THROMBOSIS Pergamon

RESEARCH 16; 47-j8 Press Ltd.1979. Printed

in Great

Britain

PROTHROMBIN MADRID : A NEW FAMILIAL ABNORMALITY OF PROTHRCMBIN

A. BEZEAl..,M.-c. GUILLIN, F. OLtlEDA*,M. QUINTANA*and

N. GOMEZ*

Service Central d'Immunologie et Hematologie, UER K. Bichat, Hspital Beaujon, 92110 CLICHY (France), and *Institute National de Hematologia y Hemoterapia, Madrid (Spain). (Received 4.4.1979; Accepted by

in

revised

Editor

C.M.

form l.j.1979. Jackson)

ABSTRACT prothrombin has been detected in a 13 yr-old girl, with history of excessive bleeding. Prothrombin time and activated partial thromboplastin time were markedly prolonged. Prothrombin activity was very low when measured by biological assay using either physiological activators (3 A by one stage assay and 20 US u/ml by two stage assay) or Taipan snake venom (3 E) ; in contrast the level was found to be normal both by immunoassay and by assaying with the venom of either Echis carinatus or Dispholidus typus. In keeping with current nomenclature practices, the condition has been designated prothrombin Madrid. The family history revealed consanguinity between the propositus' mother and father. A 50 X reduced prothrombin level was found in both parents' plasma when using physiological activators. The pattern of inheritance appears to be autosomal recessive, the propositus being homozygote and her parents heterozygotes. Immunologic properties of prothrombin Madrid seem identical with those of the normal zymogen. The electrophoretic properties and the calcium binding of the ab-normal molecule do not differ significantly from normal, as assessed by crossed irmnunoelectrophoresis. Prothrombin Madrid has been isolated by chromatography onto DEAE Sephadex and dextran sulphate agarose. The abnormal molecule was found to bind DEAE Sephadex more tightly than normal. Purified prothrombin Madrid activation by physiological activators was found to be much slower than normal. The use of SDS gel electrophoresis and alkaline disc gel electrophoresis did not allow detection of a significant difference between prothrombin Madrid and normal prothrombin.

An abnormal

47

IXTRCDL-CTIOX Inherited qualitative abnormalities of prothrombin are very rare coagulation disorders, since only eight (1-8) dysfunctional variants have been reported. They are characterized by the discrepancy observed between the level of prothrombin as determined by functional assays using physiological activators and by innunological assay. In all cases, the inheritance of the defect is autosomal recessive. Among these 8 dysprothrombinemias, four represent either homozygous (3) or heterozygous (I, 5, i) situations characterized by a normal prothrorrbin antigenic level associated with a low functional activity. Four represent more complex situations : one case (8) corresponds to a double heterozygotism for two different dysfunctional prothrombin variants and three cases (2, 4, 6) appear to correspond to a double heterozygotism for an abnormal prothrom>in and a “true” prothromhin deficiency. The present report concerns a new congenital prothrombin abnormality detected in a Spanish family. The propositus appears to be homozygote ; the parents, who are first cousins, appear to be heterozygotes.

