aa~-0ioi~ ss.oo + .ao
r~, voi . 30, xo. ~z va. ~sis-isn, i~z . r~~a ~ c3,~ sri~.
o i~x ~ t~. t.w
REVIEW ARTICLE STRUCTURAL AND FUNCTIONAL PROPERTIES OF SNAKE VENOM PROTHROMBIN ACTIVATORS JAN RosnJC
and Gismo T~Ns
Cardiovascular Research Institute Maastricht, Department of Biochemistry, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands (Received 8 AprU 1992 ; accepted 301um 1992)
and G. T~xs. Structural and functional properties of snake venom prothrombin activators . Toxicon 30, 1515-1527, 1992 .-In this review we have summarized the current knowledge about the prothrombin activating principles present in the venom of a large number of different snake species. It appears that snake venom prothrombin activators can be classified into four different groups based on their structural properties and on their functional properties in prothrombin activation. Group I activators efficiently convert prothrombin into meizothrombin and their activity is not influenced by the non-enzymatic cofactors of the prothrombinase complex (CaCl z, factor V, and phospholipid). Group II and III activators can cleave both peptide bonds in prothrombin necessary to convert prothrombin into thrombin . The prothrombin-converting activity of Group II activators is strongly stimulated by phospholipids and factor V, in the presence of CaCl z, whereas the activity of group III activators is only stimulated by CaCl z and phospholipid . Group 1V consists of snake venom professes which do not convert prothrombin into enzymatically active products but cleave peptide bonds in prothrombin, resulting in the formation of inactive precursor forms of thrombin . J. RosINO
INTRODUCTION ooxvERSION of the zymogen prothrombin into the serine protease thrombin is one of the central reactions of blood coagulation (for reviews see SuT-rte and JACKSON, 1977 ; Rosnvc and TANS, 1988 ; MAxx et al., 1990; Dwn; et al., 1991). Thrombin can be considered to be the key enzyme in thrombus formation since it is a multifunctional enzyme which among others converts fibrinogen into fibrin and which is able to activate blood platelets. In vivo prothrombin activation is the result of limited proteolysis of prothrombin by blood coagulation factor X,. However, prothrombin can also become activated through the action of enzymes from exogenous sources (mostly snake venoms). Over the past 20 years many reports have appeared in literature concerning such exogenous prothrombin activators and it turned out that a large number of these proteins display interesting effects which have greatly contributed to our understanding the mechanism of (factor X,-catalysed) prothrombin activation occurring under physiological conditions. Exogenous prothrombin activators can be obtained either from snake venoms or from bacteria and thus far there are no reports on activators from other sources (ROBING and
TIC
lsts
1516
J . ROSTNG and G . TANS
4Q s_s I~IZOTH~OMBIN FIG.
1.
PA0IEOLYTIC
CLEAVA(iHS
A3190CIATED W11H THE TFIA(kIBIN .
CONVERSION OF
PAOTF1110MBIN
INTO
For further details see text .
TANS, 1991). In this review we will restrict ourselves to snake venom prothrombin activators and focus on those activators whose structural characteristics and functional properties in prothrombin activation are well described. To appreciate the mode of action of venom prothrombin activators it will be helpful, however, to start this review with a short introduction on the mechanism of factor X,-catalysed prothrombin activation . FACTOR X,-CATALYSED PROTHROMBIN ACTIVATION
Prothrombin is a single-chain glycoprotein with a mol. wt of 72,000, which is converted into thrombin by the serine protease factor X, (SLJTTIS and JACKSON, 1977) . In order to convert prothrombin into thrombin, factor X, must cleave two peptide bonds in the prothrombin molecule that are located between Arg273-Thr274* (Fig. 1, site 1) and Arg322-I1e323 (Fig. 1, site 2) . Depending upon the order of cleavage there are two possible pathways of prothrombin activation . Primary cleavage at site 1 results in the liberation of the activation fragment 1 .2t (36,000 mol. wt) and generation of prethrombin 2. This reaction intermediate has the same mol. wt as thrombin (36,000) but lacks enzymatic activity . Active site exposure occurs after cleavage at site 2, which converts prethrombin 2 into so-called a-thrombin . a-Thrombin is a two-chain enzyme consisting of a light (A-chain, mol. wt 4600) and heavy chain (B-chain, mol. wt 32,000) that contains the active site (FsNTON et al., 1977). When site 2 in prothrombin is cleaved prior to site 1 meizothrombin is the reaction intermediate . Meizothrombin is a two-chain molecule with the same mol. wt as pro' The numbering of amino acids is based on the amino acid sequence of human prothrombin. Human prothrombin consists of 581 residues, whereas bovine prothrombin contains 582 residuea. This difference is due to a deletion in the fourth resddue in the human molecule. Consequently, the numbering of amino acids of human prothrombin differs by one residue from that of the bovine molecule. t The nomenclature used for proteolytically derived products of prothrombin is that rocommended by the International Committee on Thrombosis and Haemostasis (JACKSON, 1977) .
