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Low-molecular weight factor VIII requires a cofactor for its procoagulant activity
THROMBOSIS RESEARCH 19; 503-511, 1980 0049-3848/80/160503-09502.00/0 Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd
LOW-MOLECULARWEIGHTFACTORVlll REQUIRESA COFACTORFOR ITS PROCOAGULANTACTIVITY Th. Vukovich,
E. K o l l e r
and W. Doleschel
Department of Medical Physiology, School of Medicine, University of Vienna, Schwarzspanierstr.17, A-I090 Vienna, Austria (Received 31.3.1980; in revised form 30.7.1980. Accepted by Editor G. MUller-Berghaus)
ABSTRACT In the course of production and quantitation of the procoagulant low-molecular weight factor VIII (LMW VIII:C) produced by dis sociation of purified high-molecular weight factor VIII in 0.25 M CaCI2, we observed that LMW VIII:C was active in only one of i t s two generally accepted test systems, namely the so-called "onestage" test. In the "two-stage" test, however, an additional highmolecular weight factor was required for LMW VIII:C to approximate i t s "one-stage" test level of a c t i v i t y . Investigation revealed that the high-molecular weight factor VIII antigen (VIIIR:Ag) provides this "cofactor" effect.
INTRODUCTION The high-molecular weight human factor VIII (HMW F V I I I ) isolated from blood anticoagulated by chelation of the plasma Ca++ ions is a large glycoprotein with a molecular weight higher than ixlO D Daltons (1). This macromolecule is responsible for two d i s t i n c t hemostatic a c t i v i t i e s , a procoagulant a c t i v i t y (VIII:C) and a so-called "von Willebrand factor" a c t i v i t y (VIIIR:WF) which supports the adhesion of thrombocytes to collagen-containing tissues (2). The rare phenomenon that two d i s t i n c t a c t i v i t i e s (VIII:C and VIIIR:WF) are located on one protein is biochemically and pathophysiologically interesting since defects in the structure of HMW F VIII can cause the coagulation disorders hemophilia A and / or von Willebrand's disease. A widely accepted model for the factor's molecular organisation is that of a complex of two comKey words: Factor V I I I , LMW factor VIII:C, Factor VIIIR:Ag, Dissociation 503
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
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ponents, one of high molecular weight carrying the s i t e for the von Will ebrand a c t i v i t y and a smaller component responsible for the f a c t o r ' s procoagulant a c t i v i t y (LMW V I I I : C ) . The larger component can be characterized by immunological means as antigen (VIIIR:Ag). This model is supported by the observation of several investigators (3-13) that HMW F VIII is eluted from gels with separation ranges of about 105-106 Daltons by the use of isotonic, Ca++-free buffers with all three a c t i v i t i e s (VIII:C, VIIIR:WF, VIIIR:Ag) occuring in the void volume fractions. Through the use of hypertonic solutions (0.25 M CaCI2 or I M NaCI), however, a separation of the two hemostatic a c t i v i t i e s can be achieved, that of VIIIR:WF found in the void volume and LMW VIII:C appearing in a l a t e r volume. A reaggregation occurs i f both fragments are mixed in isotonic, Ca++-free buffers (10-13). This communication reports our observation that LMW VIII:C, produced by dissociation of HMW F VIII in 0.25 M CaCl 2, is active only in one of the two generally accepted test systems for determination of VIII:C, namely the socalled "one-stage" test. In the "two-stage" test, however, an additional highmolecular weight factor was required for LMW VIII:C to approximate i t s "onestage" test level of a c t i v i t y . MATERIALS Human F V I I I of intermediate purity and human albumin (a); specific antihuman F V I I I serum, human F V l l l - d e f i c i e n t plasma, human F IX-deficient plasma, and phospholipid-kaolin reagent (b); human vWF-deficient plasma (c); p r o t e o l y t i c i n h i b i t o r Trasylol (d); heparin-Na and protamine-sulphate (e); Ultrogel AcA22 ( f ) ; and Sephadex G 25 (g) were obtained from the given outside sources. The human blood for preparation of cryoprecipitates was obtained from healthy male donors. (a) Schwab & Co., Vienna, Austria; (b) Behringwerke, Marburg, GFR; (c) g i f t from Prof. K. Lechner, Vienna; (d) Bayer, Leverkusen, GFR; (e) Novo, Copenhagen, Denmark; ( f ) LKB, France; (g) Pharmacia, Uppsala, Sweden. Buffer solutions: " T r i s - c i t r a t e - b u f f e r " : 0.13 M NaCl, 0.02 M Tris, 0.01M Na3-citrate plus I0 units/ml Trasylol and 1 mg/ml albumin, -pH 7.4 adjusted with HCI at 20oc. "Tris-CaCl2-buffer": 0.25 M CaCI2, 0.02 M Tris plus 25 units/ml heparin, I0 units/ml Trasylol and I mg/ml albumin, -pH 7.4 adjusted with HCI at 20°C. METHODS Preparation of HMW F V l l l HMW F V I I I was purified either from a solution of the commercial F V I I I material (containing 490 mg of total protein of which 160 mg were c l o t t a b l e , 250 units V I I I : C , and 950 units VIIIR:Ag in I0 ml solution) or from a solution of cryoprecipitate. The cryoprecipitate was obtained as follows: 450 ml blood were collected in 50 ml of a solution of 0 . I I M Na3-citrate containing I00 units/ml Trasylol. Following centrifugation at 2,500 x g and 20oc for 20 minutes, the supernatant plasma was frozen in I0 ml amounts and maintained at -70oc. For cryoprecipitation as needed, frozen samples were thawed and held in a water bath at 4oc over a period of 4 hours. The resultant precipitates were sedimented by centrifugation at 2,500 x g and 4°C for 20 minutes and redissolved in t r i s - c i t r a t e - b u f f e r to one-tenth of the original plasma volume to provide the solution used for further p u r i f i c a t i o n . The solution of i n t e r mediate purity F V I I I and that of the cryoprecipitate were chromatographed on a 30 cm long, t r i s - c i t r a t e - b u f f e r - e q u i l i b r a t e d Ultrogel AcA22 column 27 mm in diameter. 4 ml samples of both F V I I I solutions were applied to the top of the
