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The mechanism of activation of human prothrombin by an activator isolated from Dispholidus typus venom
160
Biochimica et Biophsyica Acta, 537 (1978) 160--168 © Elsevier/North-Holland Biomedical Press
BBA 38029
THE MECHANISM OF ACTIVATION OF HUMAN PROTHROMBIN BY AN ACTIVATOR ISOLATED FROM DISPHOLIDUS TYPUS VENOM
M A R I E - C L A U D E GUILLIN, ANNIE B E Z E A U D and DORIS M E N A C H E Service Central d'Immunologie et Hdmatologie, U E R X. Bichat, I-I6pitalBeaujon, 92110 Clichy (France)
(Received May 25th, 1978)
Summary Purified human prothrombin was activated, both in the absence and in the presence of thrombin inhibitors (diisopropylfluorophosphate or hirudin), by a coagulant principle isolated from Dispholidus typus venom. The process of activation was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the absence of thrombin inhibitor, prolonged incubation of prothrombin with the purified venom yielded thrombin, fragment 1 (F 1) and fragment 2 (F 2). In the presence of diisopropylfluorophosphate, which in the experimental conditions used inhibited only partially the thrombin generated activity, products obtained upon activation of prothrombin by the venom were F 1 and a two~hain, disulfide-bridged protein of 58 000 daltons called meizothrombin (des F 1). In the presence of hirudin, which fully inhibited thrombin generated activity, prothrombin activation by the venom did not liberate any fragment, but prothrombin was converted to a derivative composed of two disulfide-bridged polypeptide chains of 48 000 and 37 000 daltons, called meizothrombin. These results are similar to those reported by others when studying the process of prothrombin activation by Echis carinatus venom and allow to conclude that Dispholidus typus venom cleaves a bond linking the A and B chains of thrombin, converting prothrombin into meizothrombin. This enzyme is then responsible for the cleavage of the bond linking F 1 and F 2 and the bond linking F 2 and the A chain of thrombin.
Introduction Several reports contain evidence of disseminated intra vascular coagulation following boomslang bite [1--3]. The boomslang (Dispholidus typus) is a snake Abbreviation:
SDS, sodium d o d e c y l sulfate.
161 from South Africa; its venom appears to be able to activate prothrombin [2] in plasma and has been used to assay purified prothrombin and purified acarboxyprothrombin [4]. Several snake venoms can activate prothrombin, including those of the taipan snake Oxyuranus scutellatus scutellatus [5,6], the sawscaled viper Echis carinatus [7--9] and the mainland tiger snake Notechis scutatus scutatus [10]. The process of activation of prothrombin by Talpan snake venom [6] and by tiger snake venom [10] has been shown to be similar to the activation of prothrombin by activated factor X (Xa), whereas the process of activation promoted by Echis carinatus venom has been shown to be different [ 7 - 9 ] . Little is known about the process of activation of prothrombin by Dispholidus typus venom; the purpose of this paper is to describe the activation mechanism of human prothrombin by the active c o m p o n e n t isolated from Dispholidus typus venom. Terminology The nomenclature used for prothrombin and prothrombin activation products is that recommended by the International Committee on Thrombosis and Haemostasis [11]. Proteolytic cleavage sites are designated bonds 1, 2, 3 starting from the aminoterminus. Fragment 1 (F 1) is a peptide that arises from the amino terminal region of prothrombin by cleavage at bond 1. The remaining part of the molecule is termed prethrombin 1. Fragment 2 (F 2) is an internal peptide formed by cleavage at bonds 1 and 2. When F 1 and F 2 remain covalently linked as a single peptide they are referred to as fragment 1.2 (F 1.2). Prethrombin 2 is that region of prothrombin from which the F 1 and F 2 regions have been cleaved. Thrombin is obtained from prethrombin 2 by cleavage at bond 3 and is constituted of two polypeptide chains (A and B chains) disulfide bridged. Materialsand Methods Normal prothrombin was prepared from human pooled fresh frozen acid/ citrate/dextrose plasma according to Devilee et al. [12]; 1 mM benzamidineHC1 was added in all buffers. The various preparations had a prothrombin activity ranging from 1500 to 1700 U.S. units/rag when measured by a two stage assay in the presence of physiological activators [13,14]. Prethrombin 1 was obtained by incubating, 4 h at 22°C, prothrombin (14 mg in 7 ml 0.1 M NaC1 1 mM benzamidine-HC1/0.01 M sodium + potassium phosphate buffer pH 6.8) with human thrombin (350 U.S. units of lot H1, kindly supplied by Dr. K. Miller, University of Miami, School of Medicine, Miami, Fla.), in the presence of soybean trypsin inhibitor (0.1 mg/ml final concentration). Proteolysis was stopped by the addition of 2 mM diisopropylfluorophosphate (from Sigma Chemical Co.) and the mixture was subjected to chromatography on DEAE-Sephadex A 50 (column 2.5 × 5 cm) equilibrated in 0.1 M NaC1 1 mM benzamidine-HC1]0.01 M phosphate buffer, pH 6.8. After washing the column with the equilibrating buffer (200 ml), a linear gradient of 0.1 M NaC1 1 mM benzamidine-HCl/0.01 M phosphate to 0.5 M NaC1 1 mM benzamidine-HC1/0.01 M phosphate (2 X 250 ml) was applied at a constant pH
162
