Fibrinogen Kawaguchi : An abnormal fibrinogen characterized by defective release of fibrinopeptide A

THROMBOSIS RESEARCH 37; 379-390, 1985 0049-3848/85 $3.00 + .OO Printed in the LISA. Copyright (cl 1985 Pergamon Press Ltd. All rights reserved.

FIBRINOGEN KAWAGUCHI : AN ABNORMAL FIBRINOGEN CHARACTERIZED BY DEFECTIVE RELEASE OF FIBRINOPEPTIDE

A

Michio Matsuda, Eiji Saeki*, Ayatsugu Kasamatsu**, Chizuko Nakamikawa, Shun-ichiro Manabe and Yuji Samejimat Institute Of Hematology, Jichi Medical School, Tochigi-Ken 329-04, Japan, *Department of Internal Medicine, Tokyo Women's Medical College, Tokyo 162, Japan, **Central Clinical Laboratory, The Saiseikai Kawaguchi General Hospital, Kawaguchi 332, Japan, and t Laboratory of Hygienic Chemistry, Faculty of Pharmacy, Hoshi University, Tokyo 142, Japan. (Received 21.9.1984; Accepted in revised form 10.11.1984 by Editor S. Iwanaga)

ABSTRACT A congenital dysfibrinogenemia was found in a 32year-old asymptomatic female and her immediate family. The propositus, apparently a heterozygote for the abnormality, characteristically showed defective release of fibrinopeptide A from half of her fibrinogen molecules. No fibrinopeptide A was cleaved off from the isolated abnormal molecule by thrombin or snake venoms (Reptilase and Ancrod) as evidenced by radioimmunoassay, high performance liquid chromatography and determination of the NHZ-terminal amino acids. The abnormal fibrinogen formed a solid gel solely by the release of fibrinopeptide B upon incubation with thrombin. We provisionally designate this abnormal fibrinogen as "Fibrinogen Kawaguchi", although possible identity with other abnormal fibrinogens is not excluded.

INTRODUCTION Among congenital dysfibrinogenemias associated with impaired release of fibrinopeptide A upon challenge with

Key words:

dysfibrinogenemia, abnormal fibrinogen, fibrinopeptide A, polymerization sites 379

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thrombin or snake venoms, those with defective release of the peptide are still limited in number (1-3). Here we report a congenital dysfibrinogenemia characterized by the absence of the release of fibrinopeptide A from the abnormal molecule as evidenced by radioimmunoassay (RIA), high performance liquid chromatography (HPLC) and NH2 -terminal amino acid determination.

MATERIALS AND METHODS The collection of blood samples and the purification of flbrlnogen were both carried out as described elsewhere (4). The isolated fibrinogen was found to be satisfactorily purified when examined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (5). Hemostasis and coagulation tests. Routine tests were performed essentially according to standard methods (6,7) or as described elsewhere (8), unless specifically stated, and the results obtained with the patient's plasma were compared to those obtained with pooled normal plasma. Fibrinogen concentrations in plasma were determined by the thrombin time method of Clauss (9), single radial immunodiffusion (SRID) and the turbidimetric method, as previously described (4). The thrombin and Reptilase times were performed exactly as described previously, with or without the addition of calcium ions (4). Studies on purified fibrinogen and its derivatives. The thrombin and Reptilase times were measured by the same method as used for the plasma samples. Timed release-of fibrinopeptide A was measured by RIA (10) on samples prepared essentially according to Gralnick et al. (11) with modifications of using a lower concentration of fibrinogen (0.2 mg/ml) and purified bovine thrombin (12) instead of the human one. Differential identification of fibrinopeptides A and B was carried out by HPLC as described by Kehl et al. (13). The NH2-terminal amino acids were determined by the method of Edman and Henschen (14). Aggregation of fibrin monomer was studied by the method of Gralnick et al. (ll), and cross-linking of fibrin mediated by activated blood coagulation factor XIII (XIIIa) was examined by SDS-PAGE using 5% gels. Binding study using thrombin-activated fibrinogen coupled to Sepharose 4B. Thirty mg of normal and propositus' fibrinogens were individually conjugated to 3 ml of Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), treated with 90 units of thrombin for 2hratroomtemperature and foranadditional18 hr at 4*C. The thrombin-treated insolubilized fibrinogen was then packed into a small column and successively washed with 100 volumes each of following solutions: 1) 0.2 M acetic acid; 2) 0.05 M Tris-HsPOq, pH 7.6, containing 1.0 M NaCl, 0.005 M EDTA, 5 KIU/ml aprotinin and 6 M urea (buffer-B); and 3) 0.05 M TrisH3P04, pH 7.6, containing 0.1 M NaCl, 0.005 M EDTA and 5 KIU/ml aprotinin (buffer-A). A 0.5 ml sample of normal or propositus' fibrinogen was applied to the fibrinmonomer-Sepharose column