1517
Snake Venom Prothrombin Activators TABIB 1 . TTIB EFFECT OF ACCEY70RY OO~ONEN11 ON THE RATE OF ITt011m01®IN ACTIVATION HY BLOOD OOAOLII.ATION FAChOR X,
Activator
9
RClatiVe rate
Mole prothrombin activated/min/mole X, X, X Ca'+ X Ca~+, PL X Ca2 +, V, ~ ~x+~ pL, V,
0.0046 0.0080 2.8900 10 .6600 1018 .0000
1 .0 1 .7 6.3 x 10' 2 .3 x 103 2.2 x 10 5
The rate of prothrombin activation wen represents the initial (steady-state) rate at 37°C, calculated from the kinetic parameters given in Rosins et at. (1980). thrombin, but which in contrast to prothrombin exhibits catalytic activity. Meizothrombin and thrombin have equal esterolytic and amidolytic activity (KORNALIIC and BLOi~ACIC, 1975 ; F~tANZA et al., 1975; MORITA et al., 197, but meizothrombin has less than 5% of the activity of thrombin towards fibrinogen, factor V and platelets (R061NG et al., 1986 ; DOYLE and MwxN, 1990). Meizothrombin can be further cleaved at site 1 which removes fragment 1.2 and generates a-thrombin . During factor X,~atalysed prothrombin activation both prethrombin 2 and meizothrombin have been shown to accumulate (cf. Rosnvc and TANS, 1988; TANS et al., 1991) and thus, it appears that thrombin can be formed via both pathways. A clear description of product generation during factor X,-catalysed prothrombin activation has always been seriously hampered by the fact that a number of peptide bonds in prothrombin (and in its activation products) are susceptible to proteolytic cleavage by thrombin . The major one is located at Arg15~Ser156 (site 3). Thus, thrombin and meizothrombin will readily cleave prothrombin (or meizothrombin) at site 3, resulting in the removal of fragment 1 and the generation of prethrombin 1 or meizothrombin des fragment 1 . Thrombin can also cleave site 4, which is located between Arg286 and Thr287 . The products of this reaction are a-thrombin (lacking 13 amino acid residues of the A~hain) and fragment 1.2 .3 in the case of meizothrombin, or a polypeptide consisting of 13 amino acids when this cleavage occurs in thrombin . Prothrombin activation shows a typical feature that is common for many other reactions of blood coagulation . The enzyme responsible for prothrombin activation, factor X appears to be a rather poor prothrombin activator. This is shown by the very low rate of prothrombin activation by factor X, alone (Table 1) . In vivo prothrombin activation requires the presence of the so-called accessory components calcium ions, negatively charged phospholipids (in vivo provided by activated platelets) and the nonenzymatic protein cofactor, factor V, (cf. Rosn~ro and TANS, 1988; MANN et al., 1990). These accessory components stimulate prothrombin activation in a multiplicative way and efficient prothrombin activation requires a prothrombinase complex consisting of a factor X.-factor V, complex bound to negatively charged phospholipids in the presence of calcium ions (Table 1) . The stimulatory effect of phospholipids is presumably due to the fact that prothrombin, factor X, and factor V, bind with a high affnity to negatively charged phospholipid surfaces . The coordinate binding of the prothrombinase proteins to the membrane surface is thought to promote strongly the formation of productive
1518
J. ROSING and G . TANS TARIE i. CLA.4sü7CAT10N OP PROTFIItOMBIN AC71VAliNO SNAKE VENOUS
Class
Cofactor dependence
Group I
None
Group II
PhosphoGpids plus CaCl2 plus factor V, Phospholipids plus CaClz
Group III
Best-known example Echis carinatus (saw-scaled viper) Notechis scutatus scutatus (tiger snake) Oxyuramcs scutellatus scutelJatus (Taipan snake)
enzyme-substrate complexes. This is reflected in a considerable decrease of the 11~, for prothrombin and a corresponding increase in the reaction rate (RoslrrG et al., 1980). The CaZ+-dependent binding of prothrombin and factor Xo to phospholipids requires so-called y-carboxyglutamic acid residues (or Gla-residues) which are introduced in the amino terminal domain of these proteins after post-ribosomal carboxylation of specific glutamic acid residues (cf. $LTITIE and JwcxsoN, 1977). The major function of the protein cofactor, factor Va, is to ensure a high V,o ~ of thrombin formation (Rosnvc et al., 1980 ; Klusi-nvwswwa~r et al., 1986) . This appears to be caused by at least three different effects: (1) stimulation of the binding of factor Xe to the lipid surface which increases the amount of factor X. participating in the reaction; (2) a factor VQinduced shift in the pathway of prothrombin activation from a reaction sequence producing the catalytically inactive intermediate, prothrombin 2, into one producing thrombin ; and (3) a factor VQdependent increase of a forward rate constant of the prothrombin activation pathway. SNAKE VENOM PROTHROMBIN ACTIVATORS
Within the group of snake venom prothrombin activators one encounters a rather large variety of enzymes with different structural and functional properties . In view of the mechanism of factor Xs-catalysed prothrombin activation described above it will not be surprising that a meaningful classification of prothrombin activators can be based on the possible stimulation of venom prothrombin-converting activity by the accessory components of the prothrombinase complex, i.e. Cap+, negatively charged phospholipids or factor V, (cf.1JENSON, 1976). By these criteria snake venom prothrombin activators can be divided into throe different classes, the properties of which are summarized in Table 2. Activators belonging to the first group do not require added metal ions nor is their activity affected by phospholipids or by factor V, . The well-known activator from the venom of Echis carinatus is representative for this group. The second group, the best-known example of which is the activator obtained from Notechis scutatus (tiger snake) venom, contains activators whose prothrombin~onverting activities are strongly stimulated by negatively charged phospholipids and by factor V, in the presence of calcium ions. The third group consists of venom activators that are stimulated by negatively charged phospholipids in the presence of calcium ions but not by factor Ve . The prothrombin activator present in the venom from Oxyuranus scutellatus (Taipan snake) is characteristic for this last group. The actual effects of the accessory components on rates of prothrombin activation by the crude venoms of the snakes mentioned above are summarized in Table 3. These data illustrate the lack of effect of accessory components on the prothrombin activator from E.
