The mechanism of the fibrinogen-thrombin reaction

THROMBOSISRESEARCHVol.i2,pp.953-9G. Q Pagmon Press Lrd.1973.Printd inGreat Britain

THE MECHANISM CF THE FIBRINOGEN-THROMBIN WCTION

Desmond H. Hogg and Birger BlombPck Department of Blood Coagulation Research, Karolinska Institutet, Stockholm, Sweden

(Received 20.1.1978. Accepted hy Received by Executive Editorial

Editor Office

S. Magnusson) 1.5.1978)

ABSTRACT The kineticsof the thrombin-catalysed release of fibrinopeptide A from fibrinogen, modified fibrinogen and various fragments of fibrinogen has been investigated in an attempt to elucidate the structural elements of fibrinogen which define its interaction with thrombin. These elements have been found to be contained within the structure formed by the first 51 amino acid residues of the &-chain. It is proposed that the binding which is of fundamental importance takes place within the sequence 1-23 of this chain and may even be localised to the sequence 8-16. This binding is strengthened, either directly or indirectly, by structures within the sequence 34-44 and to a lesser extent by structures within the sequence 45-51.

INTRODUCTION Thrombin catalyses the release of fibrinopeptides A and B from fibrinogen (1,2). In so doing, the enzyme cleaves only four specific arginyl-glycyl peptide bonds in the fibrinogen dimer, whereas trypsin, a closely related serine proteinase, cleaves almost all arginyl and lysyl peptide bonds in fibrinogen (3,4). To explain the difference between the specificities exhibited by the two enzymes, it has been suggested (5,6) that thrombin has an extraordinarily hidden active site which for steric reasons can react only with the N?i2-terminal portions of the Acr- and Be-chains. In a previous report (7) we concluded that the structural elements of fibrinogen which are of major importance in defining the thrombin-catalysed release of fibrinopeptide A, are located within the architecture formed by the first 51 amino acid 957

THROMEIN-FIBRINOGEN

954

REACTION

Vo1.12,No.6

residues of the h-chain. In this communication,we report findings concerning locality cf these thrombin-bindingelements within this architecture.

MATERIALS AND METHODS Bovine thronbinwas purified to an activity of 1,800 NIH units/m8 by the procedure described previously (7). Trypsin, TPCK-treated,was obtained from WorthingtonBiochemicalCorporationand sperm whale apomyoglobinwas supplied by Beckman InstrumentsInternationalS.A.

Human plasmin, having an activity

of lo-15 caseinolytic(CTAl) units/mg, was kindly supplied by Dr. Bjarn Wiman, Dept. of Medical Chemistry, University of Umei, Sweden. Tryptic digests, two dimensionalpeptide maps and amino acid analysis were carried out as described before (8). The NH2-terminalswere analysed as described in a previous communication(7). Preparationof fragments of fibrinogenused as subsrrates.

;=

The various substratesused in this investigationare listed in Table I.

TABLE I Initial rates of the thrombin-catalysedappearanceof NH2-terminal glycine in various substrates at 370C in 25 mM Tris-Xl, 0.125 M NaCl, pH 7.2. Results for substrates l-4 have previouslybeen published elsewhere (7).

Substrate Substrate No. 1 2

3 4 5

Fibrinogen Thioredoxintreated fibrinogen N-DSK Ao(l-51) AcY(l-51) BB(f-118) An(l-44) &i(l-3312 Ao(l-23) BB(l-118)

Molecular Molarity weight uM

10 (mol NI?l Zc-1)

20 25

2.00 t 0.14 2.62 + 0.13

5,500 5,500

20 40 40

1.48 2 0.17 1.05 f 0.07

12,200 4,800 7,900 2,300 12,200

40 40 20 40 40

340,000 340,000

59,000

'Footnote: CTA = Committee on ThrombolyticAgents

0.55 + 0.01 0.42 z 0.07 0.08 _=0.01 0.06 - 0.01 0

Vol.12,No.h

THROYBI4-FIBRINOGEN

REACTIOS

955

Fibrinogen, thioredoxin-treated fibrinogen, N-DSK and Acr(l-51) were prepared as described previously (7). The B8-chain of N-DSK, B8(1-118), was isolated from reduced and S-carboxymethylated N-DSK by gel-filtration (9) and this was further purified by chromatography on CM-Cellulose (9.10). Two sub-fragments of the Pa-chain of N-DSK, h(l-44)

and pCr(l-23)were pre-

pared by digesting this chain with plasmin. To a solution of 22 mg of Pa(l51) in 1 ml of 0.2 M NH4HCO3, pH 8.2, was added 0.7 mg of human plasmin. The mixture was incubated at 37' for 2 h, 0.1 ml of glacial acetic acid was then added and the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. la). The material recovered from peak 1 was chromatographed on G-100 to remove plasmin and yielded 11.5 mg of freeze-dried &(l-44).