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column and eluted with the t r i s - c i t r a t e - b u f f e r at a flow rate of 18 ml/hour at room temperature. The void volume fractions of these elutions, containing HMW F V I I I , were concentrated to a volume of 4 ml by d i a l y s i s for approximately 3 hours at 4°C against polyethylene glycol 20,000 and maintained at -70°C for subsequent use. Fractionation of HMW factor V I I I Before experimental use each 4 ml sample of frozen HMW F V I I I was thawed in a water bath at 37oc and mixed with 4 ~ I of a heparin solution containing 25,000 NIH units/ml. Addition of I I I mg of rapidly weighted, dehydrated CaCI2 to each of the heparinized samples (final concentration 0.25 M) effected the dissociation of the factor. Separation of the factor V I I I fragments To separate the F V I I I fragments derived from both F V I I I sources, the appropriate 4 ml samples were chromatographed on the Ultrogel column preequilibrated with the tris-CaCl2-buffer and eluted with the same buffer at a flow rate of 20 ml/hour at room temperature. The eluates were monitored for t h e i r UV absorbance a t ~ 256 nm and collected in 4 ml samples at 4°C. Free Caions were removed from these samples and the contained heparin neutralized as described below. Determination of V I I I : C Quantitative determinations of V I I I : C were performed with the "one-stage" test based on the technique of Langdell et al (14) and with the "two-stage" test o r i g i n a l l y proposed by Biggs and Douglas (15) in a modification of Aronson as follows: I pt. human serum diluted lO-fold with 0.066 M NaCl in 0.05 M imidazole buffer, pH 7.3; i pt. bovine serum diluted 20-fold with the same buffer mixture; I pt. 0.025 M CaCI9 solution; I pt. phospholipid-kaolin reagent; and i pt. test solution are~mixed and held for 5 minutes at 37oc. i pt. of this mixture is then mixed with I pt. of the CaCI2 solution and, at zero time, with I pt. of fresh human citrated-plasma. The coagulation time of the l a t t e r mixture is recorded. Two d i l u t i o n s of the V I I I : C samples without Al(OH)2-adsorption were tested in duplicate. Evaluation of the test samples' a c t i v i t i e s were made from a standard plasma d i l u t i o n curve. The test values of both sample d i l u t i o n s when calculated back to the a c t i v i t y of the original test sample agreed within I015% of each other. Prior to q u a n t i t a t i o n , those test samples containing 0.25 M CaCI 2 and heparin were treated as follows: 2 ml amounts of each sample were applied to Sephadex G 25 columns (0.5 x I I cm) preequilibrated with t r i s c i t r a t e - b u f f e r . To the Ca-ion-free void volume eluates s u f f i c i e n t protamine was added to neutralize the contained heparin (minimal c l o t t i n g time of the "one-stage" V I I I : C t e s t ) . Determination of VIIIR:Ag The q u a n t i t a t i v e determinations of VIIIR:Ag were made with rabbit antiserum specific for human F V I I I according to the electroimmunodiffusion method of Laurell (17). Standard plasma As reference plasma for the quantitation of V I I I : C and VIIIR:Ag from all sources a pool of 20 fresh, c i t r a t e d plasmas (9 pts. plasma + i pt. 0 . i i M Na3-citrate ) was frozen in 1 ml amounts and held at -70oc u n t i l use. The V I I I : C and VIIIR:Ag contents of once-thawed plasma were taken as I unit/ml.
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EXPERIMENTAL The l i t e r a t u r e holds divergent answers to the question whether a limited proteolysis of HMW F VIII is a prerequisite for the factor's dissociation in hypertonic salt solution. To minimize this complication we have used for our experiments HMW F VIII prepared from a commercial F VIII concentrate liable to possible proteinase a c t i v i t y during i t s preparation and our own HMW F VIII derived from cryoprecipitates of fresh plasma treated with proteinase i n h i b i t o r and subsequently purified in the presence of such inhibitors. When the chromatographic eluates of the CaCl2-fragmented HMW F VIII from both sources were tested for their UV absorbance, their VIIIR:Ag contents, and their VIII:C a c t i v i t i e s as determined by the "one-stage" method, the results obtained are those given in Fig. I. Inspection of the elution patterns from both F VIII preparations shows s i m i l a r i t y with respect to all three parameters. UV-absorbing material was present in void-volume (Vo) fractions and in t o t a l volume (Vt) eluates of both chromatograms. VIIIR:Ag was eluted exclusively in the Vo-eluates (recovery in 6 chromatographies 90-100% of that applied to the column) while VIII:C a c t i v i t i e s were displayed by fractions no 21 to 28 (recovery 25-50%) in the separation range of the given chromatographic column. The fact that in both chromatograms the VlllR:Ag-containing Vo-eluates were free of procoagulant a c t i v i t y and the LMW VIII:C-containing eluates were free of VIIIR:Ag indicates a complete dissociation of both fragments on the Ultrogel column. Since the HMW F VIII derived from a commercial source and F VIII prepared by us from fresh plasma to minimize proteolysis reacted similarly to treatment with hypertonic CaCI2, i t is improbable that proteolysis is a prerequisite for this factor's dissociation.
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H M W F VIII for the the VIIIR:Ag U/ml equilibrated with = F VIII from
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To determine possible discrepancies in the quantitation of LMW V I I I : C a c t i v i t y between the results of the one- and two-stage analytical t e s t , we tested the same eluate fractions of the chromatograms by means of the two-stage procedure. The results are given in Fig.2. No a c t i v i t y was dedectable in any f r a c t i o n despite the fact that the highest a c t i v i t y determined in the eluates with the one-stage t e s t was more than lO0-fold that of the s e n s i t i v i t y l i m i t of the two-stage test for "unfragmented" F V I I I . From these two-stage results we questioned whether the procoagulant a c t i v i t i e s in fractions no 21 to 28 of the chromatograms as shown by the one-stage test were F V l l l - s p e c i f i c , since the l a t t e r test is peculiarly sensitive to such "non-F V l l l - r e l a t e d " c l o t t i n g a c t i v i t i e s as, e.g. that of thrombin (17). To establish the F V l l l - s p e c i f i t y of the procoagulant a c t i v i t y in the given f r a c t i o n s , we tested them by the one-stage F IX assay which matches the one-stage V I I I : C test in i t s s e n s i t i ~ t y to "non-test-specific" coagulant a c t i v i t i e s . No such a c t i v i t y was detected by t h i s test (Fig.2). These results show that the procoagulant a c t i v i t i e s of fractions no 21-28 as determined by the one-stage V I I I : C test are indeed related to V I I I : C .