Snake Venom Prothrombin Activators
151 9
TAHIE 3. THE EFPEC,T OP CALCIUM IONS, NEGATIVELY CHARGED PH08PHOLIPIDB AND FACTOR V, ON THE RATE OF PROTHROMBIN ACTIVATION HY THE CRUDE VENOMS OF Echis Cali7latY.r, NOteChis BCUtatfL4 sCUlates AND OXyYlatlIL4
scuteltatur scuteltatus
Activator Venom Venom, Caz+ Venom, Caz+, PL Venom, CaZ+, PL, V,
Echis carinatur 3 nnrole prothrombin activated/min/mg venom
Notechis scutatus seutatus 9 nmole prothrombin activated/min/mg venom
Oxyurarae scvte!latus scutellatus S nmole prothrombin activated/min/mg venom
34 .2 54 .3 42 .3 48 .2
O .000l8 0 .00027 2 .04000 9234 .00000
0 .27 2 .13 968 .00 985 .00
The rate of prothrombin activation given represents the initial (steady-state) rate determined at 37°C. Final concentrations of reactants were: 0.5 PM prothrombin, 5 mM CaQ=, 50 ltM phospholipid vesicles (20% phosphatidylserine/80% phosphatidylcholine ; mole/mole), 5 nM factor V, and appropriate amounts of purified venom activator. Data based on $PEIJER et al. (1986) and YULE[SON et at. (1991) .
carinates . The venom activator from N. scutatus is stimulated by both phospholipids plus CaZ+ and by factor V,. A maximal stimulation of 3 x 106-fold was observed when all accessory components were present at the same time. Prothrombin activation by the crude venom from O. scutellatus is stimulated approximately 3600-fold by phospholipids plus CaZ+ and is not affected by the presence of factor V,. Finally, it should be mentioned that there are several snake venoms which convert prothrombin into the precursor forms of thrombin that do not exhibit catalytic activity (i.e. prothrombin 1 and prethrombin 2). These venoms are discussed in the last paragraph of this review. Group I activators Group I activators (Table 2) constitute a group of snake venoms that contain an activating principle that activates prothrombin efficiently without requiring the presence of phospholipids or the protein cofactor V,. Studies on prothrombin activation by the crude venoms belonging to this group are complicated by the fact that many of these venoms also contain thrombin-like enzymes or factor X activators (DErrsox, 1976; BRADLOW et al., 1980; HEMKER et al., 1984; HOFMANN and Box, 1987; Govl?RS-RIEMSLAG et al., 1987). Several group I prothrombin activators have, however, been purified to homogeneity and they appear to share many structural and functional properties (Table 4). The bestknown example of a group I activator is the one present in Echis carinates venom. This activator, which has been purified by several groups, is a single~hain enzyme with an estimated mol . wt of 55,0003,000 (MORITA aIId IWANAGA, 1978, 1980; R.i~e et al., 1982; FORTOVw et al., 1983). Other venom activators from this group which have been purified to homogeneity are those present in the venoms from Dispholidus typos (Gtra.Lnv et al., 1978; BRADLOW et al., 1980), Thelotornis kirtlandi (KORNALIR and TAHORSIU, 1978; ATRINSON et al., 1980), Bothrops atrox (HOFMANN and Box, 1987) and Bothrops t:euwiedi (Govtnes-R11?MSLA(i et al., 1987) . With the possible exception of Rhabdophis tigrinus tigrirtus (MoRrrw et al., 1988) group I prothrombin activators appear to be single-chain enzymes with mol . wts ranging between 50,000 and 70,000. They are not inhibited by serine protease inhibitors and most likely belong to the metalloproteases since chelating
1520
J . ROSING and G. TANS TAHIL 4 . STRUCTURAL AND FUNCTIONAL PROPF1tTIP3 OF GROUP I PROTFDtOMBIN ACTTVATORS
Source Echis carinates
Disphotides typos Rhabdophis tigrirea tigrimrs
M,
Subunits
Inhibitors
Cofactors
Product
55,000 56,000 63,000
Single chain
Chelating and reducing agents
None
MT
EDTA
None
MT
85,000 50,000 58,000
170,000
84,000 58,000 52,000
-
-
MT
Thetotornis kirttartdi capensis
56,000
Roducing agents
None
-
Bothrops atrox
70,000
Single chain Single chain
-
MT
Bothrops neewiedi
60,000
Chelating and reducing agents TPCK Chelating and reducing agents
None
MT
Single chain
The list given in this table contains activators for which the purification to homo~neity has been reported. Similar properties have been reported for several other F.chis and Bothrops species. Abbreviations: EDTA, ethylenediaminetetraacetic acid; TPCK, tosyl-phenylalanine chloromethyl~etone ; MT, meizothrombin .
