Distribution of intermediate polymers in the fibrinogen-fibrin conversion

Distribution of Intermediate Polymers in the Fibrinogen-Fibrin Conversion’+ * J. K. Backus,3 M. Laskowski, Jr.4 and H. A. Scheraga From the Department

of Chemistry,

Cornell

University,

Ithaca,

New York

and L. F. Nims From the Department

of Biology,

Brodkhaven

National

Laboratory, Upton, New York

Received June 13, 1952

Flow birefringence studies of the initial course of the fibrinogen-fibrin conversion have been carried out previously (4, 14) in the presence of hexamethylene glycol (HMG), an inhibitor which greatly influences the rate of the clotting reaction (3). These investigations have demonstrated that in the presence of HMG the early stages of the reaction involve elongated-polymer formation (4, 14), which is reversible upon dilution with the same solvent (14). More recently (5) light scattering measurements on similarly inhibited systems have confirmed this reversibility by demonstrating the dissociation of the partially polymerized fibrinogen down to the monomeric form. The present flow birefringence work was undertaken to investigate the influenbe of HMG and pH on the “observed lengths” of intermediate polymers before the gel point. By suitable control of the ionic strength and thrombin concentration in a fibrinogen-thrombin mixture it is possible to slow the clotting reaction (18) so that measurements can be made conveniently, in the absence of HMG, before gelation occurs. Sedimentation velocity measurements were made on several of the systems to aid in the interpretation of the flow birefringence results * This work was supported by the Office of Naval Research. * Part of this work was carried out at the Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission. 3 Present address: Research Department, Proctor and Gamble, Cincinnati, Ohio. 4 Public Health Service Fellow of the Xational Heart Institute. 3.54

FIBRINOGEN-FIBRIN

CONVERSION

355

EXPERIMENTAL

Materials Fibrinogen stock solutions (l-1.5$‘&) in 0.3 M KC1 were prepared by refractionating Armour’s bovine fraction I according to the procedure of Laki (6) and analyzed for per cent clottability (6) and for concentration of clottable protein (10). The protein in such preparations was invariably 95yo clottable. The preparations were stored at 2°C. and solutions more than 1 week old were not used. Parke, Davis bovine thrombin (approximately 20 r/mg.), dissolved in 0.3 &f KCl, was used to effect clotting, and HMG, recrystallized from ether, and dissolved in 0.3 M KCl, served as an inhibitor for several of the systems studied. Stock solutions of phosphate, citrate, and borate buffers were prepared from reagent-grade materials. The ionic strength in all cases was 0.6. Phosphate buffers were used over the whole pH range; in addition citrate was used at pH 6.5 and borate at pH 9.5-10.5.

Clotting Time General information about the clotting time of fibrinogen-thrombin mixtures as a function of pH and ionic strength was obtained from the data of Shulman and Ferry (18) and was checked in all cases, using their criterion for the clotting time.

Flow Birefringence In studies on uninhibited systems, clotting mixtures were prepared by mixing 2 parts stock fibrinogen solution, 1 part buffer solution of the appropriate pH, and 1 part thrombin solution at 25.O”C. The resulting solution had an ionic strength of 0.375 and contained 6.3 g./l. of fibrinogen. In most cases the thrombin concentration was 0.3 p/ml.; however, when the clotting time under these conditions was less than 8 min., it was necessary to reduce the thrombin concentration to as low as 0.01 p/ml. in order that measurements could be made before the gel point. For such low thrombin concentrations, it was necessary to use identical reaction vessels in all runs in order to obtain reproducibility. Measurements were also made on inhibited clotting systems by adding HMG to arrest the clotting reaction after it had proceeded for a given time. When determining the effect of protein concentration on the flow birefringence measurements, the above clotting mixtures were diluted immediately before measurements were made. In order to determine the effect of HMG, parallel dilutions were made with and without the inhibitor. To 20-ml. samples of the same clotting mixture were added, on the one hand, 25 ml. of a solution containing 100 g/l. HMG in 0.3 M KC1 and, on the other hand, 25 ml. of 0.3 M KCl. Each solution was further diluted with its own solvent. The resulting solutions were identical except that one series was 0.47 M in HMG. Measurements of the extinction angle, x, and magnitude of the birefringence, An, were carried out on these solutions at 25.O”C. with the apparatus previously described (13). The solvent viscosity for the aqueous solutions was 0.00894 poise, and for the aqueous HMG solutions was 0.0110 poise. Before solutions were placed in the apparatus, they were evacuated for 5 min.