of 6.8. Fractions of 2 ml were collected at a flow rate of 90 ml/h. The first peak eluted contained prethrombin 1, as assessed b y sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (molecular weight 58 000). Crude venom of Dispholidus typus, purchased from Sigma Chemical Co., was fractionated by chromatography onto DEAE cellulose. 60 mg of whole venom were dissolved in 3 ml of distilled water, dialyzed against 0.05 M. NaC1/0.02 M Tris-HC1, pH 7.5 and loaded onto a column (1.5 X 17 cm) of DE 52 Whatman, equilibrated in the same buffer. After washing the column with the equilibrating buffer, a linear gradient of 0.05 M NaCl/0.02 M Tris-HC1 to 0.15 M NaC1/ 0.02 M Tris-HCl was applied at a constant pH of 7.5. Fractions (3 ml) were collected at a flow rate of 30 ml/h and assayed for coagulant activity b y adding a 100 /11 aliquot to 100 gl of normal citrated plasma and recording the clotting times. Fractions exhibiting a clotting time of 12 s or less were pooled, lyophilized, dissolved in distilled water and dialyzed against 0.1 M NaC1/0.02 M TrisHC1, pH 7.5. A sample (1.6 ml, A280: 1.4) was loaded o n t o a column (1.5 X 90 cm) of Ultrogel AcA 44 (LKB) equilibrated in 0.1 M NaC1/0.02 M Tris-HC1, pH 7.5 and developped with the same buffer. Fractions (3 ml) were collected at a flow rate of 60 ml/h; those exhibiting a clotting time of 25 s or less were pooled and lyophilized. The coagulant principle isolated from Dispholidus typus venom migrated as a single c o m p o n e n t in SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 58 000 and consists in a single polypeptide chain since its mobility was unchanged upon reduction. It exhibited no significant fibrinolytic activity since the lysis time of a fibrin clot incubated with the purified venom exceeded 48 h. Gel electrophoresis in the presence of SDS was performed according to Weber and Osborn [15] in 10% acrylamide, 0.1% SDS. A current of 8 mA per gel was applied for 3 h. Gels were stained with Coomassie blue [15] and destained electrophoretically. Each gel was scanned at 540 nm in a Gilford Model 240 S p e c t r o p h o t o m e t e r equiped with a Model 2410 linear transport accessory. Mobility of each protein was determined from the densitometry tracing and its molecular weight calculated from a linear semi logarithmic plot of molecular weight versus mobility, drawn with four standard proteins (bovine serum albumin, chymotrypsinogen, soybean trypsin inhibitor and lysozyme). Thrombin activity was measured by adding 0.1 ml of diluted sample to 0.2 ml of 0.25% human purified fibrinogen [16] and expressed as U.S. thrombin units using human thrombin (lot B3, kindly supplied by Dr. D.L. Aronson, Bureau of Biologics, F o o d and Drug Administration, Bethesda, Md.) for the calibration curve. Experiments and Results
Proteolysis of human prothrombin or prethrombin 1 by the coagulant principle isolated from Dispholidus typus venom was studied both with or without the addition of either diisopropylfluorophosphate or hirudin (Pentapharm; specific activity: 1500 antithrombin units/mg protein). When added, diisopropylfluorophosphate was usc~l at a final concentration of 10 mM and hirudin was used at a concentration o f 2000 antithrombin units/mg prothrombin. After various incubation times, aliquots were removed for thrombin activity assay and SDS polyacrylamide gel electrophoresis.
163
The addition of the purified venom to prothrombin led to the generation of significant amounts of thrombin activity (Fig. la). Maximum conversion was reached in 2 h. The amount of thrombin generated was similar to that generated in the presence of physiological activators as measured by two stage assay (1600 versus 1700 U.S. units/mg prothrombin). The rate of prethrombin 1 activation was the same as that of prothrombin (Fig. lb). SDS polyacrylamide gel electrophoresis analysis of the venom and prothrombin mixture is shown on Fig. 2. After prolonged incubation (2 h or more), the fragments obtained seem similar to those obtained upon prothrombin activation by factor Xa: unreduced samples showed (Fig. 2a) 3 fragments with molecular weights of 41 000, 28 000 and 17 000, corresponding respectively to the molecular weights of thrombin, F 1 and F 2 [17]. Upon reduction, the band, corresponding to F 2 (mol. wt. 17 000) remained unchanged. The band corresponding to thrombin (tool. wt. 41 000) disappeared and was replaced by a band with molecular weight 37 000 corresponding to the thrombin B chain. The intensity of the F 1 band (mol. wt. 28 000) decreased and two new bands appeared with molecular weights 22 000 and 10 000. According to Franza et al. [7], the 28 000 species correspond to the one chain F 1, the 22 000 and 10 000 species correspond to the two chains of F 1 which has been split by thrombin (respectively subfragment/3 and subfragment ~ ). When the incubation was limited to 30 min, the electrophoretic pattern was different. Unreduced samples (Fig. 2a, 2nd gel) showed 3 fragments with molecular weights 58 000, 41 000 and 28 000, corresponding respectively to the molecular weights of prethrombin 1, thrombin and F 1 [17]. Fragment 2 (moh wt. 17 000) cannot be seen on the photograph but was present in trace amounts as assessed by densitometry scan. Upon reduction (Fig. 2b, 2nd gel), the F 1 band (mol. wt. 28 000) remained unchanged. Both the 58 000 daltons species and the thrombin band (mol. wt. 41 000) disappeared and two new species of molecular weights 37 000 and 22 000 became apparent. The 37 000 daltons species is apparently derived (according to the intensity of the band) from both the thrombin band and the 58 000 daltons species seen on unreduced samples: its molecular weight corresponds to that of the thrombin B chain. In order to determine whether the 22 000 daltons species was derived from F 1 or from the prethrombin 1 region of prothrombin, purified prethrom-
ilb 5
30
60 l~qD 240 incubation time ( m i n u t e s )
5
30
60 120 incubation time
240 (minutes)
Fig. 1. A c t i v a t i o n o f p r o t h r o m b i n a n d p r e t h r o m b i n 1 b y t h e c o a ~ ] a n t p r i n c i p l e i s o l a t e d f r o m Dispho|idus t y p u ~ v e n o m . P u r i f i e d v e n o m (1 #$) w a s a d d e d t o (a) p r o t h r o m b i n o r (b) p r e t h r o m b i n 1 (1 m g i n 0 . 5 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l / 0 . 0 2 M T r i s - H C l , p H 7 . 5 , i n b o t h eases). T h r o m b i n a c t i v i t y w a s meast~red a f t e r v a r i o u s i n c u b a t i o n t i m e s a n d e x p r e s s e d as U . S . t h r o m b i n u n i t s g e n e r a t e d per m g p r o t h r o m b i n o r p r e t h r o m b i n 1.