CASE REPORT The propositus is a girl born in Spain in 1964. Since early childFirst bleeding episodes were hood she has a history of excessive bleeding. noted at the age of about one year, consisting of ecchymosis after accidental falls while toddling. In 1969, an excessive and prolonged bleeding after dental extraction required blood transfusions. In 1971, an injury of one lip was followed by prolonged (IO days) bleeding. The first menses, in 1977, were profuse and prolonged : the following month, menorrhagias were so severe that the patient had to be admitted to the hospital and to be transfused with blood and vitamin K dependent clotting factors concentrates (Bebulin) . Management of the menorrhagias was attempted using oral because of a post-transfusional hepatitis, this contraceptive therapy, but, therapy was discontinued ; menses are persistently profuse and most of the time require transfusions of blood and Bebulin. Epistaxis and hematomas are frequent, but no hemarthrosis is noted. The propositus’ parents are first cousins. The father has no bleeding symptom. The mother has had two abortions, but apparently without bleeding manifestations ; her menses are profuse. The propositus has one brother, who has no bleeding history. MATERIALS AND METHODS Coagulation and immunological studies. Blood from a normal donor, from the proposrtus, her parents and brother was collected in 0.13 Y sodiun citrate (1 volume/9 volumes blood). The platelet poor plasma was either used immediately or kept at - 30°C and used after thawing at 37’C. Routine coagulation studies included : bleeding time (9), one stage prothrombin time performed using human thromboplastin, activated partial thromboplastin time performed using kaolin and human brain cephalin, assay of factors V (IO), VII + x (II), VII (12), x (13, 14), VIII, IX and XI (15) and fibrinogen (IO). Prothrombin coagulant activity was measured using different methods : - a classical one stage assay using human thromboplastin and an artificially prothrombin depleted substrate (17) - a one stage assay using Ta’ipan snake venom (Sigma Laboratories, St Louis,

~01.16,~0.1/‘2

ON PROTHROXBIN

$9

XADRID

MO) according to Magnin and Lawson (18) - a one stage assay using either Echis carinatus or Dispholidus typus ve: 0.2 mg/ml of human fibrinogen (4 mglml) nom (Sigma) performed as follows was added to 0.2 ml of plasma diluted in 0.1 M NaCllO.02 Tris-EC1 pH 7.5, and the clotting time was recorded after addition of 0.1 ml venom (1 mg/ml). - a two stage assay performed according to Ware and Seegers (19) with SOpreviously described modifications (20) - a two stage assay using purified Dispholidus typus venom as previOuslY described (2 1) Results of the one stage assays were expressed as per cent of normal by scale according reference to a calibration curve drawn on a bilogarithmic to the clotting time of various dilutions of no-1 plasma. Results of the two stage assays were expressed as US thrombin units/ml using human thrombin (lot B3, kindly supplied by Dr. D.L. ARONSON,Bureau of Biologics, Food and Drug Administration, Bethesda, “cd) for the calibration curve. Immunological studies were carried out using antiprothrombin anti-serum (Atlantic antibodies, Camden, Maine). Immunoassay was performed using the rocket technique according to Laurel1 (22) in 1 9. w/v agarose (Indubiose A 37, Industrie Biologique FranGaise, Clichy, France) COntaining antibody in 2.5 mM calcium lactate/O.075 M sodium barbital pH 8.6 ; the electrophoresis was run for 4 hours at 10 volts/cm. Double diffusion (23) was performed in 2 mM calcium lactate/O.@6 M sodium barbital pH 8.2. Two dimensional crossed immunoelectrophoresis was carried out according to Laurel1 (24). The first dimension was run in I X v/v agarose in 0.075 M sodium barbital pH 8.6 containing either disodium ethylene diamine (EDTA) was 60 mi(2 mM) or calcium lactate (2 I&) ; the time of electrophoresis nutes in the presence of EDTA and 120 minutes in the presence of calcium lactate at IO volts/cm. The second dimension was run in 1 9. w/v agarose containing antibody in 2.5 mM calcium lactate/O.075 M sodium barbital was 4 hours at IO volts/cm. pH 8.6 ; the time of electrophoresis Prothrombin was isolated according to Morrison Prothrombin purification. and Esnouf (25), starting from small amounts of fresh frozen acidcitrate-dextrose plasma (75 ml) obtained by plasmapheresis from a normal and her mother and father. The only modifications donor, the propositus were as follows : 1 mMbenzamidine - HCl was added in all buffers ; 0. I mg/ml soybean trypsin inhibitor (Sigma) was added in the buffer (0.2 M citrate pH 6.8) used for the elution of the barium citrate adsorbed proteins. The prothrombin preparation obtained by DEAE Sephadex chromatography was further chromatographed on dextran sulphate agarose according to Pepper and Prowse (26) ; 4 mg proteins were applied to a 1.5 x 10 cm column equilibrated in 0.4 2 trisodium citrate. Polyacrylamide gel electrophoresis. Gel electrophoresis in the presence of sodium dodecyl sulfate (SDS, from Sigma) was performed according to Weber and Osborn (27) in 10 % w/v acrylamide 0.1% W/V SDS. A current of 8 mu per gel was applied for 3 hours. Gels were stained with Coomassie blue and destained electrophoretically. Polyacrylamide disc gel electrophoresis at slkaline pH was performed in 7 % w/v acrylamide according to Davis (28) with minor modifications (29).

