme 0 1 ml of enzyme solution and 0 1 nil of 0 1 M CaCl, solution wcrc added simultnnconsly to 0.2 ml of a 0.5% bovine fibrinogen solution. The reactions w r ? stopped by the addition of 0.5 in1 of nn 8 Af iue:t-ZC/o SDS-0 1 M nicrc.aploe~lianolsolution.

70

R. F. DOOLITTLE

71

FIBRINOGEN TO FIBRIN CONVDRSION 100

80

20

0

0

2

4 ENZYME

6

8

10

( A R B . UNITS)

FIG.37. Disappearance of individual chains from bovine fibrin reaggregated in presence of varying amounts of guinea pig liver transamidase. (Data from scans of gels shown in Fig. 36.)

munochemical methods, and in several cases distinctive features have been reported, especially with regard to mobilities observed by immunoelectrophoresis (Table VII) . In the few cases where hydrodynamic studies have been conducted, no gross differences have been reported. A number of reports have claimed differences in carbohydrate composition, but until more is known about individual variability in this area, too much emphasis on this feature might be misleading. I n fact, firm conclusions about any suspected differences ought not to be formed until more is known of the polypeptide structures of these proteins. It ought to be noted that the exact mode of inheritance for human fibrinogens is not altogether clear. On the basis of pedigree and familial studies, Al6nachQ (1970) reports that abnormal fibrinogens are inherited as autosomal dominants. Usually, however, the abnormal fibrinogens exhibit the ability to interfere with clot formation when mixed with normal fibrinogen. As such, heterozygotes may have equal-or almost FIG. 36. Sodium dodecyl sulfate (SDS) electrophoresis gels obtained with thrombin-induced bovine fibrin which had been dispersed in 5M urea and then reaggregated in the presence of varying amounts of guinea pig liver transamidase and CaC12. (1) Control (no enzyme or CaCL); (2) control (no enzyme); (3) 1.0 enzyme unit; (4) 2.0 enzyme units; (5) 3.0 enzyme units; (6) 5.0 enzyme unit; (7) 10 enzyme units; (8) reference fibrin cross-linked by factor XIII*. Enzyme units arbitrary.

72

R. F. DOOLITTLE

FIG.35. Scans of sodium dodecyl sulfate electroplioresis gels of watcr-soluble carbodiimide-treated bovine fibrinogen (bottom) and untrcntcd control prcpara tion (top) after 96 liouis :it 4°C. Carbodiiniidc c3onc*enti:ition= 2 x J I . Noti. complete disappearance of a-chains. X, Y, and Z corrcspond to a-chain diincrs, trimers, and tctramers, respectively. Arrom indicate direction of electropliorcaia.

w

1

r

80

m

3

60

f 40

u a u.

a

20 0

0

5

10

15

[ C D I I (MoledLiter x 104)

20

0

2

4

6

8

1

0

TIME (Hours)

FIG.39. Fibrin clots rendered insoluble in 1% monocliloroacetic acid or 5 d l urra by the action of a water-soluble carbodiimide (l-etliyl-3-(3-di1netliylaininopiopy1)carbodiimide.HC1).

Left: Percent insoluble fibiin formrd a t various conrrntlntions = 1% monociiloroacetic acid; filled symbols = 5 fiI urea). Right: Percent insoluble fibrin formed as a function of tiinc at two diffcrrnt concentrations of carbodiiinide; 0 = 1.0 x 10.' A2 ; A = 2.0 X lo-' dl. All reartioils a t 4°C. of carbodiimide (open symbols

TABLE VII Some Abnormal H u m a n Fibrinogens That Are Slow lo Clot upon the Addition of Thrombin

Designation

First, &ported

Paris

1963

In

Vanconvel4 Baltimorec

1963, 1968' 1964

Zurichd Clevelande

196.5 1967

Iletroitf St. Louisa

1968 1968

Paris

196s

IIh

Bethesda'

1970

Los Angelesl Amsterdamk

1970 1971

Double diffusion precipitin lines

Immunoelectrophoret i c mobility

Sedimentation coefficient

Carbohydrate content

Fibrinopeptide release

Slightly abnormal Normal No spurring Slightly abnormal

Von Felten et al. (1966, 1069). Sherman et al. (1972).

7

Zietz and Scott (1970).

m Abnormal in plasma, but normal after purification.

Yes

Normal

No

Abnormal Abnormal

Yes Yes

Normal

Normal Slightly delayed

Normal

Abnormal Abnormal

Yes Yes

Normal

Yes

Delayed

Normal

Virtually normal Normal

Hasselback ct (11. (1963); Jackson et al. (1968). e Forman et al. (1968). Samama et al. (1969); Mester and Szabados (1968). k Janssen and Vreeken (1971). n

Inhibits clotting of normal fibrinogen

Normal

Abnormal Slightly abnormal

M6naehi. (1964).

0

Normal

?m

Polymerization of fibrin monomer

Normal

Slightly abnormal Slightly high Normal Weak, no spurs Low Normal Abnormal No spurring Normal Low Abnormal Spur Piormal Normal No spurring Ilifferent" Normal No spurring

Fibrinopeptide mobility

High sialic acid, low neutral hexose.

Yes

Abnormal Abnormal Beck ct al. (1965); Mosesson and Beck (1969). f htammen et al. (1969); M. Blombllck ct al. (1968). Gralnick ct al. (1971). Originally thought to be hypofibrinogenemia.

74

R. F. DOOLITTLE

equal-amounts of normal and abnormal fibrinogen, a situation that might actually be described as autosomal codominant. If abnormal fibrinogens can be detected in heterozygotes, then one worries about any conclusions drawn on the basis of chemical observations made on unseparated mixtures. I n a t least one case, that of fibrinogen Zurich (Von Felteii et al., 1969), the abnormal molecule was separated from the coexistent normal type by clotting the mixture with Reptilase? I n this case, the normal molecule gelled, leaving the variant behind in the clot liquor. I n the case of fibrinogen Detroit, only a single molecular species was thought to be present. Either the individual was liomozygous for thc amino acid replacement found or synthesis of the normal molecule was repressed. I n the former case, the coincidence of the same residue being replaced in both parental lines would suggest recent consanguinity. A familial disorder of a different sort than those listed in Table VII has been reported by Egeberg (1967). I n this case fibriiiogen is eonverted to fibrin by thrombin at an accelerated rate. A complete chemical characterization of this very interesting material has not yet been undertaken. A report of a fibrinogen which clots nornially but then exhibits defective cross-linking has been made (Hampton, 1968) , but the situation is still somewhat confused in this case, a i d the fibrinogen may not be the cause of the malady (Hampton and Morton, 1970). Although the fibrinogen field is still a long way from the remarkable correlation of structural changes and dysfunctions achieved in the case of hemoglobin (Perutz and Lehniann, 1968), further structural studies on those variant fibrinogens already identified as well as on others as yet undiscovered may provide critical information about those features of the molecule which are associated with specific events in the conversion to fibrin.