agents such as EDTA or o-phenantroline readily destroy the prothrombin-activating capacity of these enzymes. One of the most striking features of group I activators, at least as judged from their mode of action in prothrombin activation, is that these enzymes appear only capable of cleaving the Arg322-rie323 bond (site 2, Fig. 1) in prothrombin (MORITw et al., 1976; Guu,I,nv et al., 1978 ; ATKINSON et al., 1980; HOFMANN and Box, 1987; GOVERS-R>EMSLAG et al., 1987). So the product of prothrombin activation by these activators is meizothrombin. Due to autocatalytic removal of the fragment 1 region, prothrombin 1 and fragment 1 are also readily observed during activation . BRISI' et al. (1982) reported that the prothrombin activator from Echis carirtatus can also remove fragment 1 by cleaving at G1y158-Ser159, a peptide bond just adjacent to the one cleaved by thrombin . In some cases it has been reported that prolonged incubation of prothrombin with group I venom activators finally results in thrombin formation. Such thrombin formation has been mainly observed with human prothrombin (FRwNZw et al ., 1975 ; GUII.LIN et al., 1978 ; RIB et al., 1982) and not with bovine prothrombin (KORNALIK and BE.OMHACR, 1975 ; MORrrw et al., 1976; GovExs-RIEM3LAG et al., 1987). These differences probably resulted from sutocatalytic cleavage of bond 4 (cf. Fig. 1) by meizothrombin itself, which is a rather efficient reaction in the case of human prothrombin (RISE et al., 1982). Group II activators The prothrombin-converting activities of group II activators (Table 2), the best-known examples of which are the activators present in the venoms of several Notechis species, are strongly stimulated both by factor V, and by negatively charged phospholipids plus calcium ions (Josnv and EsxouF, 1966 ; HERRMANN et al., 1972; CI~sTER and CRAWFORD, 1982 ; MwucuAr .L and HERRMANN, 1983). Two group II activators (both from the Notechis
Snake Venom Prothrombin Activators
152 1
family) have thus far been purified to homogeneity (TANS et al., 1985 ; WILLIAMS and WIC, 1989 ; Table ~. The activator from the venom of Notechis scutatus was structurally and functionally characterized by TArrs et al. (1985) . This activator is a two-chain enzyme with a mol. wt of 54,000 that consists of a heavy chain and a light chain with mol. wts of 32,000 and 23,000, respectively, held together by one or more disulphide bridges. The active site is located on the heavy chain of the molecule, as evidenced by the incorporation of the fluorescent active site-directed chloromethylketone dansyl~lu~rly-Arg-CH ZCI. The enzyme appears to belong to the class of serine professes since it is inhibited by wellknown serine protease inhibitors . Surprisingly, the venom activator was found to contain 8 Gla-residues which by analogy with factor X, will most likely be located in the aminoterminal portion of the light chain of the molecule. The prothrombin~onverting activity of the Notechis activator is stimulated in a multiplicative way by the prothrombinase accessory components, negatively charged phospholipids, factor V, and calcium ions (Table 3). The presence of all accessory components results in an overall stimulation of more than a million-fold . The mechanism by which the accessory components stimulate prothrombin activation by the venom protease is actually quite similar to that observed in factor X,~atalysed prothrombin activation . Like factor X, the purified venom activator appears to be a very poor activator of prothrombin with very unfavourable kinetic parameters . The 1~ for prothrombin is very high (105 ~M) and V~ is only 0.13 enzyme turnovers per min. The rate enhancement observed in the presence of negatively charged phospholipids is explained by a dramatic decrease in the 1~ for prothrombin to result in a ~ of approximately 0.2 ~M with little change in the V~ of the reaction . The major effect of factor V, is a 3000-fold increase of the V~ of the reaction . The venom activator appears capable of randomly cleaving both peptide bonds necessary to be split in order to convert prothrombin into thrombin . This can be concluded from the fact that in reaction mixtures in which prothrombin is activated by the venom activator appreciable amounts of prethrombin 2, meizothrombin and thrombin accumulate . In Table 6 we have compared a number of structural and functional properties of the venom activator from N. scutatus with those of the physiological prothrombin activator, factor X,. These data illustrate that the activator present in N. scutatrts venom is an enzyme which is remarkably similar to factor X,. Tt siB 5 . S~rxucrvre~r. ~ Fuivctmrui. Paor~erms oF anour II M,
Subunita
Notechis scutatus scutatur
54,000
32,000 23,000
Nottclw ater niger
58,000
37,000 23,000
Source
PaOTrfltOèIBIN
Inhibitors
ACr1VA10RS
Cofactors
roduct
BenTSmidine PL, V Ca~+ T, MT, PT2 DFP, SBTI dansylGGACK PL, V Ca=+ -
Although no other purißcation data are as yet available, activators with similar properties have been reported to be preaeat in the venom of several other Noteclria species and of Cryptophis nigrracena, HoplooepkaGrs atepMrrü, Paerrdechis porpbyriacus and Tropid~eclris carirtotus. Abbreviations : DFP, düsopropylfluorophosphate; SBTI, soybean trypsin inhibitor, dansylGGACK, dansyl(ilu-0ly-Arg~hloromethylketo~ ; PL, phospholipids V blood coagulation factor V,; T, thrombin ; MT, meizothrombin ; P'I'2, prethrombin 2.
1522
J. ROSING aad G. TANS TABLE
6.
X, AND l'H8 ACI7VATOR Notcchis scutatus scutatus vBNOt~
Snt1LARrr~s OF BOVINB FACIOR
PURIF~D FROLi
Noteckis scutatus scalants
Factor X,
54,000 32,000 23,000 2 .00
44,000 27,000 17,000 2.40
0.16 25 .00
0.21 46.00
Mol. wt Heavy chain" Light chain GLA~ontent (%) Prothrombin activationt tip (pM) kq, (sec -')
'Both enzymes have the active site located on the heavy chain. tKinetic parameters determined in the presence of CaCh, phospholipid and factor V, . Data from RosnvG et aJ. (1980), TANS et at. (1985) and Sum and JACKSON (1977) .
A number of other venoms (all from Australian elapidae) appear to contain a group II activator (Ci-~sTeR and CxAwIroRD, 1982; M ARCr-tzt .L and HExRMANN, 1983; SPaIJaIt et al., 198 . However, none of these activators has as yet been purified or has been characterized in more detail (cf. legend to Table 5). Group III activators Group III (Table 7) consists of venom activators whose action on prothrombin is strongly stimulated by the presence of negatively charged phospholipids plus calcium ions but not by factor V,. Like group II activators, the activators belonging to group III are only fotmd in the venoms from Australian elapidae . The prothrombin activator from the venom of Oxyuranus scutellatus has been extensively characterized (WALx>;It et al., 1980; $P13IJER et al., 1986) and the data from SPI~JER et al. (1986) will be used as a guide in the further discussion of the properties of this activator . The enzyme responsible for prothrombin activation in O. scutellatus venom likely belongs to the class of serine professes since its activity is inhibited by a large number of well-known serine protease inhibitors (Table ~. It appears that the venom activators classified in group III are rather unusual in that they appear to be multiple subunit enzymes (WALKI~t et al., 1980; SPEIJER et al., 1986; MASCI et al., 1988; NAKAGAKI et al., 1992). SDS-gel electrophoretic analysis of non-reduced samples of the purified venom activator from O. scutellatus venom (SPEUER et al., 1986) shows four major bands with TARIE
7.
