the formation of fibrin variants

ESPERIMENT.41, AKD MOLECUL:AR PATHOLOGY 3, 287-z% (1944)

The Formation of Fibrin Variants’

M. &IURRAY AND L. A. GRAY, JR.?

Department of Pathology, University of Louisville School of Medicine, Louisville, Kentucky

Received Jztly 25, 1963

INTRODUCTION

In previous studies by other investigators concerning the evolution from fibrinogen of the A and B peptides, certain amorphous fractions were noted during the chromato- graphic separation of these same peptides. Laki et al. (1958) reported two fractions in addition to the A and B peptides which were interpreted as amino acids and small peptides. Blomback and Vestermark (1958) reported the isolation of an epsilon peptide and ninhydrin-reacting material.

If it is indeed true that thrombin is specific in splitting the glycyl-arginyl bond, then it would be difficult to explain the evolution of these small moieties. Where other enzymes have been linked to the catalysis of fibrin, it has been suggested that these act in a similar fashion to thrombin and split the glycyl-arginyl bond. It has been reported (Blomback and Yamashina, 1958; Blomback, 1958) that reptilase splits the A chain in the glycyl-arginyl area, and it has also been suggested that papain acts like thrombin. The interpretation of these data would lend itself to the idea that only one molecular configuration of the fibrin monomer is possible in order to proceed to polymerization. In order to test this hypothesis it is proposed in these experiments to characterize enzymatic activity upon the fibrinogen substrate by resolving the pattern of all of the split products large and small, and if reproducible patterns were determined, it might be possible to better interpret the rules by which abnormal fibrins and other biologic polymers are formed.

MATERIALS AND METHODS

Thrombin was secured from the Parke-Davis Company and diluted with saline to the desired number of units. Fibrinogen was purified according to the method of Blomback and Blomback (1956). Bovine fibrinogen was prepared from fresh whole blood secured from the Fischer Packing Company, Louisville, Kentucky, and human fibrinogen was prepared from lyophilized Fraction I, kindly provided by American National Red Cross. Staphylococcal coagulase was prepared according to the method of Murray and Gohdes (1960). Vasculokinase was prepared according to the method of Murray and Chadwick (1962). Twice crystallized papain was purchased from General Biochemical Company.

Parke-Davis thrombin was reacted with purified fibrinogen, and the experiments of Laki et al. (1958) were repeated. The supernatant solution was separated from

1 Supported by grants (HE-OS079 and HE-076SS) from the United States Public Health Service.

2 Present address: Johns Hopkins School of Medicine, Baltimore, Maryland.

286 M. MURRAY AND L. A. GRAY, JR.

protein with 10% trichloroacetic acid and chromatographed on DEAE cellulose. The A and B peptides as well as the two amorphous fractions reported by Laki were recovered. In order to better resolve the small split products, the enzymes were reacted with the purified fibrinogen for discrete periods of time. The supernatant solutions were separated from the clots by centrifugation and then freed of protein with trichloroacetic acid. The solutions were desalted and lyophilized, and aliquots of this material were placed upon a column of 12% cross-linked divinyl benzine beaded resin (25 ~1 in diameter) which was 130 cm long and 1 cm in diameter. Gra- dient elution with a citric acid-sodium citrate system was done according to the method of Piez and Morris (1960). The eluates were reacted with ninhydrin in a Technicon system, and recorded.

This system is capable of resolving amino acids, derivatives, di- and tri-peptides, and an occasional tetra-peptide. When it was decided to separate these peptides, the eluant stream from the column was divided and the eluates were collected in a fraction collector. The resolved peptides were then concentrated in a Rinco evaporator and subsequently hydrolyzed in constant boiling hydrochloric acid. The amino acids were desalted and re-chromatographed on the same resin in order to identify them.