164
a
b
72, =
_72.000
58,q
- 58[000
41,~ -37,000 ~28,000
28
.22,00D 17
~17,000 -10,000 0
30
120
240
+
0
30
120
240
÷
Fig. 2. S D S polyacrylamide gel electrophoresis of h u m a n prothrombin incubated with purifiedDispholidus typus venom. 1 pg of v e n o m was incubated with prothrombin (1 m g in 0.5 ml 0.1 M NaCI 1 m M benzamidine-HCl/0.02 M Tris-HCl, p H 7.5). After various incubation times at 22°C (indicatedin minutes at the bottom of the gels),samples were prepared by dilutingan aliquot of the incubation mixture in (a) 0.01 M sodium phosphate, p H 7, 1 % S D S (unreduced samples) or (b) 0.01 M sodium phosphate p H 7, 1 % S D S 1 % 2-mercaptoethanol. Samples loaded were in the range of 30 to 50 /~g protein.
bin 1 was incubated with the venom and proteolysis products analysed by SDS polyacrylamide gel electrophoresis (Fig. 3). Unreduced samples showed (Fig. 3a) no molecular weight change when compared to prethrombin 1 before addition of the venom. Reduction resulted (Fig. 3b) in the appearance of two components of molecular weights 37 000 and 22 000. The 37 000 dalton species correspond to the size of the thrombin B chain whereas the 22 000 dalton species must correspond to the rest of prethrombin 1 (F 2 plus thrombin A chain).
a
b
F i g . 3. S D S P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r e t h r o m b i n 1 i n c u b a t e d w i t h p u r i f i e d Diepholidus typus v e n o m . 0 . 7 ~ g o f v e n o m w a s i n c u b a t e d w i t h p r e t h r o m b i n 1 ( 0 . 7 m g in 0 . 3 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l / 0 . 0 2 M Trls-HCI, p H 7 . 5 ) . A f t e r 5 a n d S 0 r a i n i n c u b a t i o n a t 2 2 ° C ( i n c u b a t i o n t i m e s i n d i c a t e d a t t h e b o t t o m o f t h e gels), u n r e d u c e d (a) a n d r e d u c e d (b) s a m p l e s ( 5 0 # g ) w e r e s u b j e c t e d t o S D S gel e l e c t r o p h o r e s l s .
165
These results allow to conclude that the major transient intermediate product of molecular weight 58 000 obtained upon a 30 rain incubation of prothrombin with the venom is composed of 2 polypeptide chains of molecular weights 37 000 (thrombin B chain) and 22 000 (F 2 + thrombin A chain). In order to elucidate whether the appearance of both F 1 and F 2, after prolonged incubation of prothrombin with the venom, was due to proteolysis of prothrombin by the venom or was due to the thrombin generated during the activation process, the experiment was performed in the presence of a thrombin inhibitor, diisopropylfluorophosphate. After 2 h incubation, unreduced samples (Fig. 4a) showed two fragments of molecular weights 58 000 and 28 000. Upon reduction, the 28 000 dalton species persisted (Fig. 4b), whereas the 58 000 dalton species disappeared and two new bands with molecular weights of 37 000 and 22 000 appeared. According to the molecular weights of the products, it was concluded that in the presence of diisopropylfluorophosphate, activation of prothrombin by the venom yields F 1 (mol. wt. 28 000) and a fragment of molecular weight 58 000 consisting of a two-chain disulfidebridged protein. After prolonged incubation (4 h) two additional fragments were seen, with molecular weights 41 000 (thrombin) and 17 000 (F 2). These results prompted to check whether the thrombin generated in the mixture was completely inhibited. After 4 h incubation, despite the high concentration of diisopropylfluorophosphate added (10 mM), the enzyme was still measurable (70 thrombin U.S. units/mg prothrombin). The experiment was thus repeated in the presence of a more potent inhibitor: hirudin. In the presence of hirudin (2000 antithrombin units/mg prothrombin), no thrombin activity was measurable even after 4 h incubation and unreduced samples showed on SDS polyacrylamide gel electrophoresis (Fig. 5a) no molecular weight change of prothrombin. Reduction resulted (Fig. 5b) in almost complete disappearance of the 72 000 dalton species whereas two major products of molecular weights 48 000 and 37 000 became apparent. The 37 000 dalton species correspond to the thrombin B chain whereas the molecular weight of
a
b
72,000"* 58,00G--
41,000"28,000-"
w
i=o
24o 4-
F i g . 4. S D S P o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r o t h r o m b i n i n c u b a t e d w i t h p u r i f i e d D~pholldus typus v e n o m , i n t h e p r e s e n c e o f d l i s o p r o p y l f l u o r o p h o s P h a t e . 1 ~zg o f v e n o m w a s i n c u b a t e d w i t h p r o t h r o m b i n (1 m g i n 0 . 5 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l 1 0 m M d i i s o p r o p y l f l u o r o p h o s p h a t e / 0 . 0 2 M T r i s - H C l , p H 7 . 5 ) . A f t e r v a r i o u s i n c u b a t i o n t i m e s ( i n d i c a t e d i n m i n u t e s a t t h e b o t t o m o f t h e gels), u n r e d u c e d ( a ) a n d r e d u c e d (b) s a m p l e s ( 2 0 t o 4 0 # g ) w e r e s u b j e c t e d t o S D S gel e l e e t r o p h o r a s i s .
166
a
b 72.000
72.000
.-48.000 ~ 37.000
0
30
120
240
+
0
30
120
240
.
Fig. 5. S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r o t h r o m b i n i n c u b a t e d w i t h p u r i f i e d Dispholidus typus v e n o m , in t h e p r e s e n c e o f h i r u d i n . 1 ~ g o f v e n o m w a s i n c u b a t e d w i t h p r o t h r o m b i n (1 m g in 0 . 5 m l 0.1 M NaC1 1 m M b e n z a m i d i n e - H C l / O . 0 2 M T r i s - H C l , p H 7 . 5 . c o n t a i n i n g 2 0 0 0 a n t i t h r o m b i n u n i t s o f h i r u d i n ) . A f t e r v a r i o u s i n c u b a t i o n t i m e s ( i n d i c a t e d in m i n u t e s a t t h e b o t t o m o f t h e gels), u n r e d u c e d (a) a n d r e d u c e d (b) s a m p l e s ( 2 0 t o 5 0 /~g) w e r e s u b j e c t e d t o S D S gel e l e c t r o p h o r e s l s .