ST UCTURAL AND FUNCIiONAL PAOPERI'~4 OF GROUP
Source Oxyuraxus scutettatus PsetrdonaJa textitir
M, 300,000 (380,000) ~ 200,000
III
PROTIÜtOQ~IN ACfIVATOR3
Subunits
Inhibitors
Cofactors
Product
Multiple subunits Multiple subunits
Hen7amidine SBTI, dansylGGACK -
PL, Ca2 +
T, MT
PL, Ca~+
Activators with similar properties have been reported to be present in the venom of Oxyurarttts microlepittotus, Psettdonaja r~îreis (Dugite) and Pseudonaja rruchalis (Crwardar) . Abbreviations: SBTI, soybean trypsin inhibitor; dansyl-GGACK, dansyl--Glu~rly-Arg~hloromethylketone; PL, phospholipid ; T, thrombin ; MT, meizothrombin .
Snake Venom 1?rothrombin Activators
1523
220,000 Mr uMt
c E
0 r 0
4 2
a 0 0.00 0 .05 0 .10 0 .15 0 .20 0.25 0 .30 0 .35
FIG .
2.
nM factor Va or ml 220,000 Mr unit EFFF.CI' OP FACTOR V, AND THE VITiO!! COFACTOR SIJBIJNIT ON YROTIütOMBIN AC'T'IVATION HY TIC CATALYTTC SUBUNIT OF TIC OJCyUrIDJtl3 Scutellatus ACTIVATOR .
The initial rate of prothrombin activation by the catalytic subunit (mol . wt 57,000) of the venom activator was determined at 37°C in the presence of increasing amounts of factor V, or purified venom cofactor subunit (mol . wt 220,000) . The rate of prothrombin activation was determined at 0.5 pM prothrombin, 5 mM CaC h and 50 pM phospholipid vesicles (25% phoaphatidylserine/ 75% phosphatidylcholine; mole/mole). Reprinted with permisaion from $PEIJER et ol. (1986) .
mol. wts of 220,000, 120,000, 80,000 and 60,000, respectively, whereas bands with mol. wts of 110,000, 80,000 and 30,000 were observed after reduction. The catalytic site is located on the 60,000 polypeptide as evidenced by the incorporation of active site-directed fluorescent chloromethylketone, dansyl-Glu-Gly-Arg~H 2C 1 . After reduction, the fluorescent label migrated with a mol. wt of 30,000, which indicates that the subunit with mol. wt of 60,000 probably consists of two 30,000 mol. wt subunits held together by one or more disulphide bonds. Table 3 shows that prothrombin activation by the purified venom activator is strongly stimulated by negatively charged phospholipids plus calcium ions, whereas factor V, has little effect. The reason for this apparent lack of stimulation by factor V° is due to the fact that the large subunits of the activator actually act as a factor Vélike cofactor in venom-catalysed prothrombin activation . This was concluded from experiments in which the cofactor and the enzymatic unit of the venom activator were separated during gradient gel electrophoresis . The cofactor unit appeared to have a mol. wt of 220,000 and the enzymatic unit migrated at 57,000 . Compared with the native activator the 57,000 enzymatic unit exhibited a very low prothrombin-converting activity. Prothrombin activa-
1524
J. ROSING and G. TANS
Factor V,-like part
Factor X,-like part active site FIG.
3.
HYPOTHETICAL
MODEL
FOR
TfIE
VENOM
PROTHROMHIN
scutellates. For further details see text .
ACTIVATOR FROM
Oxyuranes
tion by the 57,000 unit was, however, greatly stimulated by incubation either with the 220,000 cofactor unit from the venom activator or with bovine factor V, (Fig. 2). Thus, factor V, can substitute for the 220,000 subunit present in the venom activator. These data led to a hypothetical model (Fig. 3) for the venom prothrombin activator from O. scutellatus. In this model it is proposed that the activator is comprised of a factor X,-like catalytic unit of 60,000 mol. wt and of a 220,000 factor V~like subunit. The data obtained after SDS-gel electrophoretic analysis of the purified activator (see above) suggest that the cofactor unit may actually consist of subunits with mol. wts of 110,000 and 80,000 and that the catalytic unit is composed of two subunits with a mol. wt of 30,000. TABLE
8.
VENOMS rHAr wNVIasT PROrtoeoMHnv Ihro
Source
Ineonucls wITTTOUr
cArALYrTC ACrMTY
M,
Inhibitors
CofactoTS
Product
Agkistrodort acutes AgkistrodorT rhodostoma Agkistrodhn covTtortrix Bothrops atrox
33,500 25,600 36,000
Ca2* Ca~*
PT1, PT2 PTI, PT2 PTI, PT2
Crotales odmnantees
32,700
DFP, PMSF PMSF, al-M, AT-III DFP, SBTI, AT-III, chloromethylketonea DFP, 2-ME, chloromethylketo~s
PL, CA~*
PTl PTI, PT2
Abbrcviations: DFP, düsopropylfluôrophosphate; PMSF, phenyhnethylsulfonylchlolide; arM, az-macroglobulin ; AT-III, antithrombin III; SBTI, soybean trypsin inhibitor, 2-ME, 2-mercaptoetbanol; PL, phospholipid; PTI, prethrombin 1; P'I'2, Prethtnmbin 2.