to remove dissolved :tir, another 1 min. IAng required to fill the apparatus. When clotting times were less than 20 min., the evacu:ltion was omitted. In the cast of clotting times under 5 hr., mc:lsurements were rnatlc on the same solurion as the reaction progressed in t.he apparatus. For :I particular solurion, only the extinction angles were determined, the 37~ measurements being made on a duplicate sample. If the clotting time u-as under 20 min., the me:tsurcment.s were made only at one gradient,, 6600 sec.-l, the lowest gradient usetl in these studies, in only one sense of rotation for a particular solution. The cxitinction angles were independent of the direction of rotation, the planes of tr:lnsmission of t.he Nicol prisms having been carefully determined in advance. For solutions in which the clotting times were of the order of 5-25 hr., aliquots were withdrawn from the reaction mixture at various times, evacuated, and placed in the flow birefringence apparatus for immediate meusuremcnt. In such cases, it was possible to make measurements of x and in on the same solution. Rotary diffusion const.ants were computed from the dependence (12, 15) of the extinction angle on velocity gradient, assuming that the particles, for which :L prolate ellipsoidal model is used, have an infinite axial ratio. This is certainly a justifiable assumption for a dried specimen in light of recent electron microscope observations in this lahoratory. Since the dcpcndencc of the extinction angle on the velocity gradient is very insensitive to axial ratio, this is also considered to he a good assumption for the particles in solution. Because of t,he polydispersity of the system. the length calculated from I’errin’s equation (11) depends on the velocity gradient. The length calculated at 6tXlOsec.-’ has heen used as an index of the size distribution and is herein designated as 116600. For the calculations the minor axis of the particle was taken as 38bi., the length being relatively insensit.ive to the value chosen, for highly asymmetrical part.icles. As clotting proceeds, the increased values of L 66n0ran be attributed to an increase in the concentration of a series of intermediate, elongated polymers of fibrinogen, present in solution before the gel point.

Sedimenlafion Sedimentation velocity measurement.s were made on inhibited and uninhibited systems both before and beyond the gel point. The runs on uninhibited systems were carried out so that, the rotor would reach maximum speed before the gel point. Solutions investigated in this manner had a fibrinogen concentration of 0.5% and an ionic strength of 0.3, the thrombin concentration being varied to give convenient clotting times at each pH investigated. In some cases the solution was allowed to gel in the slalionary ultracentrifuge cell, kept in such an orientation that the meniscus of the gel formed in the position it normally acquires during a run. Runs were also carried out on inhibited systems which were allowed to clot for various times at various pH values and the reaction “stopped” by addition of HMG. In the acid and neutral ranges, 0.47 .I! HMG was sufficient for inhibition, but for the alkaline range a higher inhibitor concentration (0.94 M) was required. The fibrinogen and thrombin concentrations during clotting were the same as in the uninhibited runs. .A twofold dilution was introduced by addition of the inhibitor solution just. prior to ultracentrifugation.

FIBRINOGES-FIBRIN

CONVERSION

357

Sedimentation velocity measurements were carried out at 20,000 and 60,OOO r.p.m. and room temperature with a Spinco Model E ultracentrifuge. RESULTIS