B. H u m a n Fetal, Fibrinogen The possible existence of a fetal fibrinogen, analogous to fetal hemoglobin, was first noted by Kiinzer (1961). While newborn children frequently have low amounts of other clotting factors, their fibrinogen levels are normal by adult standards (Kiinzer, 1964). Witt and her co-workers (1969) isolated fibrinogen from human cord blood, and although its overall amino acid composition was indistinguishable from the adult type, it eluted somewhat later upon DEAE-cellulose chromatography. Fingerprints of tryptic digests-a formidable task for a molecule containing approximately 190 arginines and lysines per half-moleculerevealed a t least three different peptides among the 40-50 that stained most strongly. Further studies (Witt and Ptiiller, 1970) resulted in the finding that the fetal fibrinogcii liad almost twice as much inorganic

FIBRINOGEK TO FIBRIN CONVERSION

75

phosphorus per molc as thc adult typc, and it is possible that the diffcrent peptides observed on thc fingcrprints werc due to phospliorylation of normally unphosphorylated fibrinopeptides. Fctal fibrinogen was found to have the same hexose content as adult fibrinogcn (Witt and Mullcr, 1970). As further evidcncc of the uniquciicss of fetal fibrinogen, it was reported that tlic pH-dcpcndcncc of thrombin-cntnlyzctl fibrin formation is different from the adult type, clotting being significantly rctardcd at moderately high p H (Witt et at., 1969). Mills and Karpatkin (1972) , invcstigatiiig thc notion that fctnl plasma (cord blood) is slow to clot upon tlic addition of thrombin, coiicluclcd that the delay is due to a greater content of preformed fibrin in cord ldood fi1)rinogcn preparations. Thcir findiiig that fibrinogen from cord blood, when care is taken to exclude preformed fibrin, is iiidistinguislinblc from adult fibrinogen upon SDS-gel electrophoresis and isoclcctric focusing challenges the existence of a distinctive fetal fibrinogen in humans.

C. The Influence of Calcium Ions on Fibrin Polymerization It is widely recognized that thrombin-catalyzed fibrin formation occiirs significantly fastcr in the presence of calcium ions, although the exact basis of this phenomenon remains unclear. The aggregation of fibrin nioiioiiicrs is also spceded up hy calcium ions, and, in fact, many of tlic genetically variant human fibrinogens which exhibit defective polymerization (Section V1,A) behave almost normally whcn calcium ions are prcscnt in the system. Therc are at lcast two possible explanations for these effects, and either or both may be responsible for the acceleration of polymerization. For one thing, calcium can activate latcnt factor XI11 activity, the eiisuing cross-linking pulling tlic cquilibrium ovcr past thc intcrmcdiatc polymer stage. It will lie recalled that iiitcrmcdiatc polymers formed in the prcscnce of calcium ions have the same properties as those formed in their absence exccpt that they are no longer dissociablc (Kate et at., 1953). Swoiid, calcium ions inny bc involved more dircctly, influencing localizcd clectrostatic orientations or other bonding situations of the polymerizing units thcmselves (Boyer et nl., 1972). I n this regard it has been reported that calcium ions cilube n. shift in frcquency of bands attributed to amides when fibrinogen and fibrin arc cxnniincd by infrared dichroic techniques (Knhn et at., 1970). Fibrinogen clottability is drastically impaired by trentiiiciit with EDTA (Bithell, 1964) ; the effect is complctely reversed upon the addition of calcium ions, although it is not clear whether or iiot tlic cnlciuni is interacting with fibrinogen directly or by rcinoving hound EDTA from tlic system (Elias and Iyer, 1967). The EDTA-trcatincnt a150 results in significant structural changes, which in tlic past have been attributcd to

76

R. F. DOOLITTLE

dissociation into half-molecules (Blomback et al., 1966; Capet-Antonini, 1970). There is also an increase in the amount of a-helix under these circumstances (Capet-Antonini, 1970). The structural changes are also reversed upon the addition of calcium ions. It is possible that the effect of EDTA is that of a substitute acceptor moiety for certain donor groups on fibrinogen which are involved in polymerization, the orientation of carboxyl groups in EDTA being conincidentally favorable for multiple interactions. The inhibition which ensued from such an interaction would certainly be reversed if calcium ions were present to complex the EDTA. It is even possible that the hypothetical donor groups involved might be the hydrogen bond donors suggested by Laskowski e t al. (1960) (Section IV,E). A number of ions besides calcium can influence the rate of fibrin formation, including thiocyanate and llalide ions (Edsall and Lever, 1951) and a variety of anions from organic acids (Abilgaard, 1964). I n the case of the halides, fluoride tends to accelerate polymerization wliereas bromide ions are definitely inhibitory. Abilgaard (1964) has attcmpted to correlate these observations with the ability of these ions to shift thc isoelectric point of fibrinogen as determined by the pH of minimum solubility. He believes that many of these anions bind to fibrinogen in such a way as to encourage a double-layer effect which has electrostatic benefits for polymerization.

D. Cobalt-Fibrinogen Fibrinogen has been found to form an unusual complex with cobalt (11). Krantz and Fiedler (1968) discovered that parenteral administration of cobalt salts to rabbits resulted in the formation of a cobalt-fibrinogen complex containing two to four atoms of cobalt per molecule of fibrinogen. Most other plasma proteins were not significantly affected, although a T.ii31,e VIII Some Physicochcmical Paramdi'rs of Normal and Cobalt-Fibrinogen'

Sedimentation coefficient 1)iffiisioii coefficient Molecular weight Partial specific volume Frictional ratio Percent a-helix

S?n,w

Dm.w ,If 8

flfu -

Normal fibrinogen

Cobalt-fibrinogen

7 9 2 2 340,000 0 71-0.72 2 :14 2 1- 2 2 h

11 6 : 1 340,000 0 72 1 62 45-36

Adapted from Behlke ct al. (1969) and Fiedler

rjt

a / . (1971).

I, Note that this value is somewhat lower than that listed

Mihalyi (1965).

iii

Table I as reported hy

FIBRIXOGEN TO FIBRIN CONVERSION

77

few did bind the ion. Characterization of the cobalt-fibrinogen, which is virtually nonclottablc, revealed that the molecule had undergone a remarkable conformational change to a very much more compact structure (Table VIIIj . Both the sedimentation and diffusion coefficients were significantly increased, while the molecular weight remained normal (Behlke et al., 1969), and the amount of a-helix, as measured by optical rotatory dispersion and circular dichroism, shifted from about 23% to 36% (Fiedler et al., 1971). Some heterogeneity is apparently introduced since a number of additional bands are exhibited by isoelectric focusing compared with normal fibrinogen (Krantz et al., 1970), although all the derivatives arc immunologically fully cross-reactive. Tlic enormous change in hydrodynamic properties brought about by cobalt-complexing would seem t o reflect an unusually distcnded structure in the uncomplexed molecule.

E. Evolutionary Considerations 1. Vertebrate Clotting

Blood coagulation in all vertebrates follows thc same fundamental plan, culminating in a fibrinogen to fibrin conversion that is effected by fibrinopeptide removal (Doolittle and Surgenor, 1962). Thrombin from any vertebrate species will clot the fibrinogen of virtually any other, although the clotting times involved vary morc or less inversely with the evolutionary relationship of the species involved. In extreme cases, thrombins from distantly related creatures may effect clotting by releasing only one of the two sets of fibrinopeptides (Doolittle et al., 1962; Doolittle, 196513). These heterologous interactions of thrombin and fibrinogen are classic manifestations of the general phcnonicnon loosely referred to as “species spccificity” in which interacting nincromolecules from a given organism seem to be co-adapted for maximurn effectiveness. I n spite of the general cross-reactivity of thrombins and fibrinogens from different vertebrate classes, immunological cross-reactivity is usually limited to a single vertebratc class (Kenton, 1933; Hektoen and Welker, 1927). Antibodies raised against mammalian fibrinogen do not prec4pitatc avian fihrinogens, for cxmiplc, and vice versa, iiidicatiiig that the superficial aspects of their structurcs have been prone to substantial variation during evolution. An illustration of this propensity for change has already been encountcrcd in our discussion of the fibrinopeptides (Section II,D,3j, which are among the most variable peptide structures studied. Other portions of the fibrinogen molecule are known to be much more conser~ative,however. For example, w1icrc:is the fibrinopeptidcs from human and bovine fibrinogens differ a t sixtccii of their thirty directly