the 48 000 species agrees well with the sum of the molecular weights of F 1, F 2 and the thrombin A chain. Thus, in the presence of hirudin, the prothrombin derivative formed upon the action of Dispholidus typus venom consists of a two chain disulfide-bridged protein; F 1 and F 2 remain attached to the parent protein. Discussion
The mechanism of conversion of purified prothrombin upon activation by factor Xa is well known [17--26]: in a first step, Xa cleaves off the aminoterminal fragment, fragment 1.2, to give rise to prethrombin 2; in a second step, Xa cleaves a peptide bond on prethrombin 2 to yield the two-polypeptide chains thrombin molecule; thrombin, in its turn, catalyses the scission of fragment 1.2 into fragment 1 (F 1) and fragment 2 (F 2). Although other activation pathways are possible, the ultimate products are thrombin, the inner fragment F 2 and the amino-terminal fragment F 1. Prothrombin activation by Dispholidus typus venom has previously been reported not to be dependent upon the presence of calcium ions [2]. In agreement with these data, the activation rate of prothrombin and prethrombin 1 upon the action of an activator isolated from Dispholidus typus venom have been found similar, indicating that the calcium binding region (F 1) of prothrombin is not involved in this activation process. This implies that Dispholidus typus venom, like Echis carinatus venom, may be a useful tool in assay of acarboxyprothrombin which lacks calcium-binding sites in the F 1 region of the molecule. The results obtained in. this study, when investigating by SDS polyacrylamide gel electrophoresis the activation process of human prothrombin upon the action of the venom, in the absence of thrombin inhibitor, are similar to those obtained by others when studying the activation of human [7] or bovine
167 [8,9] prothrombin by Echis carinatus venom. The ultimate products obtained are thrombin, F 2 and F 1. However, when the prothrombin activation proceeds in the presence of hirudin (which inhibits thrombin), the results indicate that the pathway of prothrombin activation is clearly different from that following Xa activation. Although the amino acid composition and the esterase activity of the intermediate products have not been determined in this study, the similarity of the fragmentation patterns observed on SDS polyacrylamide gels in the case of prothrombin activated by Dispholidus typus venom and in the case of prothrombin activated by Echis carinatus venom [9], allows the conclusion that the activation process is the same when using the two venoms. Indeed, Dispholidus typus venom, like Echis carinatus venom, cleaves the bond linking thrombin A and B chains (bond 3), giving rise to an active molecule, called meizothrombin [11]. Only in the absence of hirudin is meizothrombin able to catalyse the scission of two peptide bonds, one linking F 1 and F 2 (bond 1), and one linking F 2 and the thrombin A chain (bond 2). In this process, susceptibility of bond 1 is greater than that of bond 2 since, when prothrombin is incubated with the venom for a relatively short period of time (30 min), a transient activation product is yielded (in which bond 2 is not cleaved), corresponding to meizothrombin (des F 1) and consisting of the F 2 and A thrombin chain moieties disulfide-bridged to the B thrombin chain. Acknowledgements This work was supported by grants from Institut National de la Sant~ et de la Recherche M~dicale, CRL no. 77 2 137 7 and UER Xavier Bichat, Universit~ Paris VII, Paris (France). References 1 Spies, S.K., Malherbe, L.F. and Pepler, W.J. (1962) S. AfT. Med. J. 36,834--838 2 MacKay, N., Ferguson, J.C., Bagshawe, A., Forrester, A. and McNicol, G.P. (1969) Thromb. Diath. Haemorrh. 2 1 , 2 3 4 - - 2 4 4 3 Lakier, J.B. and Fritz, V.U. (1969) S. Aft. Med. J. 43, 1052~1055 4 Neisestuen, G.L. and Suttie, J.W. (1972) J. Biol. Chem. 247, 8176--8182 5 Denson, K.W.E., Borrett, R. and Biggs, R. (1971) Br. J. Haematol. 21,219--226 6 0 w e n , W.G. and Jackson, C.M. (1973) Thromb. Res. 3,705--714 7 Franza, B.R., Aronson, D.L. and Finlayson, J.S. (1975) J. Biol. Chem. 250, 7057--7068 8 Kornalik, F. and Blomb~ck, B. (1975) Thromb. Res. 6, 53--63 9 Morita, T., Iwanaga, S. and Suzuki, T. (1978) J. Biochem. 79, 1089--1108 10 Mann, K.G., Heldebrant, C.M., Bajaj, S.P., Fass, D.N. and But~owski, R.J. (1973) IVth International Congress on Thrombosis and Haemostasis, p. 469, Vienna, Austria, Abstract 11 Jackson, C.M. (1977) Thromb. Haemostas. 38, 567--577 12 Devilee, P.P., Hemker, H.C. and Bas, B.M. (1975) Biochim. Biophys. Acta 379,172--179 13 Ware, A.G. and Seegers, W.H. (1949) Am. J. CIin. Pathol. 19,471--482 14 Cesbron, N., Boyer, C., Guillin, M.C. and Menaehe, D. (1973) Thromb. Diath. Haemorrh. 30, 437--450 15 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 16 Kazal, L.A., Amsel, S. and Miller, O.P. (1963) Proc. Soc. Exp. Biol. Med. 113, 989--994 17 Kisiel, W. and Hanahan, D.J. (1973) Biochim. Biophys. Acta 3 2 9 , 2 2 1 - - 2 3 2 18 Lanchantin, G.F., Friedmann, J.A. and Hart, D.W. (1968) J. Biol. Chem. 243, 476--486 19 Stenn, K.S. and Blout, E.R. (1972) Biochemistry 11, 4502--4515 20 Gitel, S.N., Owen, W.G., Esmon, C.T. and Jackson, C.M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1344--1348 21 Heldebrant, C.M., Butkowski, R.J., Bajaj, S.P. and Mann, K.G. (1973) J. Biol. Chem. 248, 7149--7163
168 22 23 24 25 26
Esmon, C.T,, Owen, W.G. and Jackson, C.M. (1974) J. Biol. Chem. 249. 606--611 Kisiel, W. and Hanahan, D.J. (1974) Biochem. Biophys. Res. Commun. 59,570--577 Morita, T., Nishibe, H.. Iwanaga, S. and Suzuki, T. (1974) J. Biochem. 76, 1031--1048 Owen, W.G., Esmon, C.T. and Jackson, C,M. (1974) J. Biol. Chem. 249, 594---605 Seegers, W.H., Walz, D.A., Reuterby, J. and McCoy, L.E, (1974) Thromb. Res. 5, 829~860
Biochimica et Biophsyica Acta, 537 (1978) 160--168 © Elsevier/North-Holland Biomedical Press
BBA 38029
THE MECHANISM OF ACTIVATION OF HUMAN PROTHROMBIN BY AN ACTIVATOR ISOLATED FROM DISPHOLIDUS TYPUS VENOM
M A R I E - C L A U D E GUILLIN, ANNIE B E Z E A U D and DORIS M E N A C H E Service Central d'Immunologie et Hdmatologie, U E R X. Bichat, I-I6pitalBeaujon, 92110 Clichy (France)
(Received May 25th, 1978)
Summary Purified human prothrombin was activated, both in the absence and in the presence of thrombin inhibitors (diisopropylfluorophosphate or hirudin), by a coagulant principle isolated from Dispholidus typus venom. The process of activation was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the absence of thrombin inhibitor, prolonged incubation of prothrombin with the purified venom yielded thrombin, fragment 1 (F 1) and fragment 2 (F 2). In the presence of diisopropylfluorophosphate, which in the experimental conditions used inhibited only partially the thrombin generated activity, products obtained upon activation of prothrombin by the venom were F 1 and a two~hain, disulfide-bridged protein of 58 000 daltons called meizothrombin (des F 1). In the presence of hirudin, which fully inhibited thrombin generated activity, prothrombin activation by the venom did not liberate any fragment, but prothrombin was converted to a derivative composed of two disulfide-bridged polypeptide chains of 48 000 and 37 000 daltons, called meizothrombin. These results are similar to those reported by others when studying the process of prothrombin activation by Echis carinatus venom and allow to conclude that Dispholidus typus venom cleaves a bond linking the A and B chains of thrombin, converting prothrombin into meizothrombin. This enzyme is then responsible for the cleavage of the bond linking F 1 and F 2 and the bond linking F 2 and the A chain of thrombin.