FIG. 1

1.5-

,/‘.

: ;

1.0 -

:

-r

-,: :

Gel-filtration of plasmin digested An(l-51) on a column (82 cm x 1.8 cm*) of Sephadex G-25 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 2 ml. a) aftek a 2 h digestion; b) after an 18 h digestion, inset: thin-layer chromatography of material recovered from peak 2 with arrow indicating the spot found to contain Aa(l-23).In both diagrams, the final peak of radioactivity having no absorbance at 280 ran is due to the peptide segment 45-50 (see Fig. 4).

The smaller fragment, Pa(l-23), was obtained on prolonged digestion of Acr (l-51) with plasmin. To a solution of 36 mg of Ao(l-51) in 2 ml of 0.2 M NH4HCG3, P H 8.2, wad added 0.8 mg of human plasmin. The mixture

was incuba-

ted at 37' for 18 h and after addition of 200 ul of glacial acetic acid the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. lb). The second peak, which was pooled as indicated, yielded 15.5 mg of freeze-dried material. Thin-layer chromatography of this material on cellulose (Whatman CC41) in the medium l-butanol-pyridine-acetic acid-water (150:100:30:120v~v)

956

THROWBTN-FIBRISOGE3

REX-l-TON

vo1.12,so.6

produced the ninhydrin-positive pattern shown in the inset in Fig. lb. Amino acid and NH*-terminal analysis of the material eluted from the spots indicated that the arrowed spot in Fig. lb contained &(l-23).

To isolate this

fragment on a preparative scale, 70 )~l of an aqueous solution of 6 mg of the material obtained from the above gel-filtration were applied as a streak to a 0.5 nm thick layer of cellulose on a 20x2@ cm plate. The plate was developed in the same medium as was used above and was air dried. The band corresponding to the arrowed spot in Fig. lb was located by spraying the two vertical edges of the thin layer with a ninhydrin-collodine solution (11) and allowing this to develop for 1 h at room temperature. The appropriate band of cellulose was then scraped from the plate and the peptide material (0.8 mg) was recovered by three one-hour long extractions with 2 ml of 10% acetic acid at room temperature.

30

40

50

60

TO

80

sfacl,o!. Nurntm

FIG. 2 a) Gel-filtration cf N-bromosuccinimide treated N-DSK on a column (95 cm x 5 cm 2, of Sephadex G-50 in 10% acetic acid. Flow rate: 20 ml/h; fractions: 5 ml. b)Gelfiltration of material recovered from "a" on a column (82 cm x 5 cm2) of Bio-Gel P-10 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 5 ml. Fractions in "b" were subjected to ninhydrin analysis (12).

*or

A small dimeric fragment of fibrinogen, &~(1-33)~, the two halves being joined by the symmetrical disulphide Aa28-&28, the tryptophanyl peptide bonds (h33 nimide. Amodificationof

was prepared by cleaving

and Aa44) in N-DSK with N-bromosucci-

a procedure which has been described (13) wasused.

To minimise the modification of Pa24 His, free histidine was incorporated in the reaction mixture. To a solution of 195 mg of N-DSK and 18.7 mg of histidine in 11 ml of 70% acetic acid was added 9.5 ml of. a solution of Nbromosuccinimide (18 n&ml)

in the same solvent. This was allowed to stand

Vo1.12,No.6

THROXBIN-FIBRTNOCEX

REACTIOS

95;

at room temperature for 2 h. Following addition of 1 ml of a solution of tryptophan (24.8 mg/ml) in 70% acetic acid, the solution was allowed to stand at room temperature for an additional 16 h. This was then diluted with water to 10% in acetic acid and was freeze-dried. The residue was dissolved in 5 ml of 10% acetic acid and gel-filtered through a column of Sephadex G-50 (Fig 2a). The fractions indicated were pooled and freeze-dried and the recovered material was dissolved in 4 ml of 10% acetic acid and gel-filtered through a column of Bio-Gel P-10 (Fig. 2b). On freeze-drying the fractions indicated yielded 3.5 mg of material, the amino acid composition of which was in agreement with the structure An(l-33)2; see Table II. TABLE II Amino acid analysis of substrates 6-8 obtained after 22 h hydrolysis (7). Half-cystine was determined as S-carboxymethylcysteine. Figures in parenthesis are the theoretical number of residues and those underlined were used as integer. Peptide