78

R. F. DOOLITTLE

comparable residues (46% identity), their y-chain carboxy-terminal segments, which are the sites of y-y cross-linking, have only three differences in twenty-eight positions (89% identity) (Sharp et al., 1972). For the most part the physicochemical properties of those vertebrate fibrinogens which have been examined all seem to be very similar, and the same purification schemes which have been developed for insmmslinn

1 2

FIG. 40. Sodium dodecyl sulfate clectrophorcsis gels of individual a-, /3-, and y-chains produced upon reduction of fibrins from 26 selcctcd mammalian species representing five orders (Primates, Perissodactyls, Carnivores, Artiodactyls, and Proboscids). (1) Human, (2) chimpanzee, (3) gorilla, (4) orangutan, (5) sinmany, (6) ccbus monkey, (7) slow loris, (8)horse, (9) donkey, (10) rhinoceros, (11) tapir, (12) goldcn jackal, (13) black bear, (14) collnrcd peccary, (15) vicuna, (16) muntjak, (17) elk, (18) pronghorn, (19) sheep, (20) ibex, (21) impala, (22) Giant’s gazelle, (23) Persian gazelle, (24) yak, (25) water buffalo, (26) Indian elrphant. I n many cases the a-chain appears as a doublet; this may be due to ancillnry drgradntion during fibrin formation, or it may reflect inherent polymorphism. Most of the gelb represent individual animal specimens.

79

FIBRINOGEN TO FIBRIN CONVERSION

fibrinogens have been applied to plasmas of all vertebrate classes (Finlayson and Mosesson, 1964). A survey of thirty mammalian fibrins using SDS gel electrophoresis has revealed that the molecular weights of y- and p-chains have remained fairly constant during mammalian evolution, but a-chains vary substantially, a size range of 60,000-80,000 being observed (Fig. 40). The largest a-chains are found to exist among the equines (horses, donkeys, and mules) , increasing the overall molecular weight of an a&y2 fibrinogen unit to approximately 380,000 for these creatures (cf. Blomback and Laurent, 1958). The six-chain structure comprised of two pairs of three nonidentical chains exists even in the most primitive vertebrate extant, the lamprey eel (Doolittle, 1965a), and in this casc the SDS gels yield a-chain mokcular weights of about 100,000, leading to an overall molecular weight for fibrinogen of about 400,000 (Doolittle and Wooding, 1973). Ascidians and other protochordatcs seem not to have an extracellular protein comparable to vertebrate fibrinogen. 2. Invertebrate Clotting

Although many invertebrates havc clotting systems that are primarily large-scale agglutinations of blood cells, a few crustaceans-like the crayfish and lobster-and perhaps some insects have an extracellular circulating protein which is directly convertible into a gel similar to fibrin (Gregoire and Tagnon, 1962). I n thcsc creatures the conversion into the gel form is brought about by a calcium-dependent enzyme found in their VERTEBRATES

1

Xlll

I

PROTHROMBIN THROMBIN

FIBRINOGEN/

T H ROMBlN

FIBRIN t

XIII' Ca2+

CROSS -LINKED

* FIBRIN

PEPTIDES

LOBSTER CELL "FIBRINOGEN"

TRANSAMI DASE Calf

CROSS -LINKED FIBRIN

FIG.41. Comparison of vertcbrxte and lobster blood coagulation systems. Tlic complex sequcnce of events leading to the transformation of protlirombin to tliroinbin in vertebrates lins bcrn omitted. Similarly, tlic cventa provoking the disruption of the coagulocytes containing tlir trnnsamidnsc which clots lobster fibrinogen have not been included.

80

R. F. DOOLITTLE

TABL~C IX A Comparison of Some Properties of Lobster and Vertebrate Fibrinogens

Molecular weight Sedimentation coefficient Diffusion coefficient Partial specific volume Ex tin ction coefficient Polypeptide chains Amino-terminal end groups a

Af SP0,W

D20.w

E: z

l

Lobster"

Vertebrateb

420,000 14.5 2.9 0.71 12.5

340,000 7.9 2.2 0.71-0.72 15.8

280

-

2

Leucine

2 x 3

Variable

Fuller and Doolittle (1971a); 1)oolittle and Fuller (1972).

* See also Table I.

blood cells and certain other tissues (Glavind, 1948). These invertebrate fibrinogens are not clotted by vertebrate thrombins, and limited proteolysis is not involved in the conversion process (Fig. 41). Instead, these molecules are cross-linked directly by the formation of t- (y-glutamyl) lysine cross-bridges to give a covalently bonded gel (Fuller and Doolittle, 1971b). Furthermore, these crustacean fibrinogens are physicochemically very different from the vertebrate type, having much more compact structures, as reflected by their sedimentation and diffusion coefficients (Table I X ) and by the shapes viewed with the electron microscope (Fig. 42). The molecular weight of lobster fibrinogen is somewhat greater than that of vertebrates (Fuller and Doolittle, 1971a), and the protein appears to be composed of two polypeptide chains of molecular weight about 210,000 each (Doolittle and Fuller, 1972). Its amino acid composition indicates that it is composed of significantly more hydrophobic amino acids than the vertebrate molecule (Table X ) , an observation in keeping with the suggestion that large proteins with compact shapes nccd more nonpolar side chains to fill their inner volumes than rodlike proteins of the same molecular weight (Bigelow, 1967). The significant differences in the physical and chemical properties of the lobster and vertebrate fibrinogens led to the proposal that the two molecules are the products of independent evolution, the superficial siniilarity afforded by the utilization of t- (y-glutamyl) lysine cross-links not really being sufficient grounds for supposing any common ancestry (Fuller and Doolittle, 1971b). On the other hand, when one considers how readily the presence of cobalt ions wrought a dramatic conformational shift in the structure of vertebrate fibrinogen, perhaps i t would not require so much change to transform the lobster type into a rodlike protein. For example, supposing the two subunit chains of the lobster inoleculc

FIBRINOGEN TO FIBRIN CONVERSION

81

FIG.42. Electron iiiicrogrtiplis of iicgativcly sl:iincd lobst(-r fibrinogrn nnd fibrin prrp:u:itions. Upper lcft : High ni:ignifirntion of lobstcr fikxiiiogcn ( X 1,400.000); u ~ i ~ ) i,iglit: t~r Inrgcr field of lohtcr fibrinopcn (X650.000) ; lowcr left : c h i n s fornicd :If(cr cxposurc to clotting cnzyirie ( x210.000) ; lower riglit: :irr:ij-s of cliains forriled after longer cxpoaurc to clotting cnzyiiic (XlS0,OOO). From Fuller e l nl. (1971).

were nicked by some appropriate proteolytic eiizyrne to yield three pairs of nonidentical chains; would the molcculc tend to open up and assume a niore extended structure? Further discussion of this possibility will be deferred until we take up the problcms of biosyiitlicsis and assembly (Section V1,F). 3. Origin of the Polypeptide Chains in Fibrinogen

Are the t h e e polypeptide chains found in vertebrate fibrinogens related by a common ancestral type? The suggestion has been made previously that the a- and /?-chains might be so related, since they are both thrombin sensitive and because the limited sequence data a t the time suggested

82

R. F. DOOLITTLE

TABLE X Amino Acid Compositions of Lobster and Mammalian Fibrinogcnua

1

Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Cystine/2 Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan

t

Lobsteld

Mammalianc

9.9

12.0

7.1 8.2

6.4 7.8

10.9

11.5

5.3 6.2 5.5 1.3 6.9 1.7 5.1 9.6 3.2 4.1 4.2 4.2 4.8 1.9

5.2 9.7 4.4 2.3 4.5 2.1 4.3 6.2 3.4

3.1 2.2 7.4 5.2 2.4d

Presented as mole percent total amino acids.