EXPERIMENTAL

The action of thrombin on fibrinogen

In a series of experiments 10 units of thrombin was reacted with 200 mg of purified fibrinogen. The reaction was carried out at 37°C with various lots of fibrinogen, both bovine and human, The variable in these experiments was the time of reaction. The times utilized varied between 2 minutes and 18 hours. The protein-free supernatants were chromatographed as described previously, and the results are shown in Tables I and II. The glycine contaminant introduced during the preparation of fibrinogen was always recovered, In addition aspartic acid was consistently recovered. Serine and threonine appeared to be recovered as well. In addition to these amino acids, varying numbers of unidentified peaks were seen, and these were labeled “PI to P,.” If the mobility of these peaks are taken into consideration some consistency in their evolution is noted. Because of their configuration and their loci, it is presumed that these represent small peptides. It would then appear that the action of Parke-Davis thrombin was such that not only are the A and B peptides evolved, but the enzyme is capable of splitting other bonds than the glycyl-arginyl bond. If the resolved amino acids are compared with those derived from the A and B peptides (Table III) (Sjo- quist et al., 1960), then it would appear that serine and threonine would have to be derived from the A peptide or the fibrin monomer. If Parke-Davis thrombin is not contaminated with another proteolytic enzyme it would appear that the A peptide or the fibrin monomer might serve as its own competitive inhibitor or anti-thrombin by competing for enzyme. These experiments then serve as controls for the study of other fibrin-forming enzymes.

The action of staphylococcal coagulase on purified fibrinogen

Five mg of purified staphylococcal coagulase was reacted with 200 mg of purified fibrinogen. The total evolved products were collected as previously described, and the resulting amino acids and peptides were resolved in the amino acid analyzer. The data are shown in Table IV. Fifteen amino acids were recovered. Of these,

TABLE I THE EVOLUTION OF AMINO ACIDS AND SMALL PEPTIDES BY THE ACTION OF THROMBIN ON FIBRINoWN

Reaction: 10 units thrombin (Parke-Davis) with 200 mg fibrinogen.

TAU ASP THR SER GLU PRO GLY ALA CYS VrzL

MET ISOLEU LEU TYR PHE NH, LYS

PI P.,

P, P;

p, P, pi p,

0.08 O.D.

1.50

0.03

2.40

1.50

2.20

2 min. 5 min. 5 min. 10 min. 10 min. Bovine Bovine Bovine Bovine Bovine

0.04 0.D

2.00

0.04

0.04 0.06 0.35 0.30 0.30 0.23

0.03 O.D. 0.03 O.D. 0.02 O.D. 0.02 O.D.

0.07 0.01

0.08 0.01

0.02 2.00

0.01

0.02 0.06 0.22 0.20

0.20 0.16 0.1s

2.00 2.00 0.02

2.30 0.06 0.14 0.03 0.16 0.04

0.05 0.20 0.30 0.23' 0.0s

TABLE II THE EVOLUTION OF AMINO ACIDS AND SMALL PEPTIDES BY THE ACTION OF THROMBIN ON FIBRINOGEN

Reaction: 10 units thrombin (Parke-Davis) with 200 mg fibrinogen.

120 min. Bovine 120 min. Bovine 18 hours Human

0.02 O.D. 0.04 O.D. 0.07 O.D. 0.09 0.07 0.07

2.00 2.00

0.05 0.05 0.04 2.00 0.02 0.04 0.06 0.25 0.20 0.23 0.20

289

TAU ASP THR SER GLU PRO GLY ALA

CYS VAL MET ISOLEU LEU TYR PHE NH, LYS ARG PI P, p:< p,


cystine, isoleucine, and histidine do not occur in either the A or B peptides (Table III). This would suggest that staphylococcal coagulase either attacks the A and B chains of the fibrin monomer at a greater distance than the critical glycyl-arginyl bond and/or the C chain (Clegg and Bailey, 1962). Since our N-terminal studies indicated 3 N-terminals for coagulase fibrins (Murray, 1962), it is most probable that the C chain is also attacked. It was our interpretation that if the C chain were

TABLE III THE AMINO ACID COMPOSITION OF THE A AND B PEPTIDES

Amino acid A B Amino acid A B

TAURINE ASP THR SER GLU PRO GLY ALA CYS VAL

3 4 1 1

2

2 2 2 2 5 2

1

1 1

METHIO ISOLEU LEU 1 1

TYR so, 1

PHE 1 1

NH3 LYS 1

HIST ARG 1 2 GLUTAMINE 1

TABLE IV THE EVOLUTION OF AMINO ACIDS AND SMALL PEPTIDES BY THE ACTION OF STAPHYLOCOCCAL

COAGULASE ON FIBRINOGEN

Reaction: 5 mg staphylococcal coagulase (partially purified) with 200 mg fibrinogen.