. Amino Acid

Aci(l-44)

Half-cystine Aspartic acid Serine Glutamic acid ProTine Glycine Alanine Valine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

1.85(Z) 6.30(7) 3.11(4) 5.87(5) 1.79(2) 6.30(6) 2.71(3) 3.00(3) 1.52(l) 0.83(l) 1.76(2) 0.70(l) 1.94(2) 3.40(3)

Ao(l-23)

&X(1-33)2 - half molecule

2.24(2) 0.82(l) 2.62(3) 0.68(l) 5.99(6) 1.84(2) 1.46(3) 0.96(l)

0.30(l) 3.63(4) 2.02(3) 4.92(4) 1.11(l) 5.76(6) 2.88(3) 3.00(3) 1.32(l)

1.00(l) 2.58(3)

1.22(l) 0.86(l) 0.75(l) 3.48(3)

Kinetic Techniques The kinetics of the thrombin-catalysed hydrolysis of all substrates was investigated at 37O in 25 mM Tris-HCl, 0.125 M NaCl, pH 7.2. The procedures for following the hydrolysis of substrates l-4 (Table I) have been described in detail elsewhere (7). Of these procedures, "method C" was used in experiments involving substrates 5-8. The various methods consisted of periodically removing aliquots of the digest and subjecting these to NH2-terminal analysis

958

THROXBIN-FIBRIXOGEN

REACTIOS

vol.l2,~0.6

The PTU-amino acids thus obtained in each using 35S -phenyl-isothiocyanate. L aliquot were separated by paper chromatography, the radioactivity was eluted and this was estimated by liquid scintillation. The percentage hydrolysis at each time could thus be calculated by expressing the counts for PTH-Gly as a percentage of those obtained for either PTH-Tyr, in the case of substrates 1-3, or the sum of PTH-Ala and PTH-Asp in the case of substrates 4-8. Velocities at "time zero" were calculated by statistical analysis (14) of these results. As the Be-chain of N-DSK, substrate 9, has a blocked NH2-terminal (lS), it was not possible to measure the appearance of NH*-terminal Gly as a function of this substrate's NH2-terminal. Instead, aliquots of the digestionmixture were added to a known amount of freeze-dried sperm whale apomyoglobin and the NH 2-terminal glycine was measured as a function of the NH*-terminalof this protein, valine (16), according to "method C".

The concentrations in the reaction mixtures of substrates l-4 were determined as described before (7) and those of substrates S-9 were based on their amino acid analysis.

RESULTS AND DISCUSSION The Substrates Chemical data concerning substrates l-4 and substrate 9 are given elsewhere (8,10,15,17). The amino acid compositions of substrates 6-8 are provided in Table II and a two-dimensional peptide map of Ao(l-51) is shown in Fig. 3 FIG. 3 Two-dimensional tryptic peptide map of Ao(l-51). The ninhydrin-positive spots are numbered according to the topography of the corresponding peptides in the intact sequence (Fig. 4). 1:fibrinopeptide A, sequence l-16; 1':fibrinopeptide A where Ser 3 is phosporylated; 2:sequence 17-19; 3:sequence 20-23; 4:sequence24-29; S:sequence 30-44; 6:sequence 45-50; 7:homoserine arising from CNBr-modificationof Met 51. Spots 2 and 6 stained yellow with ninhydrin.