Introduction Several reports contain evidence of disseminated intra vascular coagulation following boomslang bite [1--3]. The boomslang (Dispholidus typus) is a snake Abbreviation:
SDS, sodium d o d e c y l sulfate.
161 from South Africa; its venom appears to be able to activate prothrombin [2] in plasma and has been used to assay purified prothrombin and purified acarboxyprothrombin [4]. Several snake venoms can activate prothrombin, including those of the taipan snake Oxyuranus scutellatus scutellatus [5,6], the sawscaled viper Echis carinatus [7--9] and the mainland tiger snake Notechis scutatus scutatus [10]. The process of activation of prothrombin by Talpan snake venom [6] and by tiger snake venom [10] has been shown to be similar to the activation of prothrombin by activated factor X (Xa), whereas the process of activation promoted by Echis carinatus venom has been shown to be different [ 7 - 9 ] . Little is known about the process of activation of prothrombin by Dispholidus typus venom; the purpose of this paper is to describe the activation mechanism of human prothrombin by the active c o m p o n e n t isolated from Dispholidus typus venom. Terminology The nomenclature used for prothrombin and prothrombin activation products is that recommended by the International Committee on Thrombosis and Haemostasis [11]. Proteolytic cleavage sites are designated bonds 1, 2, 3 starting from the aminoterminus. Fragment 1 (F 1) is a peptide that arises from the amino terminal region of prothrombin by cleavage at bond 1. The remaining part of the molecule is termed prethrombin 1. Fragment 2 (F 2) is an internal peptide formed by cleavage at bonds 1 and 2. When F 1 and F 2 remain covalently linked as a single peptide they are referred to as fragment 1.2 (F 1.2). Prethrombin 2 is that region of prothrombin from which the F 1 and F 2 regions have been cleaved. Thrombin is obtained from prethrombin 2 by cleavage at bond 3 and is constituted of two polypeptide chains (A and B chains) disulfide bridged. Materialsand Methods Normal prothrombin was prepared from human pooled fresh frozen acid/ citrate/dextrose plasma according to Devilee et al. [12]; 1 mM benzamidineHC1 was added in all buffers. The various preparations had a prothrombin activity ranging from 1500 to 1700 U.S. units/rag when measured by a two stage assay in the presence of physiological activators [13,14]. Prethrombin 1 was obtained by incubating, 4 h at 22°C, prothrombin (14 mg in 7 ml 0.1 M NaC1 1 mM benzamidine-HC1/0.01 M sodium + potassium phosphate buffer pH 6.8) with human thrombin (350 U.S. units of lot H1, kindly supplied by Dr. K. Miller, University of Miami, School of Medicine, Miami, Fla.), in the presence of soybean trypsin inhibitor (0.1 mg/ml final concentration). Proteolysis was stopped by the addition of 2 mM diisopropylfluorophosphate (from Sigma Chemical Co.) and the mixture was subjected to chromatography on DEAE-Sephadex A 50 (column 2.5 × 5 cm) equilibrated in 0.1 M NaC1 1 mM benzamidine-HC1]0.01 M phosphate buffer, pH 6.8. After washing the column with the equilibrating buffer (200 ml), a linear gradient of 0.1 M NaC1 1 mM benzamidine-HCl/0.01 M phosphate to 0.5 M NaC1 1 mM benzamidine-HC1/0.01 M phosphate (2 X 250 ml) was applied at a constant pH
162
of 6.8. Fractions of 2 ml were collected at a flow rate of 90 ml/h. The first peak eluted contained prethrombin 1, as assessed b y sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (molecular weight 58 000). Crude venom of Dispholidus typus, purchased from Sigma Chemical Co., was fractionated by chromatography onto DEAE cellulose. 60 mg of whole venom were dissolved in 3 ml of distilled water, dialyzed against 0.05 M. NaC1/0.02 M Tris-HC1, pH 7.5 and loaded onto a column (1.5 X 17 cm) of DE 52 Whatman, equilibrated in the same buffer. After washing the column with the equilibrating buffer, a linear gradient of 0.05 M NaCl/0.02 M Tris-HC1 to 0.15 M NaC1/ 0.02 M Tris-HCl was applied at a constant pH of 7.5. Fractions (3 ml) were collected at a flow rate of 30 ml/h and assayed for coagulant activity b y adding a 100 /11 aliquot to 100 gl of normal citrated plasma and recording the clotting times. Fractions exhibiting a clotting time of 12 s or less were pooled, lyophilized, dissolved in distilled water and dialyzed against 0.1 M NaC1/0.02 M TrisHC1, pH 7.5. A sample (1.6 ml, A280: 1.4) was loaded o n t o a column (1.5 X 90 cm) of Ultrogel AcA 44 (LKB) equilibrated in 0.1 M NaC1/0.02 M Tris-HC1, pH 7.5 and developped with the same buffer. Fractions (3 ml) were collected at a flow rate of 60 ml/h; those exhibiting a clotting time of 25 s or less were pooled and lyophilized. The coagulant principle isolated from Dispholidus typus venom migrated as a single c o m p o n e n t in SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 58 000 and consists in a single polypeptide chain since its mobility was unchanged upon reduction. It exhibited no significant fibrinolytic