* From Fuller and Doolittle (1971a). Q

Calculated from data of Cartwright and Kekwick (1971) for human, bovine, ovine, and porcine fibrinogens. From Henschen and BlombKck (1964), human and bovine only.

some homology (Doolittle, 1970). Since then, however, the amino acid sequences of those portions of the three chains found in the human disulfide knot have been completed (Blomback, 1971b), and some features of homology have been identified in all three chains. The alignment shown in Fig. 43 indicates that there can be as much as 2&25% identity between any two of the chains; it should be recalled that myoglobin and the various chains of mammalian hemoglobin only have about 25% identity (Dayhoff, 1969). There is one particularly striking sequence which recurs in all three chains of human fibrinogen a t the point where the three chains are likely all bound to each other by disulfide bonds. Thus, each has a pair of cysteine residues separated by the tripeptidyl sequence Pro-Thr/Ser-Thr/ Gly- (Fig. 44). Space-filling models of this fascinating linkage group reveal an unexpectedly high degree of frecdom for the individual chains, the “disulfide swivel” affording a junction where the three peptidcs could be mutually joined without crossing each other.

1

3

2

4

5

6

a j?

7

8

9

10 11 12 13 14 13 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

'Ma Asp Ser Gly Glu Gly Asp Phe Leu Ala Gln Gly Gly Gly"

1

1 E u Gly Val .4sn Asp hsn Glu Glu Gly Phe Phe Ser Ala hrg Gly His Arg Pro Leu Asp Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg Pro A h Pro Pro Pro Ile Fer Gly Giy Gly'o 41 42 43 44

1

45 46 47 48 49 50 31 52 53 51 53 56 57 58 59 60 61 62 G3 61 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

-

Val Glu Arg His Gln Ser Ala Cys Lys .4sp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp T r p .4sn T YLys ~ Cys Pro"

a

Val hrg Gly Pro Arg Val

j?

Tyr Arg A h Arg Pro Ala Lys .\la .\la .\la Thr Gln Lys L p Val Glu Arg Lys

7

81 82 83 84 85 86 87 88 89 90 91 92 93 94 93 96 97

ITITVal Ala Thr Arg Asp Asn Cys Cys Ile --

Ala Pro Asp Ala Gly Gly Cys Leu His Ala Asp Pro Asp Leu Gly Val I r u C

e

Leu Asp Glu Arg Phe Gly Ser Tyr Cys Proa -

98 99 100 101 102 103 104 105 106 107 108 109 110 111 I12 113 114 113 116 117 118 119 120

a

Srr Gli. Cys .\rg Metjl

p

Thr GIy Cys Gln Leu Glu Glu .\la Leu Gln Gln Glu Arg Pro Ile Arg

y

Thr Thr Cys Gly Ile .\la .kip Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys Thr Scr Glu Val Lys Ghi IRU~O

j?

Ser Met115

y

Ile Lys -\la Ile Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser (.\sp,Lys,Pro)Met:s

Asn Ser Val Asp Glu Leu Asn Asn .kn Val Glu Ala (Gln.Ser, Tyr.Ser, Ser, Srr. Phe.Thr)"r

121 122 123 124 123 126 127 128 129 130 131 132 133 134 133 136 137 138

FIG.43. Amino acid sequences of amino-terminal portions of human fibrinogen chains isolated from disulfide knot (DSK). The sequences have been aligned to allow maximum homology; in this regard, note particularly the sequences a t 7 W 3 . Arrows indicate thrombin cleavage points for release of fibrinopeptides. Adapted from sequences published by B l o m k c k (1971b).

E GI

8 3

8E

c)

84

R. F. DOOLITTLE

FIQ.44. “Disulfide swivel,” showing how three different chains (a-,,8-, and y-) can enter and leave a t maximum solid angles. Constructions with space-filling models (not shown) indicate considerable flexibility for such an arrangement.

If the three nonidentical chains in vertebrate fibrinogen are indeed related by common ancestry, should we assume that the original molecule was composed of equivalent chains? Monod et al. (1965) have suggested that in oligomeric proteins polypeptide equivalence implies spatial equivalence; they have extended this reasoning to the nonidentical (but clearly homologous) chains of hemoglobin. If the same principle is applied to fibrinogen, then we would expect an arrangement of the subunits that would afford each the same spatial orientation. For example, absolute equivalence would be attained if the amino-terminals of six chains were joined a t the origin of an x, y, z system of mutually orthogonal axes, the six chains proceeding therefrom to -x, +x, -y, + y , - e l and + a . On the other hand, the thrce nonidentical chains in vertebrate fibrinogen might have evolved by a slightly less direct route, whereby contiguous duplications gave rise to homologous regions in a single polypeptide chain, much the way tlic various regions of heavy chains in immunoglobulins are related (Singer and Doolittlc, 1966; Hill et al., 1966). If these chains were then subjected to limited proteolysis in such a way as to yield three separate chains, there would still be three more or less homologous chains, but the syriiinctry arguments put forth hy Monod et al. (1965) would have less bearing.

4. Relationship of Fibrinogen to Other Fibyous Proteins Fibrinogen has been assigned to the keratin-epidermis-myosin group on thc basis of early X-ray studies on fibers (Bailey e t al., 1943), and it is natural to compare it with thcse and other fiber-generating proteins to see whether there are structural elements in common. For example, col-

FIBRINOGEN TO FIBRIN CONVERSION

85

lagen has three chains (although often two are identical) and globular terminal portions a t both ends of an extended triple helix (Traub and Piez, 1971). It has a distinctive amino acid composition, including large amounts of hydroxyproline and hydroxylysine, as well as unusually high levels of glycine and proline. Neither of the hydroxylated amino acids is found in fibrinogen, and although its glycine content is somewhat higher than expected on a random basis (Fig. 8 ) , it is only about half that of collagen. The proline content of fibrinogen is perfectly average. Interestingly enough, collagen does exist in a precursor form, procollagen, which is transformed into collagen by limited proteolysis (Bellamy and Bornstein, 1971), the final molecule being capable of aggregating into fibers whereas the precursor is not. The long rodlike molecules evidently associate in a staggered overlap fashion to give the uniquely banded structure of collagen fibrils. Finally, covalent bonds involving lysine are introduced to confer additional stability on the fibers (Traub and Pice, 1971). One of these cross-linking sitcs has even been reported to be near the carboxy terminus of one of the chains (Reukrberg et al., 1972). These similaritics to the fibrinogen-fibrin system are superficial, however, and should not be considered evidence for any ancestral relationship between the two proteins. The covalent cross-links, although both involving lysine side chains, utilize a fundamentally different chemistry (Traub and Piez, 1971). Furthermore, the ends of a n extended protein are probably the best places to introduce cross-links in any system, and staggered overlaps, as any bricklayer knows, are the soundest design for any extended structure. Another protein which ought to be considered as a possiblc relativc is myosin. Myosin has a molecular weight not very different from that of invertebrate fibrinogen, a value of 430,000 being reported (Gazith et al., 1970). Its two ninjor s u h n i t s arc w70und around each other in a double helical fashion (see Fig. 49). The molecule is a very extended rod, although it does have a globular head region. Its aggregation into a polymeric form is markedly dependent on pH and ionic strength (Godfrey and Harrington, 1970). Other than these teiiuous similarities, there is no obvious relationship to fibrinogen or to the process which transforms it into fibrin. Another possibility is keratin, a protein that is exceptionally rich in cysteine. Although disulfide bonds play a very important role in fibrinogen structure, the molecule does not have an excessive number of them (Fig. 8 ) , and the coincidence of the U - p transformation of the “permanent wave” and the weak a-p transition observed during the compression of fibrin (Bailey et nl., 1943) is 1)rut)nbly only that. On the other hand, thc distribution of cystciiic residucs in com1)oncnt c of the high sulfur fraction