RESULTS Results

of

the coagulation

studp

performed

in the oropositus

are

04

50

PROTHROSfBI?i Y_-\DRID

L-01.16,No.l:~

were persistently obtained w’hen the on Table I ; the same results study was repeated at intervals during the last 5 years. The prothrombin time and partial thromboplastin time are prolonged. Prothromhin activity, as assayed by the classical one stage method, is decreased, whereas factors V, VII, VIII, IX, X, XI and fibrinogen levels are within the normal range. shown

TABLE I Coagulation

study

_--_----_____-. i

Control

or

I normal range t-___________________________. _____-___-_---. t 1 Bleeding time (min) 1 Platelet count (per mm3, I Prothrombin time (see) ) Partial thromboplastin time (set) 1 F.:-II* (Z) IF. VII+X (X) IF. VII (%) IF. X (%) ;F. V (%) 1 F. VIII (X) ; F. IX (X) IF. XI (X) Fibrinogen (mg/ 100 ml) ___-______-_________~-~~~~~~~-~. * Classical

one stage

I - 3 150 - 400,000 12 40 70 - 120 70 - 120 70 - 120 70 - 120 i 70 - 120 I 60 - 150 f 70 - 130 60 - 150 i I 250 - 400 _‘_I_--_--_--____

Proposi tus --------------3 min 3(! 180,CW 25 90 3 100 80 too IO0 100 76 60 220 -------_---_-.

assay

Various prothrombin assays were performed in the plasma of the propositus and some of the family members (Table II). In the propositus plasma, prothrombin activity is markedly depressed when measured using either physrological activators (one or two stage assay) or using Ta’ipan the level is within the normal range when desnake venom ; in contrast termined either by biological assay using Echis carinatus or Dispholidus prothromtypus venom or by immunoassay. In the plasma of both parents, bin assay reveals a 50 % reduced level only when using physiological acplasma, prothrombin activators or TaTpan snake venom. In the brother’s allow to identitivity is normal whatever the method used. These results fy the isolated prothrombin deficiency detected in the propositus as a In keeping with current nomencongenital abnormality of the molecule. the condition has been designated “Prothrombin Madrid”. clature practices,

ON PROTHROMElIN

?WDRID

TABLE II Prothrombin II iI t

according

level,

to

various

~-~~~~~_--~~--~~-~-_--_--~_~~~~~~~~~~~~~ !.Propositus He thods i Yother

__________-___-_-_-_&_-----__+-___-_ One stage assays oi : 70 - 120 %> Prothrombinase Ta’ipan snake Echis carinatus Dispholidus typus Two stage assay (S : 150 - 250 Prothrombinase

f I I i i ; I

Father

-____-___-_- , Brother j i __-_I C. ____-mm

i 3 3 94 75

i

1 I 1 I I I

US u/ml)\ f

methods

_---__-_-

20

1

52 54 84 82

43 52 100 80

134

106

I i I Immunological 103 98 assay I I I (?; : 70 - 120 X) , I ;._ _-_______-_--_________^_______~~~~~~-~-~~~~-~~_.