As would be expected, the NH2-terminal analysis of the three substrates listed in Table II showed the presence of Ala with approximately 202 Asp as NH2terminal, The presence of the latter is due to heterogeneity displayed by fibrinopeptide A (8), where the NH2-terminal Ala has been removed to reveal

THROXBIN-FIBRISOGEW

v01.12,~0.6

REACTTOS

959

Asp as the new NR2-terminal. A tryptic in Fig. larly 1-3.

peptide

map of Acz(l-44)

3, verifying

that

a corresponding NH2-terminal

ratio

of Ala + Asp:Gly:Val

that

the preparation

peptide

showed spots

digest

was 1:1:0.7.

the sequence

the spots

together

analysis

of

45-51.Simi-

showed that

with this

of An(l-19)

tol-5

corresponding

of A~z(l-23)

This,

an impurity

slightly

map performed

corresponding

electrophoretic less

Kinetics

to the

the low yield

fragment,

to a level

of

indicates

of

For reasons each of

of

approxi-

l-8

of

the first

four

conclusion cificity

substrates

all

of

are present

of

of AcY(l-51)) fibrinogen

to locate

of

reaction,

for

listed rate

for of re-

9 is a

B. The results

fibrinogen it

0

substrate (7),

the Ac!(l-51)

elements

that this

within

does

of

A was investigated

thespe-

those

which

interaction. the structure

and Au(l-23).

importance

for

the following fragment

which define

is clear

Ao(l-44)

the possible

fibrinopeptide

the initial

in defining

these

of v

this

Since

dimeric

by using

struc-

the isola-

Afl(l-33)2.

The contribution fibrinopeptide

of

the B8-chainof

A was investigated

lated

Act- and B&chain

v.

substrate

by inhibition

of

two sub-fragments,

is a dimer,

of

given at length

that

importance

more precisely

was assumed to corres-

fibrinopeptide

elements

an Rf value

the value

value

been discussed

of major

we isolated

in the region

of

is possible

the structural

a role

itself

ted fragment

it

the fibrinogen-thrombin play

In an attempt

supported

having

(7),

the v.

no

substrates

I is a measure

of release

was drawn: while

not contain

elesewhere

in Table

rate

spot

having

further.

from various

A. Similarly

the initial

dimension

This

and was not examined

which have been given

the substrates

3.

4 in Fig.

Aa(l-33)2

spot was observed

in the second

of fibrinopeptide

of fibrinopeptide

measure

spot

30-33

and S-carboxymethylated

An additional

but having

than that

of release

on reduced

to l-4.

mobility

pond to the sequence

lease

showed only

a tryptic

in the amino acid

contains

of

corresponding

30%.

A tryptic

of

of

the spots

was devoid

map of Pa(l-23)

Val and Glu found

ture

the peptide

analysis

Arg,

mately

showed only

an equimolar

of N-DSK, substrate

5 compared with of

N-DSK to the thrombin-catalysedrelease by using

thrombin

by the knowledge

that

for

by the isolated that

5,

The reduction

substrate BP-chain

fibrinopeptide

solution

4 is

B itself

of

most probably

This proposal

inhibits

of

the iso-

in the value

caused

fragment.

of

is

the actionof

TH-ROMBTN-FIBRISOGES

960

REACTION

Vo1.12,Xo.6

thrombin on fibrinogen (18) and that thrombin, in high concentration, causes release of this peptide from the isolated Bg-chain fragment (5). Thus, it is clear that thrombin binds to structures in the isolated Bg-chain. However, this results in an inefficient release of fibrinopeptide B. The addition of thrombin to a solution of fibrinogen gives rise to a rapid release of fibrinopeptide B following a lag phase during which release of fibrinopeptide A takes place (19). The rapid release of fibrinopeptide B in this case can not be explained by simply assuming that an inaccesible binding site on the BPchain of N-D%

becomes exposed on release of fibrinopeptide A. If this was

true, fibrinopeptide B would also be released from the isolated BB-chain. It is mre

likely that a conformational change occurs in the Bg-chain on release

of fibrinopeptide A and that this architecture is favourable for the thrombin-catalysed release of fibrinopeptide B. Alternatively, the BB-chain as it presents itself in native fibrinogen may be more susceptible to thrombin than the corresponding structure in the isolated chain. In the latter case, efficient release is not possible until the site has been exposed by the release of fibrinopeptide A. Removal of the carboxy-terminal sequence 45-51 from Ao(l-51) (see Fig. 4) to produce Au(l-44), substrate 6, caused a lowering of the v o value from 1.05 to 0.42. It would appear, therefore, that this carboxy-terminal sequence 1 2345610 9 10 11 12 13 14 15 Aio-A~-Ssr-Gly-Glu-Gly-Asp-Phe-Leu-Alo-Glu-Gly-Gly-Gly-~ol-Arg-Cly-Pro-

16

I7 18

t 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 -Arg-Vol-Vo~-Glu-Arg-His-Gln-Ser-Alo-Cyr-Lys-Asp-Ser-Asp-Trp-Pro-Phe-Cy9

I

I

35

4

37 30 39 40 41 42 43 44 45 46 47 40 49 -Ser-Asp-Glu-Asp-Trp-A~n-Tyr-Ly~-Cys-Pro-Ser-Gly-C~~-Arg-Met f FIG.