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P r o c o a g u l a n t a c t i v i t i e s o f the e l u a t e f r a c t i o n s as o n e - s t a g e V I I I : C test ( D / O ) , t w o - s t a g e V I I I : C test F I X test ( A ) . '~" = F V I I I f r o m c r y o p r e c i p i t a t e . c o m m e r c i a l concentrate.
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To explain the i n a c t i v i t y of LMW V I I I : C in the two stage test we considered the fact that the reagents of the f i r s t step of t h i s t e s t are, in contrast to those of the one-stage t e s t , very poor in t h e i r content of VIIIR:Ag. To study whether VIIIR:Ag is required for LMW V I I I : C to effect c l o t promotion we tested the r e a c t i v i t y of LMW V I I I : C by the two-stage test in the presence of a VlllR:Ag-rich Vo solution obtained by pooling equal amounts of fractions no 13 to 16 from the given chromatograms. Mixtures of 0 . I ml of the Vo-pool and 0.I ml of each of the individual eluate fractions no I0 to 40 were prepared and analyzed. The results, given in Fig. 3, show that in the presence of Vo eluated substances LMW V I I I : C was demonstrable in the fractions no 21 to 28. The observed a c t i v i t y in these fractions was 60 % (mean of 6 chromatograms) of
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
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that quantified by means of the one-stage test.
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F I G U R E 3 : P r o c o a g u l a n t a c t i v i t i e s o f the eluate f r a c t i o n s as d e t e r m i n e d by the two-stage test a f t e r a d d i t i o n o f the V I I I R : A g - r i c h Vo-pool. "A" = F VIII f r o m cryoprecipitate. "B" = F VIII from commercial concentrate.
To determine whether VIIIR:Ag of the Vo eluate pool is functioning as a "cofactor" for the demonstrated LMW V I I I : C a c t i v i t y , we tested all fractions from the chromatograms as mixtures (9 pts. f r a c t i o n + I pt. given plasma) with hemophilic A plasma (VIIIR:Ag = 1.2 U/ml) against mixtures with vWF-deficient plasma (VIIIR:Ag = 0.04 U/ml) in the two-stage test. The results given in Fig. 4, show that LMW V I I I : C from fractions no 21 to 28 was reactive in the twostage test in the mixture with VlllR:Ag-rich hemophilic plasma but not in the mixture with vWF-deficient plasma. The observed a c t i v i t i e s of the fractions no 21 to 28 run with hemophilic plasma were, as in the previous experiments, 60 % of those found by the one-stage test. Since, apart from t h e i r content of VIIIR:Ag, the hemophilic A and the vWF-deficient plasmas are identical with respect to t h e i r c l o t t i n g components, these results indicate that the "cofactor" for LMW V I I I : C is indeed VIIIR:Ag. DISCUSSION Several authors have reported that in solutions of high s a l t concentration HMW F V I I I dissociates into a low-molecular weight, procoagulant fragment (LMW V I I I : C ) and a high-molecular weight, inactive fragment (3-13). These fragments reaggregate in isotonic Ca++-free buffer (10-13). I t thus appears that the components of HMW F V I I I are held together by non-covalent forces. This concept of the molecular organisation of HMW F V I I I has been challenged by the observation (18) that the fragmentation by high concentrations of s a l t was preventable by the addition of the proteolysis i n h i b i t o r aprotinin. From these results i t was postulated that a limited proteolysis of HMW F V I I I is a prer e q u i s i t e for the f a c t o r , s dissociation. However, more recent work has demonstrated (9) that fragmentation of HMW F V I I I does occur under such s a l t conditions even in the presence of a v a r i e t y of p r o t e o l y t i c i n h i b i t o r s (benzamidine, soybean t r y p s i n i n h i b i t o r , heparin, diisopropylfluorophosphate,
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and aprotinin). Since in our experiments the HMW F VIII prepared from fresh, proteinase-inhibited plasma was fragmented by 0.25 M CaCI2 in the presence of aprotinin and heparin, we favor the view that proteolysis is not a prerequi~te for the factor,s fragmentation.
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F I G U R E 4 : Procoagulant activities of the eluate fractions as determined by the two-stage test after addition of either "cofactor"-rich,hemophilic-A plasma ( D / • ) or "cofactor"-poor, vWF-deficient plasma ( A ) . '~" = F VIII from cryoprecipitate. "B" = F VIII from commercial concentrate.
The requirement of VIIIR:Ag acting as a "cofactor" for the expression of the procoagulant a c t i v i t y of LMW VIII:C is the most important finding of our study. Our results indicate that both procoagulant inactive VIIIR:Ag forms, namely the inactive high salt HMW fragment and the hemophilic-A-F V I I I , act as "cofactor" for LMW VIII:C. That such a cofactor requirement was not previously recognized is understable since the LMW VlII:C a c t i v i t y was determined almost exclusively by means of the VlllR:Ag-containing one-stage test. In the only exception (13), Denson's modification (19) of the two-stage test was employed. The reagents of Denson's modification and those of Aronson's d i f f e r in that the l a t t e r ' s contains, among i t s f i r s t - s t a g e reagents, VIIIR:Ag derived from human and bovine sera in an amount of 0.03 U/ml final reaction mixture, while those employed by Denson are derived from plasma. Since thrombin-treatment of HMW F VIII increases the electrophoretic mobility of VIIIR:Ag (20) i t is probable that VIIIR:Ag from serum has been modified during coagulation as against that from plasma to yield an inactivated "cofactor". Contrarily, the plasma-derived reagents may well be contaminated with VIIIR:Ag able to act as "cofactor" with LMW VIII:C. Discrepancies between the results of quantitation of HMW VIII:C in purified form when compared with the a c t i v i t y of a standard plasma by either the oneor the two-stage test are commonly observed (21,22). One explanation for this phenomenon is "that different molecular forms of F VIII may react d i f f e r e n t l y in the two assay systems" (21). For example, i f LMW VIII:C contained in normal plasma (23) requires VIIIR:Ag as "cofactor" for clot promotion, as demonstrated for the high salt fragmented LMW VIII:C, the discrepancies between the results of both tests may be explained by the presence or relative absence of the "cofactor" in the reagents of these tests.