86

R. F. DOOLITTLE

of wool a-keratin (Elleman, 1971) follows a pattern which is very similar to that found in the “disulfide swivel” (Fig. 44). I n this case, many of the cysteines are spaced with three or four residues between them, serine and threonine being especially frequent among the intervening residues. It ought to be mentioned also that C- (7-glutamyl) lysine cross-links have recently been demonstrated in certain hair proteins (Harding and Rogers, 1971). The evolutionary origins of vertebrate fibrinogens remain a mystery, and many more sequence studies will have to be undertaken-on extracellular invertebrate fibrinogens as well as 011 the fibrinogen-like proteins involved in the agglutination of invertebrate blood cells (Solum, 1970)before we can expect a final solution.

F. Biosynthesis and Assembly Vertebrate fibrinogen is apparently made in the liver. More precisely, immunofluorescent studies have demonstrated that it is synthesized and stored in hepatocytes (Barnhart and Forman, 1963). The problem to be considered here is how the cell manages to assemble three pairs of nonidentical chains and bind them together with a complex system of di-

N INDIVIDUAL CHAINS

-SH

MOLECULE

C

HALF.MOLECULE

C

c

C

,I

*N

LN N J N N

N ,

,

C

c

PRODUCT

FIG.45. Conventional scheme for assembly of fibrinogen molecule from three nonidentical polypeptide chains. Compare with hypothetical assembly plan suggested in Fig. 46.

C

FIBRINOGEN TO FIBRIN CONVERSION

87

sulfide bonds. Since there are no data yet available 011 this problem, some conjecture is offered here based on observations made on other proteins. T o my knowledge, there has not been any previous discussion of this topic elsewhere. The final step in the process-or nearly filial step-is likely to be the joining together of two half-molecules (Fig. 45). Before that, however, the difficulties in synchronizing the syntlicsis a i d assembly of three large polypeptide chains seem formidable. Is it possible that something equivalent to profibrinogen is made first, in analogy with proinsulin (Steiner et al., 1967) , prothrombin (Magnuuson, 1971), or procollagen (Bcllamy and Bornstein, 1971), to i1a1nc just a few examples? Thus, one long chain of about 1500 arniiio acids-about the sizc of a myosin subunit (Gazith e t al., 1970) or, indeed, a lobster fibrinogen half-molcculc (Doolittle and Fuller, 1972)-could be synthesized, the disulfides formed, and the final product, either before or after the joining of half-molecules, nicked appropriately to yield the three nonidentical chains (Fig. 46). One consequence of this proposal would lie that, presuming that no large pieccs :we rcinovcd from the system, a t least two of the three final N

FIG.46. Hypothetical schcmc for sssernhl~.of fibrinogen nlolcculc starting with 1:irgc inolccular weight precursor chain whicli is snipped into constitucnt nonicicnticd chains aftcr folding. Snipping could occur eitl~crbrforc or after joining of halfmoleculcs.

88

R. F. DOOLITTLE

T.mIx X I Possiblr Scgnzrntal Arrangcnirnts for a Hypoihrlica! ProJhrinogcn fifolcculc and New End Groupa l'roducctl hi4 Iiimitrd Proirol?isis"

Precursor arrangemen tsh

Split I1

Split, I

3. P-a-7

Vnriahle- - Val _ - - -Pro Tyr- - Val Aln ----Val Th;---

4.c

_ - _ _ V a l Tyr_ _ - -

I . a-P-y

2. a-yP

j3-y-a

- - - -Pro

-

5. y-a-j3

- - - -Val

Ala Th;---

r-j3a

- - - -Ile

Variable- - -

6.

Ile

____

_ _ _ _ V a l Tyr

- - - -Val Ile

Variable-

--

- - - -'lo

Tyr---Val - - - -Val Ala Ile T h < - - -

- - - -Pro Val ----Val

Variable Ala Thr---

Only the most rommon mammalian end groups are presented; compare Table 111. Arrangement8 have amino-terminal segment on the left and carboxy-terminal segment, on right. c Note that only arrangement 4 involves neither highly variable nor prolyl end groups in the hypothetical splits.

carboxyl-terminals would have to be located near the amino-terminal cluster, unless there is a dramatic conformational shift after the (hypothetical) limited proteolysis. An examination of the end groups found in various fibrinogen chains might offer some clue to the arrangement of chains in a hypothetical profibrinogen molecule, since the cleavages effected would presumably exhibit some particular specificity and evolutionary constancy. A priori, we would not expect a trypsinlike specificity, since premature clotting and lysis would likely ensue in that case. Nor would proline be expected to be involved in the cleavage-unless it were always proline-since most proteases which hydrolyze ordinary peptide bonds have difficulty with those involving imino acids. The possible arrangements for three chains in a hypothetical profibrinogen subunit are listed in Table XI along with the new end groups which would be expected in each case. The most likely arrangements would predict the existence of an activating enzyme splitting on the carboxy side of valine residues.

VII. REEVALUATION OF VARIOUS MODELS

A . Properties of the Ideal Fibrinogen Model The idcal model of fibrinogen should be consistent with or offcr reasonable explanations of the following properties:

FIBRINOGEN TO FIBRIN CONVERSION

89

1. The scdiincntation-clifflisioii tlnta, wliich clearly indicate a particle whosc unliydratctl niolccular weight is approximately 340,000 2. The flow bircfringcncc and viscosity data, which indicate that the protciii lias an elongated strurturc undcr the conditions of measurement 3. A large cffcctivc volume, as prctlicted by the Scheraga-Mandelkern equation and ol)servctl espcrimcntally by small-angle X-ray measurements 4. A degrcc of a-liclicity equal to about 35% (Table I ) . Furthermore, tlic liclical regions should be distributed in accordance with the plasmin-derived fragments having a higher proportion than the small pieces chipped away during the digestion (Budzynski, 1971). 5. A collapsed/dried shape equivalent to a t least one of the structures observed in the electron microscope 6. A hydrated shape that can fit into the unit dimensions detected by the electron microscopy of microcrystals, the carboxy-terminal halves of a-chains notwithstanding 7. The existence of a double-disulfide knot (DDSK) 8. The pattern of fragments (“S,” “Y,” ‘TI,”and “E”) derived from plasmin degradation 9. A cobalt complex that is remarkably more compact than the native molecule 10. A swollen or dissociated structure after treatment with EDTA 11. A disposition of a-chains such that the carboxy-terminal halves are especially vulnerable to proteolysis and cross-linking of all kinds 12. An amino acid composition reflecting a high surface:volume ratio 13. A reasonable bioassembly scheme, especially with regard to the disposition of interchain disulfide bonds 14. A structure reflecting its evolutionary heritage 15. The fibrinopeptides and contact sites located in a manner suitable for triggering fibrin formation