83

94 95

I

I f

I / f

100 80

; i

198

/

96

1

I I __-_----_____-______/

The inununological characteristics of the abnormal prothrombin were studied using the double diffusion technique (Fig. 1) ; the precipitin line produced with the propositus plasma shows complete identity with normal prothrombin, as do the precipitin lines produced with the parents plasma.

FIG. plasma.

Double diffusion pattern. 2 : propositus’ plasma. 3

:

I

AS11 : antiprothrombin. mother’s plasma. 4

1 : normal : father’s plasma.

The electrophoretic properties of the abnormal prothrombin were investigated by crossed immunoelectrophoresis, using antiprothrombin antiserum (Fig. 2). .The electrophoretic migration of the abnormal molecule either in the absence or presence appears to be similar to that of normal, of calcium ions in the buffer during the first run. Nevertheless minor (but constantly observed) differences appear in the pattern of the peaks, with the particularly in the presence of calcium ions : the peak obtained broader than normal ; the peak obtained with propositus plasma is a little the precipitin line skewing the parent’s plasma is slightly asymetrical, towards the anode without joining the baseline.

E DTA

FIG.

2

Crossed immunoelectrophoresis Plasma samples were run in the presence (right) in the first dimension.

pattern using of EDTA (left)

antiprothrombin. or calcium ions

Prothrombin isolation has allowed to specify some characteristics of the abnormal molecule. Adsorption of prothrombin Madrid onto barium citrate was shown to proceed normally as assessed by the disappearance of the whole prothrombin antigenic material from titrated plasma upon the addition of IO ti barium chloride. DEAE Sephadex chromatograms (Fig. 3) were found to be different for normal and abnormal prothrombin. When a normal barium citrate eluate was applied to the column, proteins bound to DEAE Sephadex eluted upon the application of the gradient in : the shoulder contained trace one absorbance peak preceded by a shoulder amounts of prothrombin and higher molecular weight proteins ; the major peak contained prothrombin. When the propositus eluate war applied, proteins eluted in two absorbance peaks : the first peak contained trace amounts of abnormal prothrombin and higher molecular weight proteins ; the second peak contained the major part of abnormal prothrombin, which was eluted at a slightly higher ionic strength than normal. Despite this difference, prothrombin isolated from the parents plasma cannot be resolved by this chromatographic procedure into its normal and abnormal components. Prothrombin preparations obtained by DEAF Sephadex chromategraphy were contaminated with factor X, and for this reason, were further chromatographed on dextran sulphate agarose, which reduced factor X contamination to less than 2 %. The final prothrombin preparations were analysed by polyacrylamide gel electrophoresis. On SDS gel electrophoresis (Fig. 4 A) the preparations exhibit one major band corresponding +!igration of prothrombin Madrid to prothrombin and minor contaminants.

OX

PRO’TRROXBIN

Elution

volume

FIG.

?-UDRID

(ml)

3

DEAE Sephadcx chromatography of normal prothrombin (---) or prothrombin Madrid (-). Barium citrate eluate, dialysed against the equilibrating buffer, was applied to a column (I .5 x 12 cm) of DEAE Sephadex A 50 equilibrated in 0.05 3: ?IaCl/O.Oj ?l citrate titrated to pH 6.5 with Tris-Xl. Proteins were eluted with a 2 x 150 ml gradient (0.05 to 0.5 X NaC1/0.05 M citrate/Tris-HCl pt_I 6.5). (fig. 4 A, gel 2) does not differ significantly from normal (fig. 4 A, fails to separate the normal and abnorgel 1) ; SDS gel electrophoresis mal component of the parent’s prothrombin (Fig. 4 A gels 3 and 4). On 4 B) no difference in the migraalkaline disc gel electrophoresis (Fig. Madrid was observed as compared to normal ; r;sn rate of prothrombin the normal and abnormal components of the parents’prothrombin were not separated using this technique.

FIG.