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ACKNOWLEDGEMENTS The most valuable criticism and suggestions by A.L. Schade, Ph.D. are thankf u l l y acknowledged. This investigation was supported by a grant from the Austrian Foundation for Promotion of Scientific Research (Proj. Nr. 4019). REFERENCES I.LEGAZ,M.E.,SCHMER,G.,COUNTS,R.B. and DAVIE,E.W.Isolation and characterization of human F VIII (Antihemophilic Factor). J.Biol.Chem.218,3946-3955,1973. 2.BAUMGARTNER,H.R.and MUGGLI,R.Adhesion and aggregation: morphological demonstration and quantitation in vivo and in vitro. In: Platelets in biology and pathology. J.I.GORDON (Ed.).Amsterdam: North-Holland pub-Tishing Company,23-33,1976. 3.WEISS,H.J.,PHILLIPS,L.L.and ROSNER,W.Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography.Thrombos.Diathes.Haemorrh.27,212219, 1972. 4.0WEN,W.and WAGNER,R.H.Antihemophilic factor: Separation of an active fragment following dissociation by salts or detergents. Thrombos.Diathes.Haemorrh.27, 502-515, 1972. 5.RICK,M.E. and HOYER,L.W.Immunologic studies of antihemophilic factor (AHF, factor V I I I ) . Immunologic properties of AHF subunits produced by salt dissotiation. Blood.42,737-747, 1973. 6.COOPER,H.A.,REISNER,F.F.,HALL,M.and WAGNER,R.H. Effects of thrombin treatment on preparations of factor VIII and the Ca++-disso~iated small active fragment. J.Clin.lnvest.56,751-760,1975. 7.BOLHUIS,P.A.,BEESER-VISSER,N.H.and SIXMA,J.J. Molecular weight of human plasma factor VIII. Thrombos.Res.16,497-509, 1979. 8.LAVERGNE,J.M.,MEYER,D.,JENKINS,C.S.,OBERT,B.and LARRIEU,M.J.Influence of high ionic strength buffers on factor Vlll/von Willebrand factor from different species. Thrombos.Res.13,409-417, 1978. 9.SUSSMAN,J.J.and WEISS,H.J. Dissociation of factor VIII in the presence of proteolytic inhibitors. Thrombos.Haemostas.40, 316-325, 1978. IO.COOPER,H.A.,GRIGGS,T.S.and WAGNER,R.H. Factor VIII recombination after dissociation by CaCI2. Proc.Nat.Acad.Sci.U.S.A.70,2326-2329, 1973. ll.POON,M.Ch.and RATNOFF,O.D. Evidence that functional subunits of antihemophilic factor (F VIII) are linked by noncovalent bounds. Blood,48,87-94, 1976. 12.SUSSMAN,I.I.and WEISS,H.J. Spontaneous aggregation of low molecular weight factor VIII and its prevention by 2 mM CaCI2. Thrombos.Res.9,267-276,1976. 13.TRAN,T.H.,BOUNAMEAUX,H.,MARBET,G.A.and DUCKERT,F. Dissociation du facteur VIII humain et recombinaison des fragments VIII-C et VIII-AG. Schw.Med. Wschr.109,1029-1034,1979. 14.LANGDELL,R.D.,WAGNER,R.H.and BRINKHOUS,K.M. Effect of antihemophilic factor on one-stage clotting tests: A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J.Lab.Clin.Med.41,637-647, 1953.
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15.BIGGS,R. and DOUGLAS,A.S. The thromboplastin generation test . J.Clin. Pathol.1, 15-22, 1953. 16.LAURELL,C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem.15,45-52, 1966. 17.VUKOVICH,Th.,KOLLER,E. and DOLESCHEL,W. The influence of thrombin on the clotting a c t i v i t y of factor VIII. A study with insolubilized thrombin. Thrombos. Haemostas.39, 600-606, 1978. 18.BECK,E.A.,BACHMAN,P.,BARBIER,P. and FURLAN,A. Importance of protease inhibitor in studies on purified factor VIII (antihemophilic factor). Thrombos. Res.35, 186-195,1976. 19.DENSON,K.W.E. The simplified two-stage assay for factor V I I I . In: Human blood coagulation, hemostasis and thrombosis. R.BIGGS (Ed.) O x f o r d : ~ k w e l l Sc--c'i-e-ntific Publication. 2th ed3Tfon. 688, 1976. 20.ATICHARTAY~ARN,N.,MARDER,V.J.,KIRBY,E.P. and BUDZYNSKI,A.Z. Effects of enzymatic degradation on the subunit composition and biological properties of human factor VIII. Blood,51,281-297, 1978. 21.KIRKWOOD,T.B.L. and BARROWCLIFFE,T.W. Discrepances between one stage and two stage assay of factor VIII:C. Brit.J.Haematol.40, 333-338, 1978. 22.BARROWCLIFFE,T.W. and KIRKWOOD,T.B,L. An international collaborative assay of factor VIII clotting a c t i v i t y . Thrombos. Haemostas.40, 260-271, 1978. 23.ROCK,G.A.,PALMER,D.S.,TACKABERRY,E.S. and CRUICKSHANK,W.H. The presence of high and low molecular weight forms of factor VIII in heparinized plasma. Thrombos. Res. 13, 85-96, 1978.