Amino acid

TAUR ASP THR SER GLU PRO GLY ALA

CYS VAL METH ISOLEU LEU

24-hour Bovine

0.03 O.D. 0.02 0.01 0.01 2.00 0.21 0.01

0.05

0.03 0.03

Amino acid 24-hour Bovine

TYR 0.04 O.D. PHE 0.04

NH3 2 .oo LYS 0.30 HIST 0.15 ARG 0.05

Pl 0.08

P, 0.03

p3 0.05

p4 0.04

P5 0.02

PO 0.10

p7 0.02

attacked the possibility of branching fibrils would occur. We have demonstrated this by electron microscopy (Murray, unpublished data). It is clear from this experi- ment that the mode of action of staphylococcal coagulase is substantially different from that of thrombin (Murray and Gohdes, 1959), and that it is most probable that this enzyme attacks available peptide bonds closest to their ends. The occurrence of the small peptides is probably the indication of the random action of the coagulase enzyme.

The action of papain on jibrinogen

One and one-half mg of crystallized papain was reacted with 200 mg purified fibrinogen. Various incubation times were used as in the other experiments, and the

THE FORMATION OF FIBRIN VARIANTS 291

method of the preparation of the protein-free supernatant and the resolution of the evolved products was the same. Our data are shown in TabIe V. In these experiments, it can be seen that the number of split products increases with time of incubation, and that increasing numbers of amino acids and peptides are seen. It would appear

2 min. Bovine 5 min. Bovine 15 min. Bovine 45 min. Bovine

0.07 O.D. 0.35 O.D. 0.01 O.D. 0.03

0.02

0.02 O.D. 0.02

2.00 2.00 2 .oo 2.00

0.02 0.02 0.02

0.08 0.10 0.02 0.02 0.03 0.02 0.01 0.02

0.01 0.02 0.02 0.02

0.01 0.03 0.03 0.03 0.01 0.02 0.02 0.01

0.01 0.01 0.01 0.01

0.50

0.03 0.05 0.05 0.10 0.01

0.02 0.01 0.02 0.04

0.05 0.04 0.10

0.02 0.06 0.02 0.07

0.02 0.03 0.04 0.08 0.01 0.03 0.02 0.05

1 .oo 0.03 0.05 0.07 0.06 0.01

0.01 0.04

0.05 0.04

0.05 0.04

TABLE V

THE EVOLUTION OF AMINO ACIDS AND SMALL PEPTIDES BY THE ACTION OF PAPAIN ON

PURIFIED FIBRINOCEN

Reaction: 1.5 mg Papain with 200 mg fibrinogen.

TAU ASP TIiR SER GLU PRO GLY

ALA CYS VAL MET ISOLEU LEU TYR PHE

NH,

LYS HIST ARG PI P, p:< p4 p.5 PC p;

Ps p9 P 10 P 1,

that the action of papain is random and when critical bonds are split polymerization takes place, but simultaneously solubilization of both fibrinogen and fibrin continues.

The action of vasculokinase on fibrinogen

Various lots of vasculokinase prepared from bovine aortas were also studied as shown in Tables VI and VII. The method of preparation of the split products was the same. Consistent evolution of 11 amino acids was seen. With increasing incubation it appeared that greater amounts of small peptides were found (Figs. 1 and 2). The consistent finding of isoleucine, histidine, and occasional determinations of cystine would indicate that vasculokinase acts similarly to staphylococcal coagulase in its

292 M. MURRAY AND L. A, GRAY, JR.

action on fibrinogen. One might deduce that the enzyme attacks either the C chain or deeper in the A and B chains. This is consistent with the N-terminal studies where it was found that phenylalanine and glycine were N-terminal amino acids (Murray and Gray, 1962); it is also consistent with the lack of esterase activity of vasculokinase, which it shares in common with staphylococcal coagulase. It is further support that vasculokinase is an entity all its own and is not an amorphous protein contaminated with thrombin; nor is it thromboplastin which might attack contaminated prothrombin within a given fibrinogen preparation.

TABLE VI THE EVOLUTION OF AMINO ACIDS AND SMALL PEPTIDES BY THE ACTION OF VASCULOKINASE

ON FIBRINOGEN

Reaction: 6 mg vasculokinase with 200 mg fibrinogen.