Flow Birefringence The extinction angle and birefringence curves are similar to those reported previously (14) and justify the application of an orientation theory to the interpretation of the flow birefringence results. The birefringence was positive in all cases. In order to study the influence of pH on the clotting reaction of fibrinogen in the absence of HMG, the change of the apparent length, L6600, with time, t, was determined over the possible pH range. At all pH values between 5.2 and 10, an increase in. Le600was noted with increasing time after the addition of thrombin. Results of some of these measurements are shown in Fig. 1 plotted against t/t,, where t, is the clotting time at the given pH. The increasing degree of polymerization as the reaction proceeds is evident. Except for the data at pH 5.52 there is no tendency for the curves to level off at the gel point. It is also evident that the pH of the solution has a significant effect upon the reaction. Not only has the clotting time and the character of the clot formed been shown to change with pH (2, 18), but the particle length at the gel point is also dependent on pH. This effect is shown in Fig. 2. The variation of Lssoo at the gel point with pH is of the same general type as that of clotting time with pH reported by Shulman and Ferry (18). Because of the very low solubility of fibrinogen at pH 5.2 and below, it was impossible to tell by flow birefringence measurements whether or not aggregation occurred. However, experiments of Laki (6) indicate that none occurs at these low pH values, the only reaction being activation of fibrinogen by thrombin. ,4s the pH is raised, polymerization occurs more readily, and a larger concentration of longer particles is attained before the gel point. There is some doubt as to the accuracy of the value of L66ooat pH 10.4. Fibrinogen was incubated without t(hrombin at pH 10.0, 10.3, and 10.9 for 24 hr. with no increase in apparent length at pH 10.0, a small increase at pH 10.3, and the formation of a precipitate at pH 10.9. Thus, while the value of Lee,,,,at pH 10.4 is of doubtful validity, the results at and below pH 10.0 are considered reliable. The results obtained at pH 10, and shown in Fig. 3, are quite striking in that they show an increase in L 6600up to 3 hr. There is, however, little subsequent increase in length, and clotting dbes not occur within

CUITTING TIME IN MINUTES

- DH

0 5.52 A 6.15 . 7.0 0

1200 6.3 36 33 I6 31 60

7.9

o 6.99 l 9.72 A 9.92

FIG. 1. Increase in Leeo,~with reaction time in fibrinogen-thrombin at several pII values.

0 WITMOUT HEXAMETHYLENE 8 WITH HEXAMETHYLENE

1 O5

systems

GLYCOL

GLYCOL

I

I

I

I

I

6

7

6

9

IO

PH FIG. 2. Dependence of LSSO~ at the gel point on pH. For pH 2 10 no gelation

orcurred, the value plotted being the maximum length attained at these pH values.

FIBRINOGES-FIBRIN

CONVEHSIO~

359

26 hr. It appears that at this pH, fibrinogen is activated by thrombin and partially polymerized, although the reaction of the polymers to form fibrin does not take place. The concentration dependence of L 6~00is shown in Fig. 4. Dilutions were made both with and without HMG, all other nonprotein components of the solutions remaining the same. When used, the inhibitor concentration was 0.47 M. Solutions at pH 5.75 and 9.7 were allowed to react approximately 65y0 of the clotting time before dilutions were made. At pH 6.5, dilutions with HMG were made on systems allowed t.o react

REACTION TIME IN HOURS FIG.

3. Increase in LC60pat pH 10 without

subsequent gelation.

for approximately 90% of the clotting time, while dilutions without HMG were allowed to react 65$!& of the clotting time. This practice is necessary because of the rapid rate of the clotting reaction in the absence of inhibition. If a reaction were allowed to proceed for 90roof the clotting time, gelation would occur during the measurements if no HMG were added. Measurements made on systems which had reacted for 90% of the clotting time were somewhat more precise because of the longer length and therefore higher birefringence of the particles. It should be noted that the reaction is actually proceeding during measurement, the rate of reaction being much slower in the inhibited systems than in the uninhibited ones. As discussed previously (14) a length decrease due to elimination of solute-solute interaction would be expected to yield a linear plot having a smaller net decrease in length than that shown in

360

BACKTS,

LASKOWSKI,

2XHEK:iG.I

ASD

NIMS

Fig. 4. The curvature of the plot for dilutions with HMG at pH 6.5 definitely indicat.es a dissociation of polymers. -4 similar effect is suggested for all other dilutions, although the smaller particles and lower birefringence have made measurements difficult. It is clear that dilutions with HMG consistently give a shorter length than dilution without the inhibitor. At least part of this effect is due to the faster reaction occur5cQc

4Oco

7.

3ooc A L /

8 ,”

.