LOW-MOLECULARWEIGHTFACTORVlll REQUIRESA COFACTORFOR ITS PROCOAGULANTACTIVITY Th. Vukovich,
E. K o l l e r
and W. Doleschel
Department of Medical Physiology, School of Medicine, University of Vienna, Schwarzspanierstr.17, A-I090 Vienna, Austria (Received 31.3.1980; in revised form 30.7.1980. Accepted by Editor G. MUller-Berghaus)
ABSTRACT In the course of production and quantitation of the procoagulant low-molecular weight factor VIII (LMW VIII:C) produced by dis sociation of purified high-molecular weight factor VIII in 0.25 M CaCI2, we observed that LMW VIII:C was active in only one of i t s two generally accepted test systems, namely the so-called "onestage" test. In the "two-stage" test, however, an additional highmolecular weight factor was required for LMW VIII:C to approximate i t s "one-stage" test level of a c t i v i t y . Investigation revealed that the high-molecular weight factor VIII antigen (VIIIR:Ag) provides this "cofactor" effect.
INTRODUCTION The high-molecular weight human factor VIII (HMW F V I I I ) isolated from blood anticoagulated by chelation of the plasma Ca++ ions is a large glycoprotein with a molecular weight higher than ixlO D Daltons (1). This macromolecule is responsible for two d i s t i n c t hemostatic a c t i v i t i e s , a procoagulant a c t i v i t y (VIII:C) and a so-called "von Willebrand factor" a c t i v i t y (VIIIR:WF) which supports the adhesion of thrombocytes to collagen-containing tissues (2). The rare phenomenon that two d i s t i n c t a c t i v i t i e s (VIII:C and VIIIR:WF) are located on one protein is biochemically and pathophysiologically interesting since defects in the structure of HMW F VIII can cause the coagulation disorders hemophilia A and / or von Willebrand's disease. A widely accepted model for the factor's molecular organisation is that of a complex of two comKey words: Factor V I I I , LMW factor VIII:C, Factor VIIIR:Ag, Dissociation 503
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ponents, one of high molecular weight carrying the s i t e for the von Will ebrand a c t i v i t y and a smaller component responsible for the f a c t o r ' s procoagulant a c t i v i t y (LMW V I I I : C ) . The larger component can be characterized by immunological means as antigen (VIIIR:Ag). This model is supported by the observation of several investigators (3-13) that HMW F VIII is eluted from gels with separation ranges of about 105-106 Daltons by the use of isotonic, Ca++-free buffers with all three a c t i v i t i e s (VIII:C, VIIIR:WF, VIIIR:Ag) occuring in the void volume fractions. Through the use of hypertonic solutions (0.25 M CaCI2 or I M NaCI), however, a separation of the two hemostatic a c t i v i t i e s can be achieved, that of VIIIR:WF found in the void volume and LMW VIII:C appearing in a l a t e r volume. A reaggregation occurs i f both fragments are mixed in isotonic, Ca++-free buffers (10-13). This communication reports our observation that LMW VIII:C, produced by dissociation of HMW F VIII in 0.25 M CaCl 2, is active only in one of the two generally accepted test systems for determination of VIII:C, namely the socalled "one-stage" test. In the "two-stage" test, however, an additional highmolecular weight factor was required for LMW VIII:C to approximate i t s "onestage" test level of a c t i v i t y . MATERIALS Human F V I I I of intermediate purity and human albumin (a); specific antihuman F V I I I serum, human F V l l l - d e f i c i e n t plasma, human F IX-deficient plasma, and phospholipid-kaolin reagent (b); human vWF-deficient plasma (c); p r o t e o l y t i c i n h i b i t o r Trasylol (d); heparin-Na and protamine-sulphate (e); Ultrogel AcA22 ( f ) ; and Sephadex G 25 (g) were obtained from the given outside sources. The human blood for preparation of cryoprecipitates was obtained from healthy male donors. (a) Schwab & Co., Vienna, Austria; (b) Behringwerke, Marburg, GFR; (c) g i f t from Prof. K. Lechner, Vienna; (d) Bayer, Leverkusen, GFR; (e) Novo, Copenhagen, Denmark; ( f ) LKB, France; (g) Pharmacia, Uppsala, Sweden. Buffer solutions: " T r i s - c i t r a t e - b u f f e r " : 0.13 M NaCl, 0.02 M Tris, 0.01M Na3-citrate plus I0 units/ml Trasylol and 1 mg/ml albumin, -pH 7.4 adjusted with HCI at 20oc. "Tris-CaCl2-buffer": 0.25 M CaCI2, 0.02 M Tris plus 25 units/ml heparin, I0 units/ml Trasylol and I mg/ml albumin, -pH 7.4 adjusted with HCI at 20°C. METHODS Preparation of HMW F V l l l HMW F V I I I was purified either from a solution of the commercial F V I I I material (containing 490 mg of total protein of which 160 mg were c l o t t a b l e , 250 units V I I I : C , and 950 units VIIIR:Ag in I0 ml solution) or from a solution of cryoprecipitate. The cryoprecipitate was obtained as follows: 450 ml blood were collected in 50 ml of a solution of 0 . I I M Na3-citrate containing I00 units/ml Trasylol. Following centrifugation at 2,500 x g and 20oc for 20 minutes, the supernatant plasma was frozen in I0 ml amounts and maintained at -70oc. For cryoprecipitation as needed, frozen samples were thawed and held in a water bath at 4oc over a period of 4 hours. The resultant precipitates were sedimented by centrifugation at 2,500 x g and 4°C for 20 minutes and redissolved in t r i s - c i t r a t e - b u f f e r to one-tenth of the original plasma volume to provide the solution used for further p u r i f i c a t i o n . The solution of i n t e r mediate purity F V I I I and that of the cryoprecipitate were chromatographed on a 30 cm long, t r i s - c i t r a t e - b u f f e r - e q u i l i b r a t e d Ultrogel AcA22 column 27 mm in diameter. 4 ml samples of both F V I I I solutions were applied to the top of the
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