activity since the lysis time of a fibrin clot incubated with the purified venom exceeded 48 h. Gel electrophoresis in the presence of SDS was performed according to Weber and Osborn [15] in 10% acrylamide, 0.1% SDS. A current of 8 mA per gel was applied for 3 h. Gels were stained with Coomassie blue [15] and destained electrophoretically. Each gel was scanned at 540 nm in a Gilford Model 240 S p e c t r o p h o t o m e t e r equiped with a Model 2410 linear transport accessory. Mobility of each protein was determined from the densitometry tracing and its molecular weight calculated from a linear semi logarithmic plot of molecular weight versus mobility, drawn with four standard proteins (bovine serum albumin, chymotrypsinogen, soybean trypsin inhibitor and lysozyme). Thrombin activity was measured by adding 0.1 ml of diluted sample to 0.2 ml of 0.25% human purified fibrinogen [16] and expressed as U.S. thrombin units using human thrombin (lot B3, kindly supplied by Dr. D.L. Aronson, Bureau of Biologics, F o o d and Drug Administration, Bethesda, Md.) for the calibration curve. Experiments and Results
Proteolysis of human prothrombin or prethrombin 1 by the coagulant principle isolated from Dispholidus typus venom was studied both with or without the addition of either diisopropylfluorophosphate or hirudin (Pentapharm; specific activity: 1500 antithrombin units/mg protein). When added, diisopropylfluorophosphate was usc~l at a final concentration of 10 mM and hirudin was used at a concentration o f 2000 antithrombin units/mg prothrombin. After various incubation times, aliquots were removed for thrombin activity assay and SDS polyacrylamide gel electrophoresis.
163
The addition of the purified venom to prothrombin led to the generation of significant amounts of thrombin activity (Fig. la). Maximum conversion was reached in 2 h. The amount of thrombin generated was similar to that generated in the presence of physiological activators as measured by two stage assay (1600 versus 1700 U.S. units/mg prothrombin). The rate of prethrombin 1 activation was the same as that of prothrombin (Fig. lb). SDS polyacrylamide gel electrophoresis analysis of the venom and prothrombin mixture is shown on Fig. 2. After prolonged incubation (2 h or more), the fragments obtained seem similar to those obtained upon prothrombin activation by factor Xa: unreduced samples showed (Fig. 2a) 3 fragments with molecular weights of 41 000, 28 000 and 17 000, corresponding respectively to the molecular weights of thrombin, F 1 and F 2 [17]. Upon reduction, the band, corresponding to F 2 (mol. wt. 17 000) remained unchanged. The band corresponding to thrombin (tool. wt. 41 000) disappeared and was replaced by a band with molecular weight 37 000 corresponding to the thrombin B chain. The intensity of the F 1 band (mol. wt. 28 000) decreased and two new bands appeared with molecular weights 22 000 and 10 000. According to Franza et al. [7], the 28 000 species correspond to the one chain F 1, the 22 000 and 10 000 species correspond to the two chains of F 1 which has been split by thrombin (respectively subfragment/3 and subfragment ~ ). When the incubation was limited to 30 min, the electrophoretic pattern was different. Unreduced samples (Fig. 2a, 2nd gel) showed 3 fragments with molecular weights 58 000, 41 000 and 28 000, corresponding respectively to the molecular weights of prethrombin 1, thrombin and F 1 [17]. Fragment 2 (moh wt. 17 000) cannot be seen on the photograph but was present in trace amounts as assessed by densitometry scan. Upon reduction (Fig. 2b, 2nd gel), the F 1 band (mol. wt. 28 000) remained unchanged. Both the 58 000 daltons species and the thrombin band (mol. wt. 41 000) disappeared and two new species of molecular weights 37 000 and 22 000 became apparent. The 37 000 daltons species is apparently derived (according to the intensity of the band) from both the thrombin band and the 58 000 daltons species seen on unreduced samples: its molecular weight corresponds to that of the thrombin B chain. In order to determine whether the 22 000 daltons species was derived from F 1 or from the prethrombin 1 region of prothrombin, purified prethrom-
ilb 5
30
60 l~qD 240 incubation time ( m i n u t e s )
5
30
60 120 incubation time
240 (minutes)
Fig. 1. A c t i v a t i o n o f p r o t h r o m b i n a n d p r e t h r o m b i n 1 b y t h e c o a ~ ] a n t p r i n c i p l e i s o l a t e d f r o m Dispho|idus t y p u ~ v e n o m . P u r i f i e d v e n o m (1 #$) w a s a d d e d t o (a) p r o t h r o m b i n o r (b) p r e t h r o m b i n 1 (1 m g i n 0 . 5 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l / 0 . 0 2 M T r i s - H C l , p H 7 . 5 , i n b o t h eases). T h r o m b i n a c t i v i t y w a s meast~red a f t e r v a r i o u s i n c u b a t i o n t i m e s a n d e x p r e s s e d as U . S . t h r o m b i n u n i t s g e n e r a t e d per m g p r o t h r o m b i n o r p r e t h r o m b i n 1.