A

2coc

WITH 0.47M WITHOUT HMG HMG 5.75 . 0 6.5 A 0 97 . 0

DH

0 . I

IOOC

/

FIBRINOGEN

z CCJNCENTRATION

3

G-VLITER

Fro. 4. Effect of dilution on Lseoo, both in the presence and absence of HMG. Measurements at pH 6.5 in the presence of HMG were made at 90% of tC. All other measurement8 were made at 65% of t,.

ring in the uninhibited systems during measurements, but it is as yet impossible to draw definite conclusions as to the source of this decrease from the flow birefringence measurements. However, evidence from the ultracentrifuge runs described below suggests that HMG has a dissociating influence on the system. Sediment&on The runs on uninhibited systems at pH 6.5 indicate the same behavior reported for inhibited systems by Shulman and Ferry (19), i.e., the pres-

FIBRINOGEN-FIBRIN

361

CONVERSION

ence of two discrete peaks, one corresponding to fibrinogen and the other to the heavier intermediate polymers. Since a finite time was required to accelerate the rotor to maximum speed, it was not possible to make observations sooner than about 30 min. after adding thrombin to the fibrinogen solution. The clotting time of such a solution was about 55 min., but no gel formed in the bulk of the cell during the, ultracentrifuge run. A

c

D FIG. 5

FIG. 5. Schlieren FIG. 6. Schlieren

0

c FIG. 6

photographs photographs

of ultracentrifuge of ultracentrifuge

runs. runs.

See Table See Table

I I

If the solution were allowed to clot before starting the run, the gel centrifuged to the bottom of the cell during acceleration to 60,000 r.p.m. leaving behind discrete peaks corresponding to lower molecular weight components not yet bound into the gel network 1 hr. after gelation occurred. In the runs made on systems where HMG was added before placing the sample in the ultracentrifuge at pH 6.5 and at 9.0, two distinct peaks, identical with those described by Shulman and Ferry, were observed. At pH 6.5 a progressive increase of the area of the faster peak (accompanied by a corresponding decreasein the area of the slower peak) was observed as a function of time between the additions of thrombin and

362

BACKUS,

LASKOWSKI,

SCHERAGA

AND

NIMS

HMG inhibitor. A short induction period before the faster peak appeared was also noted. However, at pH 5.65, 5.9, and 6.1, both in citrate and in phosphate buffers as well as in solutions where the pH was adjusted by addition of HCl, only one peak appeared. The sedimentation constant of this peak corresponded to that of the slow or “fibrinogen” peak. No appearance .of a second fast peak was noted even if HMG was added right before the expected gel point of the system, e.g., after 18 min. when the clotting time was 1935 min. Representative schlieren photographs of several ultracentrifuge runs at 60,000 r.p.m. are shown in Figs. 5 and 6. TABLE I Conditions for Ultracentrifuge Runs Represented in Figs. 5 and 6~ Fig.

5A 5B 5c 50

PH

Clotting time

A&J

min.

min.

40 1935 44

6.5 6.5 5.65 9.0

26 18 30

55 6A 6.5 40 26 6B 6.5 35 6C 5.9 22 19 6D 5.9 0 Figure 5A represents pure fibrinogen; ail others represent fibrinogen-thrombin mixtures. b At is the difference in times between the addition of thrombin and the uddition of HMG. In Figs. 5A, 6A, and 6C no HMG was added.

The conditions of the experiments represented in these figures are given in Table I. Figure 5 compares fibrinogen to several clotting mixtures at three pH values. Figure 6 shows the effect of HMG at t,wo pH values. In uninhibited systems at pH 5.9 a more complex pattern is observed. If HMG is present at this pH, only the single peak is observed, whereas two peaks are observed at pH 6.5 in both the presence and absence of HMG. The behavior of the uninhibited system at pH 5.9 could be considered as a transition between the appearance of one and of two peaks DISCUSSIOX

The results may be interpreted in terms of the simplified, reversible equilibria suggested previously5 (7) for the clotting process as a whole. 5 The notation of the reaction intermediates with Dr. J. D. Ferry and his collaborators.

has been revised in agreement

FIBRINOGEN-FIBRIN

CONVERSION

363

(1)