4

Gel electrophoresis of prothrombin. A : SDS gel electrophoresis B : Alkaline disc gel electrophoresis ; 1 : normal prothrombin - 2 : prothrombin Madrid - 3 : Mother’s prothrombin - 4 : Father’s prothrombin. Specific activity of the prothrombin preparations was measured (Table III) by two stage assays using either physiological activators or Dispholidus typus venom. Thrombin activity produced was assayed at various incubation times. Results given correspond to the incubation time necessary for full activation of normal prothrombin. Specific activities of normal prothrombin, prothrombin Madrid, and prothrombin from both parents were found to be similar vhen assayed with Dispholidus typus venom ; in contrast, after an incubation time necessary for full activation of normal prothrombin, prothrombin Hadrid has generated 10 timesfewerunits of thrombin than normal. Prolongation of the incubation time (3 hours instead of 10 minutes) resulted in the production of as many thrombin units from prothrombin Madrid as from normal.

TABLE III Specific

artivity

r__-______-_-__-

*_ I I

_-a-_

of --_,-___

isolated

prothrombin

(US u/mg)

____________________~~~__~~~_~~~

i

Two stage assay I Prothrombin _____~__________________I L-_-__-_-__I preparation I i Dispholidus typus I Prothrombinase ~_______________________~_-_-_-_____-____~__________________~ / 820 I 2oc Elormal I ! 780 130 Madrid I i I 740 390 Mother I I f 1 810 410 Father I I____________-___L-______-__-__-_--L __-_________-_--____---

1 I

I, f I I f I

OY PROTHROMBIN M.%DRID

DISCZ’SSION The discrepancy observed in the propositus plasma between the normal prothrombin antigen level and the low functional activity detected using physiological activators has allowed us to conclude that the clotting defect is related to an abnormality of the prothrombin molecule. This abnormality is congenital since both parents, who are consanguinous, exhibit the same type of defect, at a minor degree. The pattern of inheritance, as in the other cases of dysprothrombinenia (l-8) appears to be autosomal and recessive. The mode of action of the sctivators used for the study of prothrombin nadrid conversion is now well known. Physiological activation of prothrombin (30 and references therein) occurs upon the action of Xa, with the reaction rate being dependent upon the presence of calcium ions, phospholipids and Va. Xa is the enzyme responsible for two successive peptide bond cleavages, the first bond linking fragment 1.2 and prethrombin 2 (bond 2) and the second one linking the A and B chains of thrombin (bond 3). Ta’ipan snake venoms splits the same peptide bonds, in the same order (31) with the reaction rate being dependent upon the presence of calcium ions and phospholipids but independent of the presence of Va (32). Either Echis carinatus (33) or Dispholidus typus venom (21) splits one peptide bond (bond 3), this single cleavage exposing the catalytic site of the prothrombin zymogen. With these enzymes, the reaction rate is independent upon the presence of any accessory factors. The currently knoym inherited prothrombin variants are characterized by an impairement of physiological activation, but differ each from another with regard to their ability to be activated by the venous. Conversion of prothromhin Brussels (34), Molise (2) and Quick (6) by Echis carinatus venom is abnormal while conversion of prothrombin Barcelona (35) and Padua (1) proceeds normally. Functional activity of prothrombin Madrid was found to be normal when assayed using either Echis carinatus or Dispholidus typus venom. This suggests that in the case of prothrombin Madrid as probably in the case of prothrombin Barcelona and Padua, the structural defect does not modify the susceptibility of bond 3 to the proteolytic action of these venons and does not involve the catalytic site of thrombin. Conversion of prothrombin :‘drid by physiological activators This could be related either to an impaiwas shown to proceed slowly. rement of the enzymatic action of Xa or/and to the lack of acceleration of the reaction by the accessory factors, calcium ions, phospholipids and Va. It does not seem that a lack of acceleration of the reaction by Va may explain, at least alone, the functional defect, since the activity of prothrombin Madrid was found to be abnormal when using TaTpan snake venom whose action is independent upon the presence of Va. A defect in the binding to phospholipids, which normally proceeds via calci.um ions cannot be ruied out ; nevertheless no abnormality in the calci.um bindfng of prothrombin Hadrid could be detected : migration in the presence of calcim ions was found to be similar to normal and the abnormal molecule was found to adsorb onto insoluble salts of barium as well a.5 normal. ACICh’OWLFDCEMENTS This work was supported by grants from Institut National de la Sante et de la Recherche MGdicale, CRL no78 2 I03 I, and from DER Xavier Bichat, Universite Paris VII, Paris, France.