164
a
b
72, =
_72.000
58,q
- 58[000
41,~ -37,000 ~28,000
28
.22,00D 17
~17,000 -10,000 0
30
120
240
+
0
30
120
240
÷
Fig. 2. S D S polyacrylamide gel electrophoresis of h u m a n prothrombin incubated with purifiedDispholidus typus venom. 1 pg of v e n o m was incubated with prothrombin (1 m g in 0.5 ml 0.1 M NaCI 1 m M benzamidine-HCl/0.02 M Tris-HCl, p H 7.5). After various incubation times at 22°C (indicatedin minutes at the bottom of the gels),samples were prepared by dilutingan aliquot of the incubation mixture in (a) 0.01 M sodium phosphate, p H 7, 1 % S D S (unreduced samples) or (b) 0.01 M sodium phosphate p H 7, 1 % S D S 1 % 2-mercaptoethanol. Samples loaded were in the range of 30 to 50 /~g protein.
bin 1 was incubated with the venom and proteolysis products analysed by SDS polyacrylamide gel electrophoresis (Fig. 3). Unreduced samples showed (Fig. 3a) no molecular weight change when compared to prethrombin 1 before addition of the venom. Reduction resulted (Fig. 3b) in the appearance of two components of molecular weights 37 000 and 22 000. The 37 000 dalton species correspond to the size of the thrombin B chain whereas the 22 000 dalton species must correspond to the rest of prethrombin 1 (F 2 plus thrombin A chain).
a
b
F i g . 3. S D S P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r e t h r o m b i n 1 i n c u b a t e d w i t h p u r i f i e d Diepholidus typus v e n o m . 0 . 7 ~ g o f v e n o m w a s i n c u b a t e d w i t h p r e t h r o m b i n 1 ( 0 . 7 m g in 0 . 3 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l / 0 . 0 2 M Trls-HCI, p H 7 . 5 ) . A f t e r 5 a n d S 0 r a i n i n c u b a t i o n a t 2 2 ° C ( i n c u b a t i o n t i m e s i n d i c a t e d a t t h e b o t t o m o f t h e gels), u n r e d u c e d (a) a n d r e d u c e d (b) s a m p l e s ( 5 0 # g ) w e r e s u b j e c t e d t o S D S gel e l e c t r o p h o r e s l s .
165
These results allow to conclude that the major transient intermediate product of molecular weight 58 000 obtained upon a 30 rain incubation of prothrombin with the venom is composed of 2 polypeptide chains of molecular weights 37 000 (thrombin B chain) and 22 000 (F 2 + thrombin A chain). In order to elucidate whether the appearance of both F 1 and F 2, after prolonged incubation of prothrombin with the venom, was due to proteolysis of prothrombin by the venom or was due to the thrombin generated during the activation process, the experiment was performed in the presence of a thrombin inhibitor, diisopropylfluorophosphate. After 2 h incubation, unreduced samples (Fig. 4a) showed two fragments of molecular weights 58 000 and 28 000. Upon reduction, the 28 000 dalton species persisted (Fig. 4b), whereas the 58 000 dalton species disappeared and two new bands with molecular weights of 37 000 and 22 000 appeared. According to the molecular weights of the products, it was concluded that in the presence of diisopropylfluorophosphate, activation of prothrombin by the venom yields F 1 (mol. wt. 28 000) and a fragment of molecular weight 58 000 consisting of a two-chain disulfidebridged protein. After prolonged incubation (4 h) two additional fragments were seen, with molecular weights 41 000 (thrombin) and 17 000 (F 2). These results prompted to check whether the thrombin generated in the mixture was completely inhibited. After 4 h incubation, despite the high concentration of diisopropylfluorophosphate added (10 mM), the enzyme was still measurable (70 thrombin U.S. units/mg prothrombin). The experiment was thus repeated in the presence of a more potent inhibitor: hirudin. In the presence of hirudin (2000 antithrombin units/mg prothrombin), no thrombin activity was measurable even after 4 h incubation and unreduced samples showed on SDS polyacrylamide gel electrophoresis (Fig. 5a) no molecular weight change of prothrombin. Reduction resulted (Fig. 5b) in almost complete disappearance of the 72 000 dalton species whereas two major products of molecular weights 48 000 and 37 000 became apparent. The 37 000 dalton species correspond to the thrombin B chain whereas the molecular weight of
a
b
72,000"* 58,00G--
41,000"28,000-"
w
i=o
24o 4-
F i g . 4. S D S P o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r o t h r o m b i n i n c u b a t e d w i t h p u r i f i e d D~pholldus typus v e n o m , i n t h e p r e s e n c e o f d l i s o p r o p y l f l u o r o p h o s P h a t e . 1 ~zg o f v e n o m w a s i n c u b a t e d w i t h p r o t h r o m b i n (1 m g i n 0 . 5 m l 0 . 1 M N a C I 1 m M b e n z a m i d i n e - H C l 1 0 m M d i i s o p r o p y l f l u o r o p h o s p h a t e / 0 . 0 2 M T r i s - H C l , p H 7 . 5 ) . A f t e r v a r i o u s i n c u b a t i o n t i m e s ( i n d i c a t e d i n m i n u t e s a t t h e b o t t o m o f t h e gels), u n r e d u c e d ( a ) a n d r e d u c e d (b) s a m p l e s ( 2 0 t o 4 0 # g ) w e r e s u b j e c t e d t o S D S gel e l e e t r o p h o r a s i s .
166
a
b 72.000
72.000
.-48.000 ~ 37.000
0
30
120
240
+
0
30
120
240
.
Fig. 5. S D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f h u m a n p r o t h r o m b i n i n c u b a t e d w i t h p u r i f i e d Dispholidus typus v e n o m , in t h e p r e s e n c e o f h i r u d i n . 1 ~ g o f v e n o m w a s i n c u b a t e d w i t h p r o t h r o m b i n (1 m g in 0 . 5 m l 0.1 M NaC1 1 m M b e n z a m i d i n e - H C l / O . 0 2 M T r i s - H C l , p H 7 . 5 . c o n t a i n i n g 2 0 0 0 a n t i t h r o m b i n u n i t s o f h i r u d i n ) . A f t e r v a r i o u s i n c u b a t i o n t i m e s ( i n d i c a t e d in m i n u t e s a t t h e b o t t o m o f t h e gels), u n r e d u c e d (a) a n d r e d u c e d (b) s a m p l e s ( 2 0 t o 5 0 /~g) w e r e s u b j e c t e d t o S D S gel e l e c t r o p h o r e s l s .