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column and eluted with the t r i s - c i t r a t e - b u f f e r at a flow rate of 18 ml/hour at room temperature. The void volume fractions of these elutions, containing HMW F V I I I , were concentrated to a volume of 4 ml by d i a l y s i s for approximately 3 hours at 4°C against polyethylene glycol 20,000 and maintained at -70°C for subsequent use. Fractionation of HMW factor V I I I Before experimental use each 4 ml sample of frozen HMW F V I I I was thawed in a water bath at 37oc and mixed with 4 ~ I of a heparin solution containing 25,000 NIH units/ml. Addition of I I I mg of rapidly weighted, dehydrated CaCI2 to each of the heparinized samples (final concentration 0.25 M) effected the dissociation of the factor. Separation of the factor V I I I fragments To separate the F V I I I fragments derived from both F V I I I sources, the appropriate 4 ml samples were chromatographed on the Ultrogel column preequilibrated with the tris-CaCl2-buffer and eluted with the same buffer at a flow rate of 20 ml/hour at room temperature. The eluates were monitored for t h e i r UV absorbance a t ~ 256 nm and collected in 4 ml samples at 4°C. Free Caions were removed from these samples and the contained heparin neutralized as described below. Determination of V I I I : C Quantitative determinations of V I I I : C were performed with the "one-stage" test based on the technique of Langdell et al (14) and with the "two-stage" test o r i g i n a l l y proposed by Biggs and Douglas (15) in a modification of Aronson as follows: I pt. human serum diluted lO-fold with 0.066 M NaCl in 0.05 M imidazole buffer, pH 7.3; i pt. bovine serum diluted 20-fold with the same buffer mixture; I pt. 0.025 M CaCI9 solution; I pt. phospholipid-kaolin reagent; and i pt. test solution are~mixed and held for 5 minutes at 37oc. i pt. of this mixture is then mixed with I pt. of the CaCI2 solution and, at zero time, with I pt. of fresh human citrated-plasma. The coagulation time of the l a t t e r mixture is recorded. Two d i l u t i o n s of the V I I I : C samples without Al(OH)2-adsorption were tested in duplicate. Evaluation of the test samples' a c t i v i t i e s were made from a standard plasma d i l u t i o n curve. The test values of both sample d i l u t i o n s when calculated back to the a c t i v i t y of the original test sample agreed within I015% of each other. Prior to q u a n t i t a t i o n , those test samples containing 0.25 M CaCI 2 and heparin were treated as follows: 2 ml amounts of each sample were applied to Sephadex G 25 columns (0.5 x I I cm) preequilibrated with t r i s c i t r a t e - b u f f e r . To the Ca-ion-free void volume eluates s u f f i c i e n t protamine was added to neutralize the contained heparin (minimal c l o t t i n g time of the "one-stage" V I I I : C t e s t ) . Determination of VIIIR:Ag The q u a n t i t a t i v e determinations of VIIIR:Ag were made with rabbit antiserum specific for human F V I I I according to the electroimmunodiffusion method of Laurell (17). Standard plasma As reference plasma for the quantitation of V I I I : C and VIIIR:Ag from all sources a pool of 20 fresh, c i t r a t e d plasmas (9 pts. plasma + i pt. 0 . i i M Na3-citrate ) was frozen in 1 ml amounts and held at -70oc u n t i l use. The V I I I : C and VIIIR:Ag contents of once-thawed plasma were taken as I unit/ml.
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
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EXPERIMENTAL The l i t e r a t u r e holds divergent answers to the question whether a limited proteolysis of HMW F VIII is a prerequisite for the factor's dissociation in hypertonic salt solution. To minimize this complication we have used for our experiments HMW F VIII prepared from a commercial F VIII concentrate liable to possible proteinase a c t i v i t y during i t s preparation and our own HMW F VIII derived from cryoprecipitates of fresh plasma treated with proteinase i n h i b i t o r and subsequently purified in the presence of such inhibitors. When the chromatographic eluates of the CaCl2-fragmented HMW F VIII from both sources were tested for their UV absorbance, their VIIIR:Ag contents, and their VIII:C a c t i v i t i e s as determined by the "one-stage" method, the results obtained are those given in Fig. I. Inspection of the elution patterns from both F VIII preparations shows s i m i l a r i t y with respect to all three parameters. UV-absorbing material was present in void-volume (Vo) fractions and in t o t a l volume (Vt) eluates of both chromatograms. VIIIR:Ag was eluted exclusively in the Vo-eluates (recovery in 6 chromatographies 90-100% of that applied to the column) while VIII:C a c t i v i t i e s were displayed by fractions no 21 to 28 (recovery 25-50%) in the separation range of the given chromatographic column. The fact that in both chromatograms the VlllR:Ag-containing Vo-eluates were free of procoagulant a c t i v i t y and the LMW VIII:C-containing eluates were free of VIIIR:Ag indicates a complete dissociation of both fragments on the Ultrogel column. Since the HMW F VIII derived from a commercial source and F VIII prepared by us from fresh plasma to minimize proteolysis reacted similarly to treatment with hypertonic CaCI2, i t is improbable that proteolysis is a prerequisite for this factor's dissociation.
E[ 5
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F I G U R E 1 : Chromatograms of 0.25 M CaCl2-fragmented VIII:C U/ml ( 0 ) as determined by the one-stage test; for ( m ) ; and for the OD 256 nm of eluates from Ultrogel AcA22 tris-CaCl2-buffer. "A" = F VIII from cryoprecipitate. "B" commercial concentrate.
H M W F VIII for the the VIIIR:Ag U/ml equilibrated with = F VIII from
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
507
To determine possible discrepancies in the quantitation of LMW V I I I : C a c t i v i t y between the results of the one- and two-stage analytical t e s t , we tested the same eluate fractions of the chromatograms by means of the two-stage procedure. The results are given in Fig.2. No a c t i v i t y was dedectable in any f r a c t i o n despite the fact that the highest a c t i v i t y determined in the eluates with the one-stage t e s t was more than lO0-fold that of the s e n s i t i v i t y l i m i t of the two-stage test for "unfragmented" F V I I I . From these two-stage results we questioned whether the procoagulant a c t i v i t i e s in fractions no 21 to 28 of the chromatograms as shown by the one-stage test were F V l l l - s p e c i f i c , since the l a t t e r test is peculiarly sensitive to such "non-F V l l l - r e l a t e d " c l o t t i n g a c t i v i t i e s as, e.g. that of thrombin (17). To establish the F V l l l - s p e c i f i t y of the procoagulant a c t i v i t y in the given f r a c t i o n s , we tested them by the one-stage F IX assay which matches the one-stage V I I I : C test in i t s s e n s i t i ~ t y to "non-test-specific" coagulant a c t i v i t i e s . No such a c t i v i t y was detected by t h i s test (Fig.2). These results show that the procoagulant a c t i v i t i e s of fractions no 21-28 as determined by the one-stage V I I I : C test are indeed related to V I I I : C .