Amino 15 min. 30 min. 30 min. 60 min. 120 min.

acid Bovine Bovine Human Human Bovine

TAUR

ASP

THR

SER

GLU

PRO

GLY

ALA

CYS

VAL

METHIO

ISOLEU

LEU

TYR

PHE

NH,

LYS

HIS

ARG

PI P, P 2 P,

0.01 O.D.

0.01

0.01

2 .oo

0.03

0.05

0.01

0.01

0.90

0.10

0.05

1 .oo 2 .oo

0.05 0.06

0.60 0.15

0.55 0.10

0.03 O.D.

2 .oo

0.02

0.05

0.02

0.40

0.05

0.04

0.05 O.D. 0.10 O.D. 0.02 0.02

0.03 0.03

0.10

0.05 0.03

0.02 0.03

0.04 0.06

0.03 0.03

0.03 0.04

0.30

0.13 0.07

0.10 0.05

0.01

2 .oo 0.30

0.15 0.04

0.13 0.06

0.12 0.03

Resolution of some peptides isolated during the activation of fibrinogen by vasculokinase

In order to determine whether the unidentified peaks were peptides, the column effluent stream was split and 65% of the effluent was collected in a fraction collector. Peaks 1 and 3 from a resolved supernatant of a reaction of vasculokinase and fibrinogen incubated for 150 minutes were collected. The specimens were processed as in Materials and Methods. The hydrolyzed peaks were re-chromatographed in the amino acid analyzer. The data are shown in Table VIII. Peak 1 contained aspartic acid and glutamic acid. Peak 3 contained threonine, serine, and glutamic acid. The sequence containing aspartic and glutamic acids seen in Peak 1 could be found in peptides A or B. However, a fragment containing threonine, serine, and glutamic acid is not


found in either A or B. This is further proof that vasculokinase attacks some other linkage than the glycyl-arginyl bond, or portions of the A and B peptide only, and indicates attack on other portions of the A and B chains or C chains.

120 min.

Human

0.13 O.D.

0.01

0.02

0.01

0.06

0.05

0.07

2 .oo

0.05

0.11

0.06

TABLE VII THE EVOLUTION OF AMINO ACIDS AND SMELL PEPTIDES BY THE ACTION OF VASCULOKINASE

ON FIBRIXOWX

Reaction: 6 mg vasculokinase with 200 mg fibrinogen.

TAUR ASP THR SER GLU PRO GLY AL.4 CYS \‘AL METHIO

ISOLEU LEU TYR PHE

NH3 LYS HIS ,4RG Pl

P,

P:,

p4

p,

pn

p7

ps

p9 P 10

P:; P P 13 P p::

P 16 P Ii

P 1x

150 min.

Human

0.05 O.D. 0.07 0.08

0.02

0.02

0.01

0.02

0.01

0.01

0.03

0.01 0.02

2 .oo

0.03

0.15

72 hour

Human

0.74 O.D. 0.36

0.36

0.60

0.05 2 .oo

0.15 1.10

0.60

0.40

0.99

1 .oo

2 .oo 1 .oo 0.20

0.07

0.11

0.08

0.05

0.40

0.25

0.08

0.05

0.03

0.04

0.20 2 .oo 0.10

0.05 0.09

0.04

0.24

0.15

DISCUSSION

The data as presented by other investigators would tend to indicate that the action of an enzyme upon the fibrinogen substrate which yields fibrin is a restricted reaction. It is suggested that only the glycyl-arginyl bond may be broken, and that the type of fibrin formed would depend upon whether both the A and B chains are split


or whether the A chain only is split. Nevertheless, in reviewing the data from these authors (Laki et al., 1958; Blomback and Vestermark, 1958), it can be shown that in their experiments, small split products are also evolved during the formation of the A and B peptides. These data as presented by us suggest a number of new interpretations concerning fibrin formation. Assuming that Parke-Davis thrombin is

MORLEU IN

FIG. 1. Chromatogram of split products evolved from the activation of fibrin by vasculokinase after 150 minutes of incubation at 37°C. Vasculokinase, 6 mg; fibrinogen, 200mg; CaC& (0.2 M), 0.1 ml.

not contaminated with other proteolytic enzymes, the A and B peptides may act as the competitive inhibitor in the formation of fibrin by also serving as substrate for thrombin. This reaction might be considered as the anti-thrombin reaction previously described as absorption of thrombin by fibrin (Klein and Seegers, 1950).

The multiplicity of split products seen as a result of the action of papain on fibrinogen suggests that any powerful proteolytic enzyme could conceivably form strands of fibrin during the random lysis of a population of fibrinogen molecules.

It is most interesting to note that both staphylococcal coagulase and vasculokinase yield a large complement of amino acids, some of which do not occur in the A and B peptides. It is therefore suggested that these enzymes chew away at available chains until a critical molecular configuration is reached, and then polymerization takes place.