Snake Venom Prothrombin Activators
152 5
Like the activator from the venom of N. scutatus, the O. scutellatus prothrombin activator was found to contain Gla residues ($PEIISR et al., 198 . By analogy with the function of Gla residues in factor X,-catalysed prothrombin activation, it is likely that these residues play an important role in the calcium-dependent binding of the venom activator to the negatively charged phospholipid membranes. The prothrombin activation products observed with the venom activator of O. scutellatus venom are meizothrombin and thrombin (Spit et al., 1986). This indicates that group III activators can also cleave the two peptide bonds necessary for the conversion of prothrombin into thrombin . Venoms that convert prothrombin into products without enrymatic activity There are several snake venoms that can cleave peptide bonds in prothrombin without converting it into products with catalytic activity (i.e. thrombin or meizothrombin) . These venoms contain enzymes that are capable of limited proteolysis of prothrombin at or near bond 1 or 3 (Fig. 1). This results in the formation of products which are similar or identical to prethrombin 1 and prethrombin 2, the enzymatically inactive intermediary products observed during factor X,-catalysed prothrombin activation . The enzymes thus far reported for this group are actually thrombin-like enzymes. There are, however, relatively few data in literature on the action of these enzymes on prothrombin, since prothrombin is not considered to be their target substrate. Moreover, some caution should be exerted as to which enzyme in the venom is actually responsible for the conversion of prothrombin, since the crude venom may contain more than one enzyme with thrombin-like activity . For example, the prothrombin-converting enzyme in Agkistrodon rhodostoma venom, ARHy (Lor i.wit et al., 1987) is not the same enzyme as the well-known fibrinogenolytic enzyme snood, an enzyme that has no activity towards prothrombin (hIRICLB et al., 1976). In Table 8 we have only summarized those venoms for which the prothrombinconverting activity has been well defined. These include enzymes present in the venoms of A. acutus (OUYANG et al., 1971 ; SEeoets et al., 1980), A . contortrix (PIRKLE et al., 197, A. rhodostoma (LotLwit et al., 1987), Bothrops atrox (NIEVVIAROWSKI et al., 1979) and Crotales adamanteus (PIRRLE et al., 1976). As far as the mechanism of prothrombin activation is concerned these enzymes may be less interesting. They may, however, serve as useful tools in the production and purification of the non-enzymatic prothrombin derivatives prethrombin 1 and prethrombin 2. REFERENCES Almvsox, P. M ., Hnwntaw, B. A ., Wnrre, J . A . M ., Gn~a, H . H. W. sad (}wrr r " Qn, M. C. (1980) Clinical features of twig snake (Thelotorrtis cgpacrir) eaveaomation. S. Afr. med. 1. SB, 1008-1011 . Bnwnt.ow, B. A ., A~t~sox, P. M ., Croup~tl3, E . D . and Gwn .r wQn , M . C. (1980) Studies on the coagulant effect of boomslang (Displroltdus types) venom. Cllr. lab . Haemat. 2, 317-331 . Bnmr, E ., NOYZD, C. M., Roa~rta, H. R and fviau+lrnx, M . J. (1982) Cleavage and activation of human prothrombin by Echir carbratrrs venom. Tlrrornb . Res . 27, 591-b00. C,~~st~t, A . and Ctuwt~n, ß . P. M . (1982) In vitro coagulant properties of venoms from Australian snakes. Toxlcarr 20, SOI-504. Dwvm, E. W ., Ftr~awww, K. and Kts~., W . (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10,363-10,370 . Dwsot+, K. W. E . (1976) Clot-inducing substances present in snake venoms with particular reference to Echis carirwtur venom. 77tmmb . Res . 8, 351-360 .
1526
J . ROSING and G. TANS
DoYt.a, M . F. and Mwrtx, K. G . (1990) Multiple active forms of thrombin . IV . Relative activities of meizothrombins. J. biol. Chem . 266, 10,693-10,701 . Fexrox, J . W., Fwsoo, M . J ., STwctcxow, A. B., Axoxsox, D . L., Youra, A. M . and Ftxr.wYSOx, J . S. (1977) Human thrombins . Production, evaluation and properties of a-thrombin. J. biol. Chem. 252, 3587-3598 . FORTOVA, H., Dvx, J . E ., Voutuztcw, Z . and Kottxwt.tx, F . (1983) Isolation of the prothrombin-converting enzyme from fibrinogenolytic enzymes of Echù carirtater venom by chromatographic and electrophoretic methods . J. Chromat. 