R. Conditions Attached to the Ideal Scheme of Fibrin Formation The ideal schcme of fibrin formation ought t o bc consistent with or offer reasonable explanations of the following conditions: 1. The geometry of the fibrinogen molecule 2. Fibrinopeptide release as a triggering event 3. The existence of intermediate polymers 4. An exothermic process 5 . Sensitivity to tlic solution environnicnt, particularly pH and ionic strength

90

R . F. DOOLITTLE

6. An orientation of units consistcnt with thc clcctric bircfringcncc data (these data are independent of the interpretations dealing with the transient dipole) 7. 7-7 Dimcr formation involving all y-chains 8. a-Chain multimers 9. The reluctance of p-chains to becomc involvcd in cross-linking processes of any kind 10. The periodicity observed by X-ray and electron microscopy 11. The inhibitory properties of certain fibrinogen degradation products, particularly “Y” and “D” (Latallo et al., 19621)) 12. The observed defects in variant human fibrinogens

Doubtless other requirements could be added in either the case of the fibrinogen model or the scheme of fibrin formation, but the aspects listed have all been discussed in previous sections, and the basis for their inclusion hopefully has been made clear. Now let us see how the various models proposed in the past measure up to these demands.

C . Reevaluation of Models from Electron Microscopy 1. The Hall and Slayter Model

The most enduring model of fibrinogen structure offered by electron microscopists has been that proposed by Hall and Slayter (1959) in which the molecule is depicted as being composed of three nodular balls held together by very slender strands (Fig. 15). The calculated volume of the molecule-taken as the sum of the volumes of the three spherical nodules-is very close to the molecular volume determined from the partial specific volume (Table I ) , emphasizing the fact that the observed image corresponds to a collapsed and/or compact form of the molecule. If the hydrated molecule has any unusual open-mesh or expanded structure, as is suggested by the hydrodynamic data, then it must have been lost during preparative procedures. On the other hand, compact domains are common in other proteins, and the three nodules may actually depict the solvated molecule as well. In this case, the bulk of the accompanying water might be loosely caged between adjacent globules. It ought to be restated, however, that the small angle X-ray scattering experiment of Lederer (1972), as well as recent high shear rate viscosity measurements (Lederer and Schurz, 1972) , are not coiisistent with the Hall and Slayter model. In many other respects the Hall and Slayter model holds up remarkably well. One of its strongest points is the excellent agreement with

FIBRINOGEN TO FIBRIN CONVERSION

91

the pattern of fragments prodiiccd b y plasmin degradation. The recently demonstrated correspondence of fragmcnt %previously deduced to be a central nodule-with the disulfide knot produced upon cyanogen bromide digestion, clearly positions all six amino-terminal sections in the middle sphere of the model. On the basis of mass considerations alone, the terminal spheres would be the most likely locations for the carboxy terminals of the various chains, and the plasmin degradation studies bear this out also. Finally, a very logical scheme of polymerization can be developed with a Hall and Slayter-like model, satisfying many of the demands cited above.

6. Other Nodular Models The models of Kay and Cuddigan (1967) and Bang (1964) also postulate nodular structures (Fig. 15). I n the first case, the Kay and Cuddigan model seems much too long to accommodate the hydrodynamic data comfortably, especially if account is taken of likely degrees of hydration. The main attraction of Bang’s model lies in its simple explanation of the banding observed in the electron microscopy of fibrin. Other than that, it is pretty much a shortened, streamlined rendition of the Hall and Slayter model. Neither of these models fits the unit dimensions obtained from the electron microscopy of microcrystals as well as the Hall and Slayter model, Kay and Cuddigan’s being too long and Bang’s too short. 3. Koppel’s Model

The highly original model of Kijppel (1966, 1967, 1970) has provoked a number of interesting experiments and considerable discussion. Among its most exciting aspects are its novelty and pleasing symmetry. The model itself takes the general form of a pentagonal dodecahedron formed from the interwoven strands of the six constituent polypeptide chains (Fig. 47). The edge lengths, which are the observational basis for the model, are about 80 A. Such a molecule would offer an immediate explanation for the anomalous hydration estimates previously obtained with the Scheraga-Mandelkern equation, since the effective volume of the swollen particle would be almost ten times that calculated from the dry weight alone. The model can accommodate the sedimentation and diffusion data without too much juggling. To emphasize this point, Lederer and Finklestein (1970) constructed aluminum models of this sort and dropped them through fluids of various viscosities, comparing them with t,he sedimentation of comparable spheres. As far as physicochemical data are

92

R. F. DOOLITTLE

Fro. 47. Kijppcl’s open structure model bawd on the gromrtry of n pentagonal dodecahedron. Note (arrows) where three nonidentical chains come into contact, consistent with separate locations for two disulfide knots. (Drawn from a photograph of the model kindly supplied by G. Kijppd.)

concerned, the most difficult point to explain has to with the characteristic flow birefringence of fibrinogen solutions. One possibility is t h a t thc cngclike isotropic structure postulatcd by Koppel is dcfornicd into n long and distended shape by the high shearing forces involved, yielding an unnatural anisotropy. The suggestion has also been made that the characteristic flow birefringence results from a small fraction of the molecules being aggregated in linear chains (Lederer and Schurz, 1972). The Koppel model offers an excellent arrangement for two widely separated disulfide knots, the three nonidentical chains coming into contact near their amino terminals (Fig. 47). In this case also, the carboxy terminals of one of the chains would be located very near the aminoterminal clusters, a good arrangement for shielding a carboxy-terminal contact site by a n amino-terminal fibrinopeptide (Fig. 47). On the other hand, the Koppel model cannot readily be adapted to the idea of all six amino terminals being in a single cluster, nor does it correlate directly with the plasmin degradation products in the way the Hall and Slrtyter model does.

93

FIBRINOGEN TO FIBRIN CONVERSION Th

I

Th

I

FIG. 4s. Original schcmntic proposal locating disulfitlc knots at lhc rnds of an elongated molecule corresponding to the Hall and Slayter model. From R. Blomback et nl. (1968).

D. Comments on Schematic Depictions Derived from Bioorganochemical Observations 1. Disulfide Knots and Double Disulfide Knots

The original findings on the nature of the disulfide knot (DSK) seemed to be in good accord with both the physical chemistry-including the electric birefringence data-and the electron microscopy of Hall and Slayter (Fig. 48). When it was discovered that the DSK was actually dimeric, a new arrangement was devised by Blomback (1971b) which imparts a definite head-tail geometry to the molecule (Fig. 49). Although the postulated structure is intended to be diagrammatic, it does offer an intriguing parallel to the structure of myosin. Ironically, the

FIG.49. Sclicmntic proposnl for vcrtcbrntc fibrinogm molrcvle which positions two thsulfidc knots nrnr endl other nt onc rnd of the molrculc (BlombPck, 197111). The sketch of inyosin (right) is proiidrd as an csaiiiple of n molcculc known to have such a directional configuration.