34

50

36 I

51

I 4

The amino acid sequence of the M-chain of N-DSR, Aa;(l-51). Filled arrow: peptide bond cleaved in the thrombin-catalysed release of fibrinopeptide A; unfilled arrows: carboxy-terminals of substrates 8, 7 and 6, respectively. (From Blomblck et al., see ref. 8.) plays some part in the thrombin-catalysed release of fibrinopeptide ASurthetmore, this function may be the why and wherefore this very sequence has been strongly preserved during marmnalian evolution (20,Zl). Since this segmentcon-

\-ot.

12,X0.6

THROYBIW-FTBRTliC’GEN

tains

two of

that

modification

Thus,

as

was

fibrinogen

the

rate of

by the

0.06

in

region

that of

connection, substrate

2 that

a significant

An attempt

Arg16-Gly17

i.e.

the

were

several

ded,

that

highly

sequence

some

lower

obtained as

at

the

for

a substrate

there

the

8-16)

of

has

residue

thrombin-susceptible

not

in

former

lower

nine

that

sequence

are

lower

than

may exist in

either

the or

segment,

of is

the

situated bond

both

of

the

fact

suggestion

in

and

in

the

the

that

of

the

this

and

thrombin-

were

examined,

these

out

the

was

same the

for

this

order

and

of

Furthermore,

(25)

than

In

a

se-

con-

portion(remammalian

importance since

prior Phe

con-

the

21-23.

during

residues

magni-

fibrinopeptide

carboxy-terminal

secretin

value of

to of

is

the

specificity

by much more

stable

conclu-

voobtainedfor Since

l-11

using

substrates It

to account of

structure

nine

Per se.

the

release

rather

(19,24).

for

do not,

Acz(l-23),extrinsic

that

be

this

carried

that

segments

sequence

An(l-51)

In

been

fibrinogen in

remained

substrate

both

for

this

33.

thrombin-catalysed

A has

enzyme

that

of

fibrinogen.

conceivable

elements

structure

around

value

is

thevacan

necessary

which

since it

for

for

a factorof

is

imPOr-

different

subsites

constants

than

0.08.

A (7).

has

interaction.The

it

considerable

fibrinogen

sequence

specificity

this

fibrinopeptide

prompted

association

nylalanine

the

concentration

function

may reside

evolution rapid

thrombin

than thatforfibrinogenby

is

with

sidues

of

outside

lO’.Thus

12-20,which

These

site amino

yet

linked of

event.

active

all,

being

v o obtained

of

a v.

value

of

on

In

have

of

fibrinopeptide

bond.

to

value

bonds

acid

reduction

course

the

of

the

residues.

a v.

dimeric

this

for to

Furthermore,

release

magnitude

Aa(l-23)

quence

in

from

disulphide

Kmforfibrinogen(23),

of

nection

role

have

significantly the

0

found

be of A.

A.

values

significant

to

must

v

two halves

the

nor

inferred

fibrinogen-thrombin

factor

A.

of

factors

specific

a direct

peptide

12-20,

orders

Ao(l-23)is

stant

based

not

24-33

the

map the

substrates

susceptible

tude

to

are

sequence

been

in

34-44

fibrinopeptide the

was

found

expected

half-cystine

more

Acr(l-33),

be

susceptibility

these

fibrinopeptide

symmetrical

role

(22)

synthetic

was

already

the

of

substrates

of

A?(l-23)

was

would

in

reduced

an even A:

sequence

release

plays

has

caused

of

release

of

961

it

difference from

Aa28-Aa28,

the

fibrinogen

the

the

resulted

version

the

two

neither

it

(7),

fibrinopeptide

that the

A:r(l-51),

influence

Al-chain

disulphide

these

in

carboxymethylation

a dimeric

this

for

v.

concluded

play

of

effecting

of

by

release

from

would

may have

the

and

found

suggested

of

symmetrical

tance

these

about

of

follows

lues

of

shortening

value

halfcystines

previously

brought

Further

It

four

and Aa(l-51)

thrombin

in

the

REACTION

to 9 is

for

a phethe held

to

THROMBIN-FIBRINOGEN

962

be

a

thrombin-binding

is provided tide

in

covering

thrombin

than

the

site finding

(24.26). by

8-23

the

sequence

the

Corresponding

support

Additional

Scheraga

is

~01.12,~~.6

REACTIOX

(27)

a much

sequence

that

better

for

this

PeD,

a synthetic substrate

for

X0-23.