FIG. 2. Chromatogram of the split products of fibrinogen during fibrin formation by the activation of vasculokinase after 72 hours of incubation at 37°C. Vasculokinase, 6 mg; fibrinogen, 200 mg; CaCI, (0.2 M), 0.1 ml.

TABLE VIII THE COMPOSITION OF Two SMALL PEPTIDES ISOLATED FROM THE .~CTIVATION OF FIBRINOGEN

BY VASCULOIXNASE

Incubation time: 150 minutes.

Amino acid

TAUR ASP THR SER GLU PRO GLY .4LA. CYS V.4L METHIO ISOLEU LEU TYR PHE

NH3

Peak I

0.05 O.D.

I .oo

Peak III

0.09 O.D. 0.05 0.05


This is further suggested by the replicable N-terminal amino acids which are found during the study of this reaction. The ability of staphylococcal coagulase to activate fibrinogen by itself establishes its integrity as a proteolytic enzyme. Similarly vasculokinase is established as a proteolytic enzyme.

Since fibrin is found in a multiplicity of pathologic reactions in GVO, and since there is no adequate explanation for the formation of these fibrins through the action of thrombin, these model systems might serve as prototypes for the formation of fibrin variants or other glycoprotein polymers.

SUMMARY

Purified fibrinogen was reacted at optimal conditions with various purified enzymes, including thrombin, papain, staphylococcal coagulase, and vasculokinase. These reactions were allowed to proceed for various lengths of time. The protein-free supernatants were resolved in an amino acid analyzer on 12% cross-linked divinyl benzine. The amino acids and small peptides were resolved. It was shown that Parke-Davis thrombin could form aspartic acid, and occasionally serine and threonine, in addition to small peptides. These ostensibly could be derived from the A and B peptides. Papain evolved a multiplicity of amino acids and peptides in increasing numbers with the time of incubation. Staphylococcal coagulase and vasculokinase produced a relatively discrete number of amino acids (15 and 11, respectively), as well as numbers of small peptides. Some of these amino acids could not be found in the A and B peptides, indicating that the fibrinogen was split differently than by the action of thrombin. Resolution of some of the vasculokinase-produced peptides indicated that they were also derived from loci other than the A and B peptides.

REFERENCES

BLOMBACK, B. (1958). Studies in the action of thrombic enzymes on bovine fibrinogen as measured by N-terminal analysis. Arkiv Kemi 12, 321-335.

BLOMBACK, B., and BLOMRACK, M. (1956). Purification of human and bovine fibrinogen. Avkiv Kemi 10, 415-443.

BLOMBACK, B., and VESTERMARK, A. (1958). Isolation of fibrino-peptides by chromatography. Arkiv Kemi 12, 173-183.

BLOMBACK, B., and YAMASIIINA, L. (1958). On the N-terminal amino acids in fibrinogen and fibrin. Arkiv Kemi 12, 299-319.

CLEGG, J. B., and BAILEY, K. (1962). The separation and isolation of the peptide chains of fibrin. Biochim. Biophys. Acta 66, 525-527.

KLEIN, P. D., and SEEGERS, W. H. (1950). The nature of plasma antithrombin activity. Blood 5, 742-‘152.

LAKI, R., GLADNER, J. A., FOLK, J. E., and KOMINZ, D. R. (1958). The mode of action of thrombin. Thromb. Diath. Haemorrhag. 2, 205-217.

MURRAY, M. (1962). Terminal amino acid analysis of various types of fibrin. Federation Proc. 21, 6.

MURRAY, M., and CHADWICK, M. (1962). Methods of purification of bovine vasculokinase. Biochim. Biophys. Acta 56, 338-342.

MURRAY, M., and GOHDES, P. (1959). The role of coagulase reacting factor in the activation of fibrinogen. /. Bacterial. 76, 450-451.

MURRAY, M., and GOHDES, P. (1960). Purification of staphylococcal coagulase. Biochim. Biophys. Acta 46, 518-522.

MURRAY, M., and GRAY, L. (1962). Terminal amino acid analysis of vasculokinase activated fibrin. Nature 194, 681.

Pmz, K. A., and MORRIS, L. (1960). A modified procedure for the automatic analysis of amino acids. Anal. Biochem. 1, 187-201.

SJOQUIST, J., BLoMnACK, B., and WALLEN, P. (1960). Amino acid sequence of bovine fibrino- peptides. Arkiv Kemi 16, 425-436.

the formation of fibrin variants

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