56

OX

PROTI-IHOMBIN M.ADRID

The authors are grateful to MS Veronique Foulon secretarial assistance.

YO1.16,SC.1/2

for

skilful

REFERENCES 1.

GIROLAMI A., BAREGGI G., BRUNETTI A. and STICCHI A. Prothrombin Padua: "new" congenital dysprothrombinemia. J. Lab. Clin. Med., 84, 654, 1974

a

2. GIROLAMI A., COCCHERI S., PALARETI G., POGGI M., BURUL A. and CAPPELLATO G. Prothrombin Molise : a "new" congenital dysprothrombinemia, double heterozygosis with an abnormal prothrombin and "true" prothrombin deficiency. Blood, 52, 115, 1978 3. JOSSO F., MONASTERIO de SANCHEZ J., LAVERGNE J.M., MENACHE D. and SOULIER J.P. Congenital abnormality of the prothrombin molecule (factor II) in four siblings : prothrombin Barcelona. Blood, 38, 9, 1971 4. JOSSO F., RIO Y. and BEGUIN S. Prothrombin Metz : a new variant of human prothrombin. Double heterozygosity for congenital hypoprothrombinemia and dysprothrombinemia. XVIIth Cong. Int. Sot. Hemat. and KVth Cong. Int. Sot. Blood Transf. Paris, 1978, Abst. book p. 860 5. KAHN M.J.P..and GOVAERTS A. Prothrombin Brussels, a new congenital defective protein. Thromb. Res., 5, 14i, 1974 6. OWEN C.A., HENRIKSEN R.A., McDUFFIE F.C. and MANN K.G. Prothrombin Quick. A newly identified dysprothrombinemia. Mayo. Clin. Proc., 53, 29, 1978 7. SHAPIRO S-S., MARTINEZ J. and HOLBURN R.R. Congenital dysprothrombinemia : an inherited structural disorder of human prothrombin. J. Clin. Invest., 48, 2251, 1969 8. SHAPIRO S.S. Prothrombin San Juan : a new complex dysprothromhinenia. In : Prothrombin and related factors, l!.C.Hemlcer and J.J. Veltkamp (Eds), Leiden University Press, 1975, p. 205 9. DUKE W.W. The relation of blood platelets to hemorrhagic disease. Description of a method for determining the bleeding time and coagulation time and report of three cases of hemorrhagic disease relieved by transfurion. 3. Amer. Med. Ass., 55, 1185, 1910 10. OWREN P.A. The coagulation of blood, investigations on a new clotting factor. Acta Med. Stand., Supp. 194, I, 1947 II. OWREN P.A. La proconvertine. Rev. Hemat., 7, 147, 1952 12. HOUGIE C., BARROW E.M. and GRAHAM J.B. Stuart clotting defect. I. Segregation of an hereditary hemorrhagic state from the heterogeneous group heretefore called "Stable factor" (SPCA, proconvertin, factor VII) deficiency. J. Clin. Invest., 36, 485, 1957 13. BACHMANN F., DUCKERT F., GEIGER M., BAER P. and KOLLER F. Differenciation of the factor VII complex. Studies on the Stuart Prover Factor. Thromb. Diath. HaemOrrh., 1, 169, 1957

ON PROTHROMBIN

MADRID

14. DIDISHBIM P., LOEB J., BLATRIX C. and SOULIER J.P. Preparation of a human plasma fraction rich in prothrombin, proconvertin, Stuart factor and PTC and a study of its activity and toxicity in rabbits and man. J. Cab. Clin. Med., 53, 322, 1959

lj.