259, 47379 . Fxwxzw, B . R ., Ateortsox, D. L. and Fnvuvsox, J. S . (1975) Activation of human prothrombin by a procoagulant fraction from the venom of Echù carinates. J. biol. Chem. 250, 7057-7068 . Govetess-Rtt~rst.wa, J . W . P., Kxwrt=.x, M . J. H ., Twxs, G., ZWML, R . F . A . and Rostra, J . (1987) Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi. Biochim. Biophys. Acts 916, 388-401 . Gun.t,nv, M . C ., BE7Ewup, A . and MatvwcttE, D . (1978) The mechanism of activation of human prothrombin by an activator isolated from Dùpholides typos venom . Biochim . Biophys. Acts 537, 160-168 . Hesrto:a, H . C., vwx Dwt~-Mtetews, M . C . E . and Devtt.~, P . P . (1984) The action of Echù carinates venom on the blood coagulation system . Demonstration of an activator of factor X . 77uomb. Res. 35, 1-9 . Ht:axlrwxx, R . P ., Dwvtar, M . G . and Stcrotaoxt=., P. H . (1972) The coagulation defect after envenomation by the bite of the dugite (Demansia mtchalù affinis), a Western Australian brown snake . Med. J. Aunt. 2, 183-186 . Hot~rwxx, H . and Box, C. (1987) Blood coagulation induced by the venom of Bothrops atrox. 1 . Identification, purification, and properties of a prothrombin activator . Biochemùtry 26, 772-780. Jwctcsox, C . M. (1977) 77tromb . Haemost. 38, 567-577 . Joetx, F. and EsrouF, M . P. (1966) Coagulant activity of tiger snake (Notechù scutates scetates) venom. Nature 211, 873,875. Kotexwt.tte, F . and Br-o~xcx, B. (1975) Prothrombin activation induced by Ecarin-a prothrombin converting enzyme from Echù carinates venom . Thromb. Res. 6, 53 3 . Kotsrtwt nc, F . and Twuottsttw, E . (1978) Procoagulant and defibrinating potency of the venom gland extract of Thelotornù kirtlandi. Thromb . Res . 12, 991-1000. Kxtstn~twswwrnr, S ., Mwxx, K. G. and NFSt~tM, M . E . (1986) The prothrombinase~atalysed activation of prothrombin procceds through the intermediate meizothrombin in an ordered, sequential reaction. J. biol. Chem . 261, 8977984. Lot.t-wx, P., Pww~e, C . G., Kwssrtstet, P. J., Ltzwtt.t.px, R . D. and Fwss, D . N . (1987) Degradation of coagulation proteins by an enzyme from Malayan pit viper (Agkùtrodon rhodostoma) venom. Biochemùtry 26, 7627-7636. Mwxx, K . G ., Nlst~tur, M . E., Cttuxcty W . R ., Hwt.t:v, P . and Ktetst~nvwswwt~rsr, S . (1990) Surface-dependent reactions of the vitamin K-dependent enzyme complexes . Blood 76, 1-16. Mw4VHAi .L, L . R. and Ht~xt~twrx, R. P . (1983) Coagulant and anticoagulant actions of Australian snake venoma. Thromb . Kaemost. 50, 707-711 . Mwsct, P . P., WEa'rwxt:x, A. N. and Dtirt:ast=.u, J . (1988) Purification and characterization of a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilù textilù). Biochem . Int . 17, 825-835. Moxrrw, T. and Iwwxwaw, S . (1978) Purification and properties of prothrombin activator from the venom of Echù carinates . J. Biochem . 83, 559-570. Moterrw, T . and Iwwxwaw, S. (1980) Prothrombin activator from Echù carinates venom . Meth . Enzym . 80, 303-311 . Moxrrw, T., IWANAGw, S . and Suzutu, T . (1976) The mechanism of activation of bovine prothrombin by an activator isolated from Echù carinates venom and characterization of the new active intermediates. J. Biochem . 79, 1089-1108 . Moxrrw, T ., Mwrsuttoro, H ., IWANAGA, S . and Swtw, A . (1988) A prothrombin activator found in Rhabdophù tigrim~s tigrirets (Yamakagashi snake) venom. In: Hemostasù and Animal Venoms, pp. 556 (Pmtci .t:, H. and Mwx~r-wxn, F . S., Jte Eds). New York : Marcel Dekker . Nwxwawta, T., Ltx, P. and Ktst~ ., W . (1992) Activation of human factor VII by the prothrombin activator from the venom of Oxyeranes scetcllates (Taipan snake) . Thromb. Res . 66, 105-116 . NtEwtwxowstu, S ., KtxsY, E. P., Bxwzvtvs~t, T. M . and Sam, K . (1979) Thrombocytin, a serine protease from Bothrops atrox venom . 2 . Interaction with platelets and plasma-clotting factors. Biochemùtry 18, 3570-3577 . OuYwxa, C ., HONG, J . S . and Ttaxa, C: M . (1971) Purification and properties of the thrombin-like principle of Agkùtrodort sector venom and its comparison with bovine thrombin. 77womb. Diath . Haemorrh . 26, 224-234 . PrRr~ H ., Mwnt~uxn, F . S . and Tt~monott, I . (1976) Thrombin-like enzymes of snake venoms : actions on prothrombin . Thromb. Res. 8, 61927 . Rtma, M-J ., Monxts, S. and Kasow, D. P. (1982) Role of meizothrombin and meizothrombin-(des Fl) in the conversion of prothrombin to thrombin by the Echù carbates venom coagulant . Blochemùtry 21, 3437-3443. Rostra, J . and Twxs, G. (1988) Meizothrombin, a major product of factor Xa~atalysed prothrombin activation. Thromb. Haemost. 60, 355-360 .