The data presented here is consistant with the proposal that the structural elements of fibrinogen which define the thrombin-catalysed release of fibrinopeptide A are contained within pU(l-51). These elements are confirmed to three regions of this structure, namely, the sequences l-23, 34-44 and 4445, the latter being of least significance. We are not at liberty to decide whether these complete sequences or only parts of them are factors determining the release of fibrinopeptide A. For example, it is evident from the above discussion of An(l-23) that the sequence 8-16 plays a significant role in this process but it is not known if this sequence contains all of the contributing structural elements within Pa(l-23).

It is proposed here that the binding between fibrinogen and thrombin which is fundamental for the subsequent release of fibrinopeptide A, takes place within the sequence l-23. The region operative in this binding may even be confined within the sequence 8-16. This interaction is strengthened either directly or indirectly by structural elements within the sequence 34-44 and to a lesser extent by elements within the sequence 45-51. The strengthening may be due to elements tlithin these two sequences being offered as binding sites for thrombin, or it may be caused indirectly by these elements interacting with structures within the sequence l-23 to hold this in a particular conformation which is preferred by thrombin.

ACKNOWLEDGEMENTS This work was supported by grants from The Swedish Medical Research Council (No. 13X-2475-1OC) and the National Institutes of Health, Bethesda, Md. (No. HL 07379-11). One of us, DHH, wishes to acknowledge the support provided by a Wellcome Trust Travelling Research Fellowship. The excellent technical assistance of Mrss. Elvy Andersson, Helga Messel, Sonja Soderman, Lisbeth Therkildsen, Lena WikstrBm, Wolfgang Finkbeiner, Nils Grondahl and Peter Wolf is acknowledged.

Vo1.12,80.6

THHOMBTN-FIBRINOGEN

REACTTOY

943

REFERENCES 1.

BAILEY, K. and BETTELHEIM, F.R. Nature of fibrinogen-thrombin reaction. Brit. med. Bull. 11, 50, 1955.

2.

BLOMBACK, B. and YAMASHINA, I. On the N-terminal amino acids in fibrinogen and fibrin. Arkiv Kemi 12, 299, 1958.

3.

WALLiN, P. and IWANAGA, S. Differences between plasmic and tryptic digests of human S-sulfo-fibrinogen. Biochim. Biophys. Acta 154, 414, 1968.

4.

PECHET, L. and ALEXANDER, B. The effect of certain proteolytic enzymes on the thrombin-fibrinogen interaction. Biochemistry 1, 875, 1962.

5.

BLOMB&K, B., BLOMBiiCK,M., HESSEL, B. and IWANAGA, S. Structure of Nterminal fragments of fibrinogen and specificity of thrombin. Nature 215, 1445, 1967.

6.

ANDREATTA, R.H., LIEM, R.K. and SCHERAGA, H.A. Mechanism of action of thrombin on fibrinogen. Proc. Nat. Acad. Sci., U.S. 68, 253, 1971.

7.

HOGG, D.H. and BLOMBACK, B. The specificity of the fibrinogen-thrombin reaction. Thrombosis Research 5, 685, 1974.

a. BLOMBLCK, B., HESSEL, B., IWANAGA, S., REUTERBY, J. and BLOMEACK, M. Primary structure of human fibrinogen and fibrin.I.Cleavage of fibrinogen with cyanogen bromide. Isolation and characterization of NH terminal fragments of the o("A")-chain. J. Biol. Chem. 247, 1496,1972 9.

BLOMBACK, B., GRBNDAHL, N.J., HESSEL, B., IWANAGA, S. and WALLEN, I'. Primary structure of human fibrinogen and fibrin. II. Structural studies on NH2-terminal part of y-chain. J. Biol. Chem. 248, 5806,1973.

10.

HESSEL, B., MARINO, M., IWANAGA, S. and BLOMB&CK, B. Primary structure of human fibrinogen and fibrin. In preparation.

11.

LEVY, A.L. and CHUNG, D. Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25, 396, 1953.

12.

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