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The adsorbed proteins were eluted with buffer-A. employing pH (7.6 to 4.1), NaCl (0.1 to 1.0 M) and A and B, and l-ml urea (0 to 6 M), using 20 ml each of buffers fractions were collected.

equilibrated by gradients

RESULTS Case history

and family

study.

A 32-year-old female was examined for hemostasis and coagulation studies. She had experienced neither excessive bleeding The examination revealed a markedly nor thrombotic tendency. prolonged thrombin time, profound hypofibrinogenemia as determined by the thrombin time method of Clauss, but normal fibrinogen levels by other methods, and elevated serum fibrinogen/fibrin degradation products (FDP). Similar results were obtained in some of her relatives, although they are all asymptomatic. No consanguinity was known in the family (FIG. 1). These findings prompted us to further investigate the propositus' plasma, especially with respect to the functional aspects of fibrinogen molecules.

40 252

0

Male

0

40 245

Female

?? @

Tested

a

Deceased-not

@

?? @

- normal

Routine hemostasis

FIG. 1

Tested -affected

/

0

tested

0

Living-not

Prwositw

Pedigree of Fibrinogen Kawaguchi.

tested

and coagulation

studies.

The bleeding time, whole blood clotting time, platelet counts and platelet aggregation were normal, but the one-stage prothrombin time, 19.1 set (control, 11.4 set), was moderately prolonged. The activated partial thromboplastin time, 29.9 set (control, 24.8 set), was also slightly prolonged. Blood coagulation factors including factor XIII were all found to be within normal ranges. Levels of plasminogen, antithrombin III and aqplasmin inhibitor were normal in quantity as well as in activity (data not shown). The FDP in the serum was, however,

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TABLE 1 Thrombin and Reptilase Times and Fibrinogen Concentrations Determined on Propositus' Plasma

Studies

Propositus

Thrombin time, set Without Ca ions With Ca ions Reptilase time, set Without Ca ions With Ca ions Fibrinogen, mg/dl Thrombin time method Turbidimetric method SRID

Normal or Control

55.1 20.6

13.0 7.6

114.2 62.6

23.6 22.1

(40 230 262

200-400 200-400 200-400

markedly elevated at more than 640 Dg/ml. The thrombin and Reptilase times were markedly prolonged, but could be partially corrected by the addition of CaC12, as has been shown in most of the hitherto described dysfibrinogenemic plasmas. These results are summarized in TABLE 1. Studies on purified fibrinogen. Molecular weights of purified Gross molecular StrUCtUre. fibrinogen and its subunit polypeptides and cross-linking profile mediated by XIIIa were all identical with those of normal fibrinogen when examined by SDS-PAGE (profiles not shown). The propositus' fibrinogen was also indistinguishable from the normal one by immunoelectrophoresis (pattern not shown). Thrombin and Reptilase times. As shown in TABLE 2, the thrombin and Reptilase times were markedly prolonged. Upon addition of CaC12 (final 0.01 M), however, the clotting times were corrected substantially, but still only partially. Timed release of fibrinopeptide A. As shown in FIG. 2, only half the normal amount of fibrinopeptide A, i. e., 1 mole per mole of fibrinogen, was released by thrombin from the The rate of release was, however, propositus' fibrinogen. apparently normal. The decreased release was also noted when challenged with Reptilase (picture not shown). In the latter experiments, only half the protein formed clots on the basis of the absorbance measured at 280 nm of the fibrinogen solution and that of the clot liquor. Analyses

of fibrinopeptides

by HPLC.

FIG. 3 illustrates

elution

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383

TABLE 2 Thrombin

and Reptilase

Times of Purified Fibrinogen Propositus

Studies Thrombin time, set Without Ca ions With Ca ions Reptilase time, set Without Ca ions With Ca ions *

Normal

262" 21.8"

10.9 7.4

>300 63.7*

20.3 12.6

Clots were formed but they were just fragile.

profiles of thrombin-cleaved fibrinopeptides examined by HPLC. Although retention times were essentially identical between corresponding components derived from the propositus' fibrinogen and the normal one, amounts of fibrinopeptides A, i. e., AP, A and AY, derived from the propositus' fibrinogen were nearly half the amounts of those derived from normal fibrinogen. The amounts of fibrinopeptides B were nearly identical between this fibrinogen and the normal one. For the patient's fibrinogen, the ratio of fibrinopeptides A versus B was nearly half of that in the normal one. Since only half the propositus' fibrinogen was found to be

'0

5 10

20

30 Time

60 ,r120 (min)

FIG. 2 Timed release of fibrinopeptide

A determined

by RIA.