94

R. F. DOOLITTLE

dcpiction is unrcalistic primarily bcrnusc it docs not agree with tlic clcctric bircfringence data a t all, over and beyond thc observations concerning a transient dipole, sincc a head-tail gconictry with the fibrinopeptides all a t one end would result in a fibrin monomer with a very large permanent dipole moment along its major axis, something all thc cxperimcntors in that field agree does not exist. 2. Fibrinogen Degradation Products

As noted above, the patterns of fragments obtained from the plasmincatalyzed degradation of fibrinogen have been very consistent with a Hall and Slayter-type model. In particular, fragment “E” was thought to correspond to the central sphere (Fig. 12). It was subsequently found that fragment “E” was immunologically cross-reactive with the disulfide knot, indicating that the amino-terminal clusters must be in the central sphere, not a t the ends of the molecule as had been supposed. This conjecture was doubtless an added stimulus for the reinvestigation of the molecular weight of the DSK and the revelation that it is indeed a dimer. Taken pretty much on their own, howevcr, the results of the extensive SDS-gel studies on plasmin-induced fragments lead t o a schematic depiction of the chains in the native fibrinogen molecule which seems entirely reasonable (Fig. 5 0 ) , whether or not the Hall and Slayter model turns out to be precisely correct. 3. Cross-Linking Contact 8ites

Studies on the arrangement of cross-linked chains resulting from fibrin stabilization have also resulted in schematic depictions of the starting molecule. The major premises on which these arrangements have been predicated in the past included (a) that all y-chains are packed together in such a way that reciprocal bonds can be formed between their carboxyterminal segments and (b) that substitute donors are prevented from incorporation into these sites until after the removal of the fibrinopeptides A (Fig. 27). These suppositions, combined with the notion that the ychain cross-linking sites are also primary contact sites, led to the depiction outlined in Fig. 51, originally devised when the electric birefringence data and terminal disulfide knots were thought to reflect the c

d C

7

C

+

+

. ’

r---

d 4

N N

N

\ d L N _-NJ-

L

d

C

N+bC L -C -

FIG.50. Schematic dcpiction of fibrinogen dcrivcd from sodium dodccogl sulfate gel electrophoresis studies of plasmin-generated fragments (Pizzo et al., 1972 ; Mills, 1972; Furlan and Beck, 1972).

95

FIBRINOGEN TO FIBRIN CONVERSION

1,

::J~L----

cccc

-

lXLL

~ :c

YNi

q

yJq-::=;=$y

FIG.51. S(.licniatic illustration of Iiow fil~rinop~ptitlcs could shicld both init i d polymerization sites and cross-linking sites on y-chains. The diagrammatic chain arrangement in this particular depiction was devised when it was grnemllp accepted that the fibrinopeptides were situated a t the ends of the fibrinogen molecule. From Doolittlc et al. (1972).

true situation. The transposition of the fibrinopeptides to the middle of the molecule and the demonstration that the bulk of the 7-chainprobahly including the carboxy-terminal segments-resides in the “D” fragment, not only makes that schematic arrangement untenable but also underlines the unwarranted assumption that the fibrinopeptides have to he spatially near the cross-linking sites. If, as now appears likely, these two moieties exist in widely separated domains, then an alternative explanation of the inaccessibility of cross-linking sites must be sought. One possibility is that there is a conformational change transmitted throughout the molecule upon removal of the fibrinopeptides, the distal cross-linking sites being exposed in the process. Alternatively, the crosslinking sites may become available after a local rearrangement of the contact sites themselves during the process of polymerization. I n other words, it is possible that the association of y-chain carboxy-terminal segments is itself the basis for exposing their acceptor side chains and that the accessibility of these sites is therefore only secondarily related to the release of the fibrinopeptides, an event which allows the association to take place. Two schematic depictions of how this might occur are offered in Fig. 52, one showing a general shift from a-helicity to pstructure, as has been suggested previously by many investigators, and a

96

R. F. DOOLITTLE

I

n A

i

I

+

-(

1-

+\

Fro. 52. Simple sketches of possible contact site readjustments ocrurring ns a result of polymerization. T o p : Shift from a-helix to intrrmolecular p-structure. Bottom: Shift from intramolecular p-structure to a-helices. In thc latter rase, B and B' designate potential cross-linking sites.

second going from p-structure to a-helicity. In either case-or in a variety of other possible situations-it is not difficult to imagine that the particular glutamine residue involved in cross-linking could become available during the contact operation.

E. Implementation of the Hall and Slayter Model It is still beyond our means to construct a model of fibrinogen with sufficient detail to satisfy all the conditions listed in Section VII1,A. It is possible, however, to consolidate many of the wide-ranging observations collected in preceding sections into a more detailed rendition of the Hall and Slayter model, particularly with regard to the general arrangement of the six polypeptide chains. The arrangement is based mainly on the cyanogen bromide and plasmin degradation studies, although consideration has been given to subsequent polymerization and cross-linking events also. The shortcomings and criticisms of the Hall and Slayter model discussed in previous sections must be kept in mind, but on balance it offers the most attractive choice at present. Given the three general domains of the Hall and Slayter model, there seems little doubt that the middle nodule must contain all six amino

FIBRINOGEN TO FIBRIN CONVERSION

97

terminals, and therefore all four fibrinopeptides. That the central globule must be very tightly packed is attested to by the failure of attempts to produce half-molecules by mild reduction procedures. The orientation of the chains is likely to be such that all four fibrinopeptides extend away from a specific location describing an arc of 180" or less (Fig. 53), much like a horsetail (cf. Fig. 49). The basis for this arrangement lies not only within the limits imposcd by disulfide linkages (Figs. 11, 43, and M ) , but also consideration of accessibility for thrombin attack and an appreciation of the small permanent transverse dipole moment found in fibrin honomer. This arrangement also proves important in the formation of intermediate polymers, giving each unit a front-side and a back-side. The strands between the nodules are probably triple helices composed of all three nonidentical chains and capable of imparting a rodlike rigidity to thc molecule as might be expected from its characteristic flow birefringence and viscosity. Cohen (1961) has previously suggested that the connecting strands might be the coiled coils that give rise to the a-patterns observed by the X-ray diffraction studies of Astbury's group (Bailcy e t al., 1943). It should be kept in mind, however, that the residual fragments after plasmin digestion have proportionately more ahelix than does the starting molecule, suggesting that the chewed away portions are nonhelical (Budzynski, 1971). If we accept the last two points-about the central nodule containing all six amino terminals and the connecting strands being triple helicesthen it follows logically that the carboxy terminals of the three nonidentical chains must reside in the terminal nodules. Moreover, the ar*

c"

I

I

I

FIG.53. Implrmrntntion of Hall and Slnytrr modrl to includc polypeptide c h i n

arrangcinent consistcnt wit11 inany of tlic biochemical data prcscntly available.

98

R. F. DOOLITTLE

rangement within the terminal globules ought to take account of three well established observations. First, the carboxy-terminal half of the a-chain is especially vulnerable to proteolytic enzymes of diverse specificities, and it is also extremely prone to get involved in cross-linking reactions of a variety of kinds. Furthermore, its presence is evidently an obstacle to crystallization (Tooney and Cohen, 1972). Second, the carboxy-terminal segment of y-chains, involving 15-20 residues, uniformly becomes involved in a reciprocal cross-link situation with neighboring molecules. Finally, ,&chains are protected in some way so that they do not get involved in cross-linking very readily and are not prematurely degraded by various proteases. An effort is made in Fig, 53 to accommodate these three points. The carboxy-terminal half of the a-chain is depicted as a free-swimming expendable appendage, unnecessary for subsequent polymerization reactions. The y-chains have been depictcd more or less axially with the carboxy-terminal segments hooked out a t the very extremities, although as noted in Fig. 30 this is not a rigid requirement. The p-chain is drawn as if it were on the same side of the molecule as the fibrinopeptide cluster, occupying a position more or less equivalent to the a-chain but lacking the loose appendage. An alternative provision might have been to adorn this region of the p-chain with carbohydrate to protect i t from proteolysis and wayward cross-linking. The arrangement is intended to provide a working model and ought not to be taken too literally. Its immediate purpose is to give us a starting framework on which to hang questions and answers regarding events during fibrin formation. Certainly one of the major points to be established experimentally is the specific location of the various carboxy terminals.