F;f+P nf *f,

(2)

mf, S fibrin

(3)

where F is fibrinogen, P is Lorand’s fibrino-peptide (9), T is thrombin, f is activated fibrinogen (6), and f, is a series of intermediate polymers of varying n which give rise to the observed flow birefringence and sedimentation behavior. On the basis of the experimental results reported here information may be obtained about the effect of HMG and pH on the distribution of polymers, f,, and also about the stability of polymers of particular molecular weights. Viscosity (19), flow birefringence (14), and light scattering (5) results show that the second step of the reaction is reversible in the presence of HMG at pH 6.5. The flow birefringence results (supported by the ultracentrifuge data) reported here indicate that this reversibility seems to exist at other pH values both in the presence and absence of HMG since the polymers tend to decrease in length on dilution. The nature of the distribution of polymers is pH dependent, there being an appreciable quantity of long polymers from about pH 6 to 10, while, outside of this pH range, the distribution appears to be shifted toward lower molecular weight polymers. The flow birefringence results at a particular pH represent an average over the distribution of sizes present in the solution and also over the relative rates of interconversion of n-mers into n’-mers. It is also possible that the values of LEEO,,at t, include the effect of the rate of step 1, i.e., the distribution at t, could be a function of the rate of production of f. Of course, no information about step 3 is obtainable from such experiments except for the situation at pH 10. Here, no clotting occurs and yet increased values of Lseoo are observed. It is apparent that steps 1 and 2 occur at this pH whereas step 3 is inhibited. It appears from Laki’s observations (6) that at pH 5 only step 1 occurs.g As already indicated it has not been possible to investigate the region higher than pH 10 for possible inhibition of step 2. The sedimentation data also shed light on the existence of such reversible equilibria in step 2. The presence of only two peaks at pH 6.5 or 9.0 is not to be interpreted in terms of only two distinct species (monomer and n-mer). Neither is the presence of a single peak below pH 6.1 indicative of only one species, (the monomer), even though 6 Further

proof

has been reported

recently

(8).

364

BACKUS,

LASKOWSKI,

SCHERAGA

AND

NIMS

it is possible to relegate the possible presence of the other components to slight base-line distortion. The flow birefringence results show increased values of Lseooincompatible with an interpretation based upon the existence in solution below pH 6.1 of only f-monomers. The explanation of the sedimentation behavior also appears to lie in these reversible equilibria. The sedimentation of systems in such an equilibrium has been treated by Pedersen (20). If the equilibrium is a very rapid one, the whole system sediments as one peak with a sedimentation constant which is a function of those of the individual n-mers and their equilibrium distribution. This type of behavior has been observed in several protein systems in rapidly reversible equilibria, e.g., the chymotrypsin monomerdimer equilibrium (16, 17). If the equilibrium is attained only very slowly and the sedimentation constants are sufficiently different, all the components appear as separate peaks. In caseswhere the equilibrium is attained with intermediate rapidity, some complicated distribution of the material arises. The behavior observed in inhibited clotting systems is probably a reflection of the presence of several rapid and several slow steps (analogs of step 2) so that a particular range of polymers appears to be favored at a given pH. For example, the two peaks present at pH 6.5 and 9.0 could arise from apparent stabilization of polymers in the range of sedimentation constant near that of fibrinogen (the slow peak) and of polymers in a higher range of sedimentation constant (the fast peak). This is in agreement with the high values of Le60”in this pH range. Below pH 6.1 stabilization of polymers having a range of high sedimentation constants is not favored and the fast peak does not appear. Only the slow peak appears which is a composite of several distinct polymers which also are responsible for the intermediate value of L 6600.The fact that there exists a range of stable polymers (fast peak) is not due to the addition of the HMG since it appears also in the uninhibited system (Fig. 6A). In fact, the two-peak type of distribution is more favored in the uninhibited system (see Figs. SC and SD), i.e., even though the flow birefringence results could not give an unambiguous answer at pH values other than 6.5, the ultracentrifuge results do indicate a dissociating effect of HMG. Thus, depending on the pH, we have particular n-mers stable with respect to analogs of step 2. Below pH 6.1 we must assume that conditions favor a greater distribution between many rapidly reversible intermediates rather than stabilization of one particular n-mer. Therefore, the results reported here give an indication of the existence