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clottable with snake venoms (Reptilase and Ancrod), we could easily separate the abnormal molecules by removing fibrin clots formed with purified Ancrod: 50 mg of propositus' fibrinogen in 50mlof 0.3 M NaClwas clotted with 50 units of purified Ancrod (a kind gift from Dr. M. Fukuda, Mochida Pharmaceutical Co., Tokyo, Japan), and the clot liquor was harvested by removing fibrin clots after 20 hr incubation (2 hr at 37'C and an additional 18 hr at 4'C). The protein was recovered from the clot liquor by precipitation with ethanol at 8%, dissolved in a small volume of 0.3 M NaCl and dialyzed against sufficient volumes of the same solution. The subunit polypeptides of fibrinogen thus prepared were identical with those of the normal one as judged by SDS-PAGE (pattern not shown). The abnormal fibrinogen molecules thus prepared could very slowly form a solid but apparently transparent gel when incubated with thrombin. The clot liquor was subjected to HPLC and the peptides were analyzed. As shown in FIG. 4, no fibrinopeptides A were identified, while nearly a normal amount of

‘ropositus

Fibrinogen Des

(abnormal

ArgE3

Kawaguchi molecules

1 8’

Des Arg

S

1

lormal A

ontrol

Dar Arg B

AP ,

I

10

I

1

I

15

20

25

Elution

Time

1 31

Iminl

FIG. 3 Analysis by HPLC of fibrinopeptides released by thrombin.

c

L 5

10

15 Elution

20 Time

25

3(

(min)

FIG. 4 Analysis by HPLC of fibrinopeptides derived from thrombintreated abnormal fraction of fibrinogen Kawaguchi. For details, see text.

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fibrinopeptides B altogether was noted, though they were all either des Arg B or probably its further degraded fraction denoted as B*. Thus the abnormal molecule formed a solid gel by liberating solely fibrinopeptide B and liberated no fibrinopeptide A at all. Markedly altered aggregation was Aggregation of fibrin monomer. observed in the propositus' fibrin monomer when compared with the normal one. As has been demonstrated in other abnormal fibrinogens, the alteration depends on the ionic strength, and virtually no aggregation occurred when the ionic strength was raised to 0.12. When the propositus' fibrin monomer was added to normal one, substantial inhibition was observed (pictures not shown). Bindinq study using insolubilized fibrin monomer derived from The column packed with propositus' or normal fibrinogen. insolubilized and thrombin-treated fibrinogen derived from the propositus was found to maximally adsorb about 5 mg of normal fibrinogen. This amount was half as much as the amount of fibrinogen adsorbed to a normal fibrin monomer-Sepharose column. A representative experiment, in which 10 mg of normal

x

2

8 0 .5

4

0

P 0

10

20

30

E 65 0

Fraction

FIG.

10

20

30

40

Number

5

Elution profile of normal fibrinogen from insolubilized normal or the propositus' fibrinogen activated with thrombin (fibrin monomer). When 10 my of normal fibrinogen was applied to the column packed with the propositus' fibrin monomer-Sepharose, nearly a half amount of the applied fibrinogen was not adsorbed as denoted by P-l. The residual half of the fibrinogen was adsorbed and eluted in nearly identical fractions (P-2) as most of the fibrinogen adsorbed to and eluted from the normal fibrin monomer-Sepharose (N).