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P r o c o a g u l a n t a c t i v i t i e s o f the e l u a t e f r a c t i o n s as o n e - s t a g e V I I I : C test ( D / O ) , t w o - s t a g e V I I I : C test F I X test ( A ) . '~" = F V I I I f r o m c r y o p r e c i p i t a t e . c o m m e r c i a l concentrate.
(•),
To explain the i n a c t i v i t y of LMW V I I I : C in the two stage test we considered the fact that the reagents of the f i r s t step of t h i s t e s t are, in contrast to those of the one-stage t e s t , very poor in t h e i r content of VIIIR:Ag. To study whether VIIIR:Ag is required for LMW V I I I : C to effect c l o t promotion we tested the r e a c t i v i t y of LMW V I I I : C by the two-stage test in the presence of a VlllR:Ag-rich Vo solution obtained by pooling equal amounts of fractions no 13 to 16 from the given chromatograms. Mixtures of 0 . I ml of the Vo-pool and 0.I ml of each of the individual eluate fractions no I0 to 40 were prepared and analyzed. The results, given in Fig. 3, show that in the presence of Vo eluated substances LMW V I I I : C was demonstrable in the fractions no 21 to 28. The observed a c t i v i t y in these fractions was 60 % (mean of 6 chromatograms) of
508
VIII R: Ag "COFACTOR" FOR LMW VIII: C
Vo1.19, No.4/5
that quantified by means of the one-stage test.
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F I G U R E 3 : P r o c o a g u l a n t a c t i v i t i e s o f the eluate f r a c t i o n s as d e t e r m i n e d by the two-stage test a f t e r a d d i t i o n o f the V I I I R : A g - r i c h Vo-pool. "A" = F VIII f r o m cryoprecipitate. "B" = F VIII from commercial concentrate.
To determine whether VIIIR:Ag of the Vo eluate pool is functioning as a "cofactor" for the demonstrated LMW V I I I : C a c t i v i t y , we tested all fractions from the chromatograms as mixtures (9 pts. f r a c t i o n + I pt. given plasma) with hemophilic A plasma (VIIIR:Ag = 1.2 U/ml) against mixtures with vWF-deficient plasma (VIIIR:Ag = 0.04 U/ml) in the two-stage test. The results given in Fig. 4, show that LMW V I I I : C from fractions no 21 to 28 was reactive in the twostage test in the mixture with VlllR:Ag-rich hemophilic plasma but not in the mixture with vWF-deficient plasma. The observed a c t i v i t i e s of the fractions no 21 to 28 run with hemophilic plasma were, as in the previous experiments, 60 % of those found by the one-stage test. Since, apart from t h e i r content of VIIIR:Ag, the hemophilic A and the vWF-deficient plasmas are identical with respect to t h e i r c l o t t i n g components, these results indicate that the "cofactor" for LMW V I I I : C is indeed VIIIR:Ag. DISCUSSION Several authors have reported that in solutions of high s a l t concentration HMW F V I I I dissociates into a low-molecular weight, procoagulant fragment (LMW V I I I : C ) and a high-molecular weight, inactive fragment (3-13). These fragments reaggregate in isotonic Ca++-free buffer (10-13). I t thus appears that the components of HMW F V I I I are held together by non-covalent forces. This concept of the molecular organisation of HMW F V I I I has been challenged by the observation (18) that the fragmentation by high concentrations of s a l t was preventable by the addition of the proteolysis i n h i b i t o r aprotinin. From these results i t was postulated that a limited proteolysis of HMW F V I I I is a prer e q u i s i t e for the f a c t o r , s dissociation. However, more recent work has demonstrated (9) that fragmentation of HMW F V I I I does occur under such s a l t conditions even in the presence of a v a r i e t y of p r o t e o l y t i c i n h i b i t o r s (benzamidine, soybean t r y p s i n i n h i b i t o r , heparin, diisopropylfluorophosphate,
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VIII R: Ag "COFACTOR" FOR LMW VIII: C
509
and aprotinin). Since in our experiments the HMW F VIII prepared from fresh, proteinase-inhibited plasma was fragmented by 0.25 M CaCI2 in the presence of aprotinin and heparin, we favor the view that proteolysis is not a prerequi~te for the factor,s fragmentation.
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F I G U R E 4 : Procoagulant activities of the eluate fractions as determined by the two-stage test after addition of either "cofactor"-rich,hemophilic-A plasma ( D / • ) or "cofactor"-poor, vWF-deficient plasma ( A ) . '~" = F VIII from cryoprecipitate. "B" = F VIII from commercial concentrate.
The requirement of VIIIR:Ag acting as a "cofactor" for the expression of the procoagulant a c t i v i t y of LMW VIII:C is the most important finding of our study. Our results indicate that both procoagulant inactive VIIIR:Ag forms, namely the inactive high salt HMW fragment and the hemophilic-A-F V I I I , act as "cofactor" for LMW VIII:C. That such a cofactor requirement was not previously recognized is understable since the LMW VlII:C a c t i v i t y was determined almost exclusively by means of the VlllR:Ag-containing one-stage test. In the only exception (13), Denson's modification (19) of the two-stage test was employed. The reagents of Denson's modification and those of Aronson's d i f f e r in that the l a t t e r ' s contains, among i t s f i r s t - s t a g e reagents, VIIIR:Ag derived from human and bovine sera in an amount of 0.03 U/ml final reaction mixture, while those employed by Denson are derived from plasma. Since thrombin-treatment of HMW F VIII increases the electrophoretic mobility of VIIIR:Ag (20) i t is probable that VIIIR:Ag from serum has been modified during coagulation as against that from plasma to yield an inactivated "cofactor". Contrarily, the plasma-derived reagents may well be contaminated with VIIIR:Ag able to act as "cofactor" with LMW VIII:C. Discrepancies between the results of quantitation of HMW VIII:C in purified form when compared with the a c t i v i t y of a standard plasma by either the oneor the two-stage test are commonly observed (21,22). One explanation for this phenomenon is "that different molecular forms of F VIII may react d i f f e r e n t l y in the two assay systems" (21). For example, i f LMW VIII:C contained in normal plasma (23) requires VIIIR:Ag as "cofactor" for clot promotion, as demonstrated for the high salt fragmented LMW VIII:C, the discrepancies between the results of both tests may be explained by the presence or relative absence of the "cofactor" in the reagents of these tests.