SOULIER J.P. and LARRIEU M.J. Nouvelle &thode de diagnostic de l'h&ruophilie.Dosage des facteurs antihgoophiliques A et B. &e_ Sang, 24, 205, 1953

16. INGRAM G.I.C. The determination of plasma fibrinogen by the clotweight method. Biochem. J., 51, 583, 1952 17. SOULIER J.P. and LARRIEU M.J. Etude analytique des temps de Quick allonges. Dosages de prothrombine, de proconvertine et de proaccelerine. Le Sang, 23, 549, 1952 18. MAGNIN A.A. and LAWSON W.B. Simultaneous determination of plasma prothrombin and antithrombin 111 (heparin co-factor). Thromb. Res., 7, 555, 1975 19. WARE A.G. and SEEGERS W.H. Two stage procedure for the quantitative determination of prothrombin concentration. Amer. J. Clin. Path., 19, 471, 1949 20. CESBRON N., BOYER C., GUILLIN M.C. and MBNACHE D. Human coumarin prothrombin. Chromatographic, coagulation and immunologic studies Thromb. Diath. Haemorrh., 30, 437, 1973 21. GUILLIN MC., BEZEAL'D A. and MENACHE D. The mechanism of activation of human prothrombin by an activator isolated from Dispholidus typus venom. Biochim. Biouhys. Acta, 537, 160, 1978 22. LAURELL C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem., 15, 45, 1966 23. OUCHTBRLONY 0. Diffusion in gel methods for immunological analysis. In : Progress in Allergy. ?. Rallos and B.H. Waksman (Eds) Larger, Basel, 6, 30, 1962 24. LAURBLL C.B. Antigen-antibody crossed electrophoresis. Anal. Biochem. 10, 358, 1965 25. MORRISON S.A. and ESNOUP M.P. The nature of the heterogeneity of prothrombin during dicoumarol therapy. Nature New Biology, 242, 92, 1973 26. PEPPER D.S. and PROWSE C. Chromatography of human prothrombin complex on dextran sulphateagarose. Thromb. Res., 11, 687, 1977 27. WBBER K. and OSBORN M. The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem., 244, 4406, 1969 28. DAVIS B.J. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 404, 1964

29. GUILLIN M.C., ARONSON D.L., BEZEAL?) 2..,MENACHE D., SCHLEGEL T. and AMa Y. The prurification of human acarboxyprothrombin. Characterization of its derivatives after thrombin cleavage. Throrcb. Res., 1, 223, 1977 30. SUTTIE J.W. and JACKSON C.M. Prothrombin structure, activation and biosynthesis. Physiological Reviews, 57, 1, 1977 31. OWEN W.G. and JACKSON C.M. Activation of nrothrombin with Oxyuranus scutellatus scutellatus (Tarpan snake) venom. Thromb. Res., 3, 705, 1973 32. DENSON K.W.E., BORRETT R. and RIGGS R. The specific assay of prothrombin using the Ta'ipan snake venom. Brit. J. Haematol., 21, 219, 1971 33. MORITA T., IWANAGA S. and SUZU'RIT. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermediates. J. Biothem., 79, 1089, 1976 34. KAHN M.J.P. Prothrombin Brussels. In : Prothrombin related factors H.C. Hemker and J.J. Veltkamp (Eds), Leiden University Press, 1975, p. 223 35. RABIET M-J., ELION J., BENAROUS R., LABIE D. and JOSS@ F. Activation of prothrombin Barcelona. Evidence for active high molecular weight intermediates. Biochim. Biophys:Acta, in press.