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fibrinogen was applied, is shown in FIG 5. These results indicate that only a half population of the insolubilized propositus' fibrinogen could expose the polymerization site "A" which is complementarytothe site assigned on the D domain of a fibrinogen molecule. The results may also provide supporting evidence that the release of fibrinopeptide B, per se, has no relevance to the exposure of the "A" site. Determination of the NHz-terminal amino acid sequence of propositus' fibrin and fibrinogen. The thrombin-induced.fibrin and the Ancrod-nonclottable fraction (abnormal molecules in the prOpOSitUS' fibrinogen) were analyzed for the NHZ-terminal amino acid sequence for eight cycles. Although three amino acids or more were expected for the fibrin and two for the abnormal fibrinogen fraction at each cycle, the assignment of the amino acids to each polypeptide chain was not necessarily difficult because the entire amino acid sequence of human fibrinogen has been elucidated (15). The assigned sequence for the NH2terminal eight cycles of the a-chain of thrombin-induced fibrin was entirely identical with either that of the Aw.-chain of normal fibrinogen; Aa 1 through 8, or that of the cc-chain of fibrin; Aci 17 through 24. Completely identical sequences were obtained for both the S- and y-chains with the sequences of those of normal, i. e., BS 15 through 22 and y 1 through 8, respectively. The result indicates that only a part of the propositus' fibrinogen liberated fibrinopeptide A upon treatment with thrombin, whereas all the molecules released fibrinopeptide 8. When the abnormal fibrinogen fraction, i. e., the Ancrodnonclottable fraction, was analyzed for the NH2-terminal eight cycles, the asigned sequences were entirely identical with those Nodatawere of the two chains of the normal molecule. available for the BP-chain because its NH2-terminal amino acid has been shown to be pyroglutamic acid (15). DISCUSSION Congenital dysfibrinogenemias characterized by totally defective release of fibrinopeptide A from the abnormal molecule have been described. Although an increasing number of dysfibrinogenemias associated with impaired release of fibrinopeptide A has been reported, those with totally defective release of fibrinopeptide A from the abnormal molecule are still limited in number (l-3). Among them are fibrinogens Metz (1) and Zurich I (2), in which an amino acid substitution of the Aa16 arginine (Arg) by cysteine or cystine (Cys) has been recently identified (3). The abnormal fibrinogen reported here as fibrinogen Kawaguchi is apparently a heterozygous form of abnormality consisting of one half normal and one half abnormal molecules. containing one normal Thus, the presence of a hybrid molecule and one abnormal Acr-chains in each half of the molecule seems to The abnormal molecule was found to liberate no be unlikely. fibrinopeptide A upon treatment with thrombin or snake venoms (Reptilase and Ancrod), as verified by RIA, HPLC and determination of the NH2 -terminal amino acids of thrombin-clotted fibrin.

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Hence, separation of the abnormal molecules from the normal molecules was relatively simple. The NH2 -terminal sequence study indicates that the Acr-chain of the abnormal fibrinogen is indistinguishable from that of the normal molecule at least up to the 8th amino acid, phenylalanine. The result also suggests that the mutation resides most probably in the COOH-terminal half of fibrinopeptide A or its conjunct segment of the Act-chain of the abnormal molecule. When treated with thrombin, the abnormal fibrinogen formed a solid but apparently transparent gel by releasing solely fibrinopeptide B as confirmed by HPLC (FIG. 4). Since the release of fibrinopeptide A was found to be totally defective in the abnormal molecule, the polymerization site "A" (16,17) would not have been exposed and thus would not have participated in Elevated FDP at more than 640 the assembly of fibrin monomers. pg/ml in the propositus' serum would thus be accounted for by the presence of intact abnormal fibrinogen or non-polymerized fibrin monomer devoid of solely fibrinopeptide B, or both, in This assumption could be verified by the the blood sample. finding that the amount of fibrinogen adsorbed to the insolubilized and thrombin-activated fibrinogen derived from the propositus was nearly half normal as depicted in FIG. 5. Thus the assembly of fibrin monomers devoid of fibrinopeptide B alone may have been modulated by another polymerization sites, most probably by the "B" site which is suggested to be exposed upon the removal of fibrinopeptide B (17). As has been shown in fibrinogen Metz (3), the release of fibrinopeptide A, per se, was also found to be dispensable for the initiation of fibrin monomer aggregation in the abnormal fraction of fibrinogen Kawaguchi. We reported recently that the polymerization site "bb", which is exposed on the linearly aligned DD domain of two adjacent fibrin molecules, functions abnormally in fibrinogen Tokyo II (4). This "bb" polymerization site has been assigned to be complementary to the "B" site (17). Since the impaired "bb" site gives rise to an altered aggregation of fibrin monomers in fibrinogen Tokyo II, the "B"-"bb" set of Olexa and Budzynski (17) seems to function independently in an essential part of the aggregation mechanisms of fibrin monomers. Fibrin clots formed with thrombin from the abnormal fraction of fibrinogen Kawaguchi may therefore provide clues for the elucidation of the "B"-"bb" site-modulated mechanism of fibrin monomer assembly. By utilizing HPLC, an increasing number of amino acid substitutions have recently been identified (3,18). Among them are Au7-aspartic acid (Asp) by asparagine (Asn) for fibrinogen Lille (19), Ac12-glycine (Gly) by valine (Val) for fibrinogen Rouen (3,18), and Act16 Arg by histidine (His) for a relatively large number of abnormal fibrinogens including Petoskey (20) and Manchester (21). From these fibrinogens, fibrinopeptide A has been slowly but eventually completely released when challenged with thrombin, and abnormal fibrinopeptides could be separated by HPLC. From fibrinogen "Metz", however, no fibrinopeptide A has been liberated, and the substitution was found to be CySteine or cystine (Cys) against Arg in position 16 of the Acrchain (3). The same substitution has been identified in a half population of fibrinogens Zurich I(3) and Schwarzach (22), from