F. Fibrin Formation with a HalE and Slayter Model Although the Hall and Slayter model of fibrinogen has stood up to biochemical scrutiny remarkably well over the years, their original notions about fibrin formation must certainly be mistaken (Fig. 15). The overlapping system described by Ferry (1952) and elaborated upon by Stryer et al. (1963) and Bang (1964) has considerable support and ought to be accepted as a general feature. In this section an attempt is made to detail some of the interactions involved a t various stages of the polymerization process, keeping in mind that somc aspects of the chain arrangement in the starting unit are arbitrary. After fibrinopeptide removal, the first polymerization event is depicted in Fig. 54 as involving the formation of overlapping dimers. The pairing ought to be reciprocal, and thc topography of the Hall and Slayter-type

99

FIBRIKOGEN TO FIBRIN CONVERSION

model demands that two widely separated contact sites are involved. One of these ought t o be near the amino terminal of the a-chain on the central nodule, not only because of its exposure by release of the fibrinopeptides A, but also because of the defective polymerization exhibited by fibrinogcn Detroit (M. Blomhiick et al., 1968). The othcr site involved in this interaction has been arbitrarily assigned to the carboxy-terminal region of the P-chain, partly because of the cxpendahle nature of the carboxy-terminal half of the a-chain (Fig. 54). After the formation of an overlapping dimer, a new set of contacts comes into play in all subsequent additions to the polymer by way of the abutting ends. As the model has been formulated here, y-y dimer formation capabilities appear for the first time a t this stage. The ychains could have been depicted in the fibrinogen model in such a way that cross-linking could have occurred a t the stage of initial dimers, but only by distorting the original Hall and Slayter dimensions considerably. Experimentally, it should be recalled that Haschemeyer (1963) detected a true end-to-end dimer under dissociating conditions (Table VI). If an occasional cross-link had been introduced, the resulting covalent dimer would indeed be end-to-end as drawn, even if the original polymer had been formed by a system of staggered overlaps. On the other hand, Gollwitzer e t al. (1970) observed cross-linked fibrin under dissociating

e DIMER STAGE (Reciprocal

a-p)

POLYMER STAGE

(y-;

DIMER)

FIG.54. Polvmrrization scliemc using n Hall and Slaytcr-typc fibrinogcn molrriilc as bstsic unit. Note two distinct contact sitcs involved in formation of intermediate polymers, one being brought into operation hterally during dinier formation, and a, second involving end-to-end contacts coming into play a t the trirnerizsttion and subsequent steps.

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conditions and concluded that the swollen fibrils were two molecules thick, suggesting that the y-7 dimerization might take place laterally as opposed to end-to-end. The final point to be made about fibrin formation and the involvcmcnt of individual chains has to do with the formation of a-chain multimers. Evidence has been prcsented (Section V,E) that this phenomenon is associated with the later events in fiber formation involving thc latcrnl aggregation of intermediate polymers or events leading to coarse fibers. I n the schematic depiction the carboxy-terminal halves of a-chains have been assigned the function of becoming indiscriminately involved, the ultimate consequence being an open network that is quite exposed and susceptible to proteolytic attack. VIII. CONCLUDING REMARKS The principal aim of this review was to gather together data from a number of different fields involved in the study of the fibrinogen-fibrin conversion and to focus on their major disagreements. I n an effort to be comprehensive, I have had to venture into areas where at best I could only report without comment the conclusions of investigators. For the most part, however, I have tried to make this a critical review, bearing down on what I think are the major inconsistencies existing between different groups and different fields. I n this regard I have tried to be objective, presenting the different sides of various controvcrsies in as favorable a light as possible. I would have been remiss, however, if after all this consideration I had not offered my personal opinion on those points about which I feel most strongly. Thc fuiidamcntal qucstion which pcrvatlcs tliis rcl-iew still has to do with the general shape of the native fibrinogen molecule. From my point of view, the question remains unanswcred in a scientific sense, i t . , that a n unequivocal demonstration of the structure has been accomplishcd. On the other hand, I think the weight of the evidence favors an asymmetric moleculc which in solution occupies a cylinder of influence whose dimensions are about 450 A long and 90 A in diameter, a volume element consistent with the recent studies on microcrystals (Tooney and Cohen, 1972). How open or compact the domains are within that cylindrical volume is still anybody’s guess, my own feeling being that they cannot be as compact as Hall and Slayter’s micrographs would have them, but that there must be a t least three discrete macrodomains as the Hall and Slayter model describes them, and as indicated by the plasmin degradation studies. Beyond the considerations of gross structure, much of this review has been concerned with the arrangement of the three pairs of nonidentical

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polypeptide chains which comprise the molecule, and especially with efforts to describe the relative locations of the amino and the carboxyterminals. Similarly, considerable attention has been devoted to a discussion of polymerization with an eye to finding out which chains participate and what portions of them have contact sites. Although significant progress has been made in answering these questions, in some regards we arc still confronted with an “either-or” proposition, two completely different lines of logic applying, depending on the initial premise. The key point rests with whether or not the two amino-terminal clusters are in fact joined by disulfide bonds in the native molecule or whether this is an artifact of an unusual disulfide exchange. For if all six amino terminals are grouped together, then the conclusions drawn from the electric birefringence results must be rejected. The difference is as fundamcntal as having the fibrinopeptides a t the ends of the molecule or a t the middle. Not only does this make a basic difference in developing a logical scheme for polymerization, but it also has consequences for understanding the cross-linking events that occur after the fact. At present the evidence clearly favors the idea of a central cluster of all six ainiiio terminals, the carboxy terminals of the various chains being located a t the extremities of the molecule. The best immediate hope for obtaining a real picture of fibrinogen lies with image reconstruction techniques and their application to the electron micrographs of ordered microcrystals. It is possible that this approach will yield an outline of the native molecule and establish the general mode of how the units are packed in fibrin. Determination of the amino acid sequences of all three chains should be informative, if not conclusive, and characterization of cross-linked and derivatized chains should pin down the rclativc locations of tlic carboxy terminals. Experiments on the assembly of fibrinogen during biosynthesis, and a search for the prevertebrate ancestral molecule may provide clues which can help put all these observations into B reasonable context. Finally, appropriate conditions for crystallizing the native molecule in a mode suitable for diffraction studies may someday be found and ultimately thc entire threedimensional structure determined. By that time all the disagreements cited in this review will long since ha.ve been forgotten. ACKNOWLEDGMENTS thank iny wife, Francrs, for Iicr help in iweparing and typing this manuscript. I n crrtain of thc cxprrimcnfs described hcrr for thr first time I was assisted by Iirnn6 Chrn. Larry Doolittle. Rill Feaster, Yuan Lin. Mark Weinstein, and Gretchen Wooding. Tllis work T ~ siqrported S by XIH Grant HE-12,759. I am also gr:itcful to Drs. S. J. Singrr ant1 J. J. S l i n q ) for rcading the manuscript, and offering critical comments on it.

I would like

10

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