the 48 000 species agrees well with the sum of the molecular weights of F 1, F 2 and the thrombin A chain. Thus, in the presence of hirudin, the prothrombin derivative formed upon the action of Dispholidus typus venom consists of a two chain disulfide-bridged protein; F 1 and F 2 remain attached to the parent protein. Discussion
The mechanism of conversion of purified prothrombin upon activation by factor Xa is well known [17--26]: in a first step, Xa cleaves off the aminoterminal fragment, fragment 1.2, to give rise to prethrombin 2; in a second step, Xa cleaves a peptide bond on prethrombin 2 to yield the two-polypeptide chains thrombin molecule; thrombin, in its turn, catalyses the scission of fragment 1.2 into fragment 1 (F 1) and fragment 2 (F 2). Although other activation pathways are possible, the ultimate products are thrombin, the inner fragment F 2 and the amino-terminal fragment F 1. Prothrombin activation by Dispholidus typus venom has previously been reported not to be dependent upon the presence of calcium ions [2]. In agreement with these data, the activation rate of prothrombin and prethrombin 1 upon the action of an activator isolated from Dispholidus typus venom have been found similar, indicating that the calcium binding region (F 1) of prothrombin is not involved in this activation process. This implies that Dispholidus typus venom, like Echis carinatus venom, may be a useful tool in assay of acarboxyprothrombin which lacks calcium-binding sites in the F 1 region of the molecule. The results obtained in. this study, when investigating by SDS polyacrylamide gel electrophoresis the activation process of human prothrombin upon the action of the venom, in the absence of thrombin inhibitor, are similar to those obtained by others when studying the activation of human [7] or bovine
167 [8,9] prothrombin by Echis carinatus venom. The ultimate products obtained are thrombin, F 2 and F 1. However, when the prothrombin activation proceeds in the presence of hirudin (which inhibits thrombin), the results indicate that the pathway of prothrombin activation is clearly different from that following Xa activation. Although the amino acid composition and the esterase activity of the intermediate products have not been determined in this study, the similarity of the fragmentation patterns observed on SDS polyacrylamide gels in the case of prothrombin activated by Dispholidus typus venom and in the case of prothrombin activated by Echis carinatus venom [9], allows the conclusion that the activation process is the same when using the two venoms. Indeed, Dispholidus typus venom, like Echis carinatus venom, cleaves the bond linking thrombin A and B chains (bond 3), giving rise to an active molecule, called meizothrombin [11]. Only in the absence of hirudin is meizothrombin able to catalyse the scission of two peptide bonds, one linking F 1 and F 2 (bond 1), and one linking F 2 and the thrombin A chain (bond 2). In this process, susceptibility of bond 1 is greater than that of bond 2 since, when prothrombin is incubated with the venom for a relatively short period of time (30 min), a transient activation product is yielded (in which bond 2 is not cleaved), corresponding to meizothrombin (des F 1) and consisting of the F 2 and A thrombin chain moieties disulfide-bridged to the B thrombin chain. Acknowledgements This work was supported by grants from Institut National de la Sant~ et de la Recherche M~dicale, CRL no. 77 2 137 7 and UER Xavier Bichat, Universit~ Paris VII, Paris (France). References 1 Spies, S.K., Malherbe, L.F. and Pepler, W.J. (1962) S. AfT. Med. J. 36,834--838 2 MacKay, N., Ferguson, J.C., Bagshawe, A., Forrester, A. and McNicol, G.P. (1969) Thromb. Diath. Haemorrh. 2 1 , 2 3 4 - - 2 4 4 3 Lakier, J.B. and Fritz, V.U. (1969) S. Aft. Med. J. 43, 1052~1055 4 Neisestuen, G.L. and Suttie, J.W. (1972) J. Biol. Chem. 247, 8176--8182 5 Denson, K.W.E., Borrett, R. and Biggs, R. (1971) Br. J. Haematol. 21,219--226 6 0 w e n , W.G. and Jackson, C.M. (1973) Thromb. Res. 3,705--714 7 Franza, B.R., Aronson, D.L. and Finlayson, J.S. (1975) J. Biol. Chem. 250, 7057--7068 8 Kornalik, F. and Blomb~ck, B. (1975) Thromb. Res. 6, 53--63 9 Morita, T., Iwanaga, S. and Suzuki, T. (1978) J. Biochem. 79, 1089--1108 10 Mann, K.G., Heldebrant, C.M., Bajaj, S.P., Fass, D.N. and But~owski, R.J. (1973) IVth International Congress on Thrombosis and Haemostasis, p. 469, Vienna, Austria, Abstract 11 Jackson, C.M. (1977) Thromb. Haemostas. 38, 567--577 12 Devilee, P.P., Hemker, H.C. and Bas, B.M. (1975) Biochim. Biophys. Acta 379,172--179 13 Ware, A.G. and Seegers, W.H. (1949) Am. J. CIin. Pathol. 19,471--482 14 Cesbron, N., Boyer, C., Guillin, M.C. and Menaehe, D. (1973) Thromb. Diath. Haemorrh. 30, 437--450 15 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 16 Kazal, L.A., Amsel, S. and Miller, O.P. (1963) Proc. Soc. Exp. Biol. Med. 113, 989--994 17 Kisiel, W. and Hanahan, D.J. (1973) Biochim. Biophys. Acta 3 2 9 , 2 2 1 - - 2 3 2 18 Lanchantin, G.F., Friedmann, J.A. and Hart, D.W. (1968) J. Biol. Chem. 243, 476--486 19 Stenn, K.S. and Blout, E.R. (1972) Biochemistry 11, 4502--4515 20 Gitel, S.N., Owen, W.G., Esmon, C.T. and Jackson, C.M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1344--1348 21 Heldebrant, C.M., Butkowski, R.J., Bajaj, S.P. and Mann, K.G. (1973) J. Biol. Chem. 248, 7149--7163
168 22 23 24 25 26
Esmon, C.T,, Owen, W.G. and Jackson, C.M. (1974) J. Biol. Chem. 249. 606--611 Kisiel, W. and Hanahan, D.J. (1974) Biochem. Biophys. Res. Commun. 59,570--577 Morita, T., Nishibe, H.. Iwanaga, S. and Suzuki, T. (1974) J. Biochem. 76, 1031--1048 Owen, W.G., Esmon, C.T. and Jackson, C,M. (1974) J. Biol. Chem. 249, 594---605 Seegers, W.H., Walz, D.A., Reuterby, J. and McCoy, L.E, (1974) Thromb. Res. 5, 829~860