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which apparently half of the normal amount of fibrinopeptide A is released. They are thus apparently heterozygous forms of the abnormality. Although we have not completed the sequence study of the NHp-terminal segment of the abnormal Acr-chain including fibrinopeptide A, our preliminary data indicate that the NHZ-terminal 1-8 segment of the abnormal Acr-chain was identical with that of the normal molecule. This finding together with the lack of fibrinopeptide A release from the abnormal Aa-chain seems to suggest that the amino acid substitution may reside in the critical position for the cleavage by thrombin, most probably at position 16 of the Aa-chain, such as in fibrinogens Zurich I and Schwarzach. ACKNOWLEDGEMENTS The authors are indebted to Dr. Y. Ohno, Special Reference Laboratory, Tokyo, Japan, for measuring fibrinopeptide A by radioimmunoassay. Thanks are also due to Dr. Stephanie M. Jung for reading the manuscript and Miss M. Takano for clerical work. This work was supported in part by a research grant from the Public Health Bureau, the Ministry of Health and Welfare of the Government of Japan. REFERENCES C. :

1. SORIA, J., SORIA, C., SAMAMA, M., POIROT, E. and KLING, Fibrinogen Troyes-fibrinogen Metz ; two new cases of Thromb. Diath. Haemorrh. congenital dysfibrinogenemia. ..___ _____..._..... .._ . ..__~ -.. 2_7:619-633, 1972.

2. HOFMANN, V., GATI, W.P. and STRAUB, P.W. : Fibrinogen Zurich I: Impaired release of fibrinopeptide A. ,Th,rombos. Haemostas. 41j709-713, 1979. 3. HENSCHEN, A., LOTTSPEICH, F., KEHL, M. and SOUTHAN, C. : Ann. N.Y._..___ Acad. Sci. Covalent structure of fibrinogen. _-._^_ __._ _._ . .4U8j28-43, 1983. 4. MATSUDA, M., BABA, M., MORIMOTO, K. "Fibrinogen Tokyo II". An abnormal impaired polymerization site on the fibrin molecules. J. Clin. Invest.

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gel

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zur 17;237-

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of 12. LUND&AD, R.L. : A rapid method for the purification bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry ~----I_-.-.__._10;25012505, 1971. F. and HENSCHEN, A. : Analysis of 13. KEHL, M., LOTTSPEICH, human fibrinopeptides by high-performance liquid chromatoHoppe-Seyler's Z. Physiol. Chem. 36231661-1664, graphy. 1981. 14. EDMAN, P. and HENSCHEN, ed. by S.B. Needlemann. 232-279.

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M.H. FINLAYSON, J.S. and MENACHE, Asp Asn. Thrombos. Haemostas.

D. :

20. HIGGINS, D.L. and SHAFER, J.A. : Fibrinogen Petoskey, a dysfibrinogenemia characterized by replacement of Arg-Acrl6 by a histidyl residue. Evidence for thrombin-catalyzed hydrolysis at a histidyl residue. J. Biol. Chem. 256:1201312017, 1981. 21. SOUTHAN, C., KEHL, M., HENSCHEN, A. and LANE, D.A. : Fibrinogen Manchester : identification of an abnormal fibrinopeptide A with a C-terminal arginine histidine substitution. Br. J. Haematol. 54:143-151, 1983. _.... -_.-__-. 22. HENSCHEN, A., KEHL, M. and DEUTSCH, E. : Novel structure elucidation strategy for genetically abnormal fibrinogens with incomplete fibrinopeptide release as applied to fibrinogen Schwarzach. Hoppe-Seyler's Z. Physiol. Chem. 3_6_4:1747-1751, 1983.