FIBRINOGEN-FIBRIN

COIWERBION

365

of various distributions of polymers at various pH values with the possible existence of apparently stabilized n-mers (for particular values of n) before the gel point (at t,). These polymers persist for a considerable time beyond the gel point and are probably ultimately bound into the gel network. The source of these pH effects is probably similar to that for the dependence of clotting time on pH and, as discussed by Shulman and Ferry (18), is probably of electrostatic origin. Also, in light of the recent work of Bailey et al. (1) who showed that a free amino group is liberated upon activation of fibrinogen by thrombin, it would appear that the equilibrium between -NHz+ and -NH2 may be a significant factor. SUMMARY

The effect of pH and hexamethylene glycol (HMG) on the initial course of the thrombin-fibrinogen reaction has been studied by means of flow birefringence, supplemented by sedimentation velocity measurements. As the polymerization proceeds the distribution of intermediate polymers depends on the pH, there being a significant number of relatively long particles before the gel point between pH 6 and 10 and shorter ones out,side of this range. These polymers persist, for long periods of time beyond the gel point but are probably ultimately bound into the gel network. Hexamethylene glycol appears to have a dissociating effect on the intermediate polymers. There appear to be many rapidly reversible equilibria between thrombin-activated fibrinogen and intermediate polymers of this activated material. However, not all these equilibria lead to the formation of stable intermediates, the range of stable intermediates depending on the pH. This phenomenon is thought to arise from the sameelectrostatic effects suggested by Shulman and Ferry (18) to explain the dependence of clotting time on pH in similar systems. REFERENCES 1. BAILEY, K., BETTELHEIU, F. R., LORAND, L., AND MIDDLEBROOH, IV. R., Nature 167,233 (1951). 2. FERRY, J. D., AND MORRISON, P. R., J. Am. Chem. Sot. 69, 388 (1947). 3. FERRY, J. D., AND SHULUAS, S., J. Am. Chem. Sot. 71,3198 (1949). 4. FOSTER, J. F., SAMSA, E. G., SHULXIN, S., AND FERRY, J. D., Arch. Biochem. Biophys. 34, 417 (1951). 5. KATZ, S., GIJTFREUND, Ii., SHULBIAN, S., AND FERRY, J. D., Amer. Chem. Sot. Abstracts, 121st meeting, p. 3%. Milwaukee, April, 1952.

366

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LASKOWSKI,

SCHERAGA

AND

NIMS

6. LAKI, K., Arch. Biochem. Biophys. 32, 317 (1951). 7. LASKOWSKI, M., JR., RAKOWVITZ, D. H., AND SCHERAGA, H. A., J. Am. Chem.

Sot. 74, 280 (1952). 8. LASKOWSKI, M., JR., SCHAPIRO, B. L., DONNELLY, T. H., AND SCHERAGA, H. A., Conference on the Fibrinogen-Fibrin reaction. National Research Council, Wash., D. C., May 21, 1952. 9. LORAND, L., Nature 167, 992 (1951). 10. MORRISON, P. R., J. Am. Chem. Sot. 69, 2723 (1947). 11. PERRIN, F., J. Phys. Radium, [7], 6, 497 (1934). 12. PETERLIN, A., AND STUART, H. A., Hand- u. Jahrb. d. Chem. Phys., Bd. 8, Abt. IB, Becker and Erler, Leipzig, 1943. 13. SCHERAGA, H. A., AND BACKUS, J. K., J. Am. Chem. Sot. 73.5108 (1951). 14. SCHERAGA, H. A., AND BACKUS, J. K., J. Am. Chem. Sot. 74, 1979 (1952). 15. SCHERAGA, H. A., EDSALL, J. T., AND GADD, J. O., JR., J. Chem. Phys. 19, 1101 (1951). 16. SCHWERT, G. W., J. Biol. Chem. 179, 655 (1949). 17. SCHWERT, G. W., AND KAUFMAN, S., J. Biol. Chem. 190, 807 (1961). 18. SHULMAN, S., AND FERRY, J. D., J. Phys. & Colloid Chem. 64, 66 (1950). 19. SHULMAN, S., AND FERRY, J. D., J. Phys. & CoZZoidChem. 66, 136 (1951). 20. SVEDBERG, T., AND PEDERSEN, K. O., The Ultracentrifuge, p. 28. Oxford Univ. Press, London, 1940.