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ACKNOWLEDGEMENTS The most valuable criticism and suggestions by A.L. Schade, Ph.D. are thankf u l l y acknowledged. This investigation was supported by a grant from the Austrian Foundation for Promotion of Scientific Research (Proj. Nr. 4019). REFERENCES I.LEGAZ,M.E.,SCHMER,G.,COUNTS,R.B. and DAVIE,E.W.Isolation and characterization of human F VIII (Antihemophilic Factor). J.Biol.Chem.218,3946-3955,1973. 2.BAUMGARTNER,H.R.and MUGGLI,R.Adhesion and aggregation: morphological demonstration and quantitation in vivo and in vitro. In: Platelets in biology and pathology. J.I.GORDON (Ed.).Amsterdam: North-Holland pub-Tishing Company,23-33,1976. 3.WEISS,H.J.,PHILLIPS,L.L.and ROSNER,W.Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography.Thrombos.Diathes.Haemorrh.27,212219, 1972. 4.0WEN,W.and WAGNER,R.H.Antihemophilic factor: Separation of an active fragment following dissociation by salts or detergents. Thrombos.Diathes.Haemorrh.27, 502-515, 1972. 5.RICK,M.E. and HOYER,L.W.Immunologic studies of antihemophilic factor (AHF, factor V I I I ) . Immunologic properties of AHF subunits produced by salt dissotiation. Blood.42,737-747, 1973. 6.COOPER,H.A.,REISNER,F.F.,HALL,M.and WAGNER,R.H. Effects of thrombin treatment on preparations of factor VIII and the Ca++-disso~iated small active fragment. J.Clin.lnvest.56,751-760,1975. 7.BOLHUIS,P.A.,BEESER-VISSER,N.H.and SIXMA,J.J. Molecular weight of human plasma factor VIII. Thrombos.Res.16,497-509, 1979. 8.LAVERGNE,J.M.,MEYER,D.,JENKINS,C.S.,OBERT,B.and LARRIEU,M.J.Influence of high ionic strength buffers on factor Vlll/von Willebrand factor from different species. Thrombos.Res.13,409-417, 1978. 9.SUSSMAN,J.J.and WEISS,H.J. Dissociation of factor VIII in the presence of proteolytic inhibitors. Thrombos.Haemostas.40, 316-325, 1978. IO.COOPER,H.A.,GRIGGS,T.S.and WAGNER,R.H. Factor VIII recombination after dissociation by CaCI2. Proc.Nat.Acad.Sci.U.S.A.70,2326-2329, 1973. ll.POON,M.Ch.and RATNOFF,O.D. Evidence that functional subunits of antihemophilic factor (F VIII) are linked by noncovalent bounds. Blood,48,87-94, 1976. 12.SUSSMAN,I.I.and WEISS,H.J. Spontaneous aggregation of low molecular weight factor VIII and its prevention by 2 mM CaCI2. Thrombos.Res.9,267-276,1976. 13.TRAN,T.H.,BOUNAMEAUX,H.,MARBET,G.A.and DUCKERT,F. Dissociation du facteur VIII humain et recombinaison des fragments VIII-C et VIII-AG. Schw.Med. Wschr.109,1029-1034,1979. 14.LANGDELL,R.D.,WAGNER,R.H.and BRINKHOUS,K.M. Effect of antihemophilic factor on one-stage clotting tests: A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J.Lab.Clin.Med.41,637-647, 1953.
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15.BIGGS,R. and DOUGLAS,A.S. The thromboplastin generation test . J.Clin. Pathol.1, 15-22, 1953. 16.LAURELL,C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem.15,45-52, 1966. 17.VUKOVICH,Th.,KOLLER,E. and DOLESCHEL,W. The influence of thrombin on the clotting a c t i v i t y of factor VIII. A study with insolubilized thrombin. Thrombos. Haemostas.39, 600-606, 1978. 18.BECK,E.A.,BACHMAN,P.,BARBIER,P. and FURLAN,A. Importance of protease inhibitor in studies on purified factor VIII (antihemophilic factor). Thrombos. Res.35, 186-195,1976. 19.DENSON,K.W.E. The simplified two-stage assay for factor V I I I . In: Human blood coagulation, hemostasis and thrombosis. R.BIGGS (Ed.) O x f o r d : ~ k w e l l Sc--c'i-e-ntific Publication. 2th ed3Tfon. 688, 1976. 20.ATICHARTAY~ARN,N.,MARDER,V.J.,KIRBY,E.P. and BUDZYNSKI,A.Z. Effects of enzymatic degradation on the subunit composition and biological properties of human factor VIII. Blood,51,281-297, 1978. 21.KIRKWOOD,T.B.L. and BARROWCLIFFE,T.W. Discrepances between one stage and two stage assay of factor VIII:C. Brit.J.Haematol.40, 333-338, 1978. 22.BARROWCLIFFE,T.W. and KIRKWOOD,T.B,L. An international collaborative assay of factor VIII clotting a c t i v i t y . Thrombos. Haemostas.40, 260-271, 1978. 23.ROCK,G.A.,PALMER,D.S.,TACKABERRY,E.S. and CRUICKSHANK,W.H. The presence of high and low molecular weight forms of factor VIII in heparinized plasma. Thrombos. Res. 13, 85-96, 1978.