Snake Venom Prothrombin Activators
1527
Rasata, J . and Teas, G . (1991) Inventory of exogenous prothrombin activators. Report for the subcommittee on nomenclature of exogenous hemostatic factors of the scientific and standardization committee of the international society on thrombosis and haemoatasis. Thromb . Nacmost . 66, 62730 . ROBING, J ., Teas, G., Goveßs-R~etsuo, J . W . P ., Zweet ., R. F . A. and Hnricm, H . C. (1980) The role of phospholipids and factor V, in the prothrombinase complex . J. biol. Chem . 256, 274-283 . Roan~o, J ., ZwMI., R . F . A . and TwNS, G. (1986) Formation of meizothrombin as intermediate in factor X,~atalysed prothrombin activation . J. biol. Chem . 261, 4224-4228 . S»aizes, W ., H., TEtao, C: M . and Novoe, E . (1980) Pt+eparation of bovine prethrombin 2 : use of Awtin and activation with prothrombinase or Eearin . Thromb. Res. 19, 11-20. Bream, H ., GovEas-Ru~tsLeo, J. W . P., Zww,+L, R . F . A. and RosINa, J . (1986) Prothrombin activation by an activator from the venom of Oxyuramv scutellatus (Taipan snake) . J . biol. Chem . 261, 13,258-13,267 . Soma:, J . W . and JecICSON, C. M . (1977) Prothrombin structure, activation and biosynthesis. Physiol . Rev . 67, 1-65 . Teas, G., GovIIts-R~uG, J. W. P., v~u~t RuN, J. L . M . L. and Rasnva, J . (1985) Purification and properties of a prothrombin activator from the venom of Nottchis scutarus scutattu . J. biol. Chem . 2611, 9366-9372. Teas, G ., J~xs.~x-C~.w~x, T ., HER, H . C ., Zwwwt, R. F . A . and Rosnvc, J . (1991) Meizothrombin formation during factor X,~atalysed prothrombin activation. Formation in a purified system and in plasma . J. biol. Chem. 266, 21,864-21,873 . Ww. .s~a, F . J ., Owslv, W . G. and ESMON, C. T . (1980) Characterization of the prothrombin activator from the venom of Oxyurarrus scutcllatus scutellatus (Taipan venom) . Biochemistry 19, 1020-1023 . Wn.r ut~ts, V . and W~, J . (1989) Purification and properties of a pracoagulant from peninsula tiger snake (Notechis attr niger) venom. Toxicon 27, 773-779. YuxsISON, L. Ye., TANS, G ., TIIOI~sssN, M . L . C . G . D., Hrttxea, H . C . and Rosnvc, J . (1991) Procoagulant activities in venoms from central Asian snakes. Toxicon 29, 491-502.
r~, voi . 30, xo. ~z va. ~sis-isn, i~z . r~~a ~ c3,~ sri~.
o i~x ~ t~. t.w
REVIEW ARTICLE STRUCTURAL AND FUNCTIONAL PROPERTIES OF SNAKE VENOM PROTHROMBIN ACTIVATORS JAN RosnJC
and Gismo T~Ns
Cardiovascular Research Institute Maastricht, Department of Biochemistry, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands (Received 8 AprU 1992 ; accepted 301um 1992)
and G. T~xs. Structural and functional properties of snake venom prothrombin activators . Toxicon 30, 1515-1527, 1992 .-In this review we have summarized the current knowledge about the prothrombin activating principles present in the venom of a large number of different snake species. It appears that snake venom prothrombin activators can be classified into four different groups based on their structural properties and on their functional properties in prothrombin activation. Group I activators efficiently convert prothrombin into meizothrombin and their activity is not influenced by the non-enzymatic cofactors of the prothrombinase complex (CaCl z, factor V, and phospholipid). Group II and III activators can cleave both peptide bonds in prothrombin necessary to convert prothrombin into thrombin . The prothrombin-converting activity of Group II activators is strongly stimulated by phospholipids and factor V, in the presence of CaCl z, whereas the activity of group III activators is only stimulated by CaCl z and phospholipid . Group 1V consists of snake venom professes which do not convert prothrombin into enzymatically active products but cleave peptide bonds in prothrombin, resulting in the formation of inactive precursor forms of thrombin . J. RosINO
INTRODUCTION ooxvERSION of the zymogen prothrombin into the serine protease thrombin is one of the central reactions of blood coagulation (for reviews see SuT-rte and JACKSON, 1977 ; Rosnvc and TANS, 1988 ; MAxx et al., 1990; Dwn; et al., 1991). Thrombin can be considered to be the key enzyme in thrombus formation since it is a multifunctional enzyme which among others converts fibrinogen into fibrin and which is able to activate blood platelets. In vivo prothrombin activation is the result of limited proteolysis of prothrombin by blood coagulation factor X,. However, prothrombin can also become activated through the action of enzymes from exogenous sources (mostly snake venoms). Over the past 20 years many reports have appeared in literature concerning such exogenous prothrombin activators and it turned out that a large number of these proteins display interesting effects which have greatly contributed to our understanding the mechanism of (factor X,-catalysed) prothrombin activation occurring under physiological conditions. Exogenous prothrombin activators can be obtained either from snake venoms or from bacteria and thus far there are no reports on activators from other sources (ROBING and
TIC
lsts
1516
J . ROSTNG and G . TANS
4Q s_s I~IZOTH~OMBIN FIG.
1.
PA0IEOLYTIC
CLEAVA(iHS
A3190CIATED W11H THE TFIA(kIBIN .
CONVERSION OF
PAOTF1110MBIN
INTO
For further details see text .
TANS, 1991). In this review we will restrict ourselves to snake venom prothrombin activators and focus on those activators whose structural characteristics and functional properties in prothrombin activation are well described. To appreciate the mode of action of venom prothrombin activators it will be helpful, however, to start this review with a short introduction on the mechanism of factor X,-catalysed prothrombin activation . FACTOR X,-CATALYSED PROTHROMBIN ACTIVATION
Prothrombin is a single-chain glycoprotein with a mol. wt of 72,000, which is converted into thrombin by the serine protease factor X, (SLJTTIS and JACKSON, 1977) . In order to convert prothrombin into thrombin, factor X, must cleave two peptide bonds in the prothrombin molecule that are located between Arg273-Thr274* (Fig. 1, site 1) and Arg322-I1e323 (Fig. 1, site 2) . Depending upon the order of cleavage there are two possible pathways of prothrombin activation . Primary cleavage at site 1 results in the liberation of the activation fragment 1 .2t (36,000 mol. wt) and generation of prethrombin 2. This reaction intermediate has the same mol. wt as thrombin (36,000) but lacks enzymatic activity . Active site exposure occurs after cleavage at site 2, which converts prethrombin 2 into so-called a-thrombin . a-Thrombin is a two-chain enzyme consisting of a light (A-chain, mol. wt 4600) and heavy chain (B-chain, mol. wt 32,000) that contains the active site (FsNTON et al., 1977). When site 2 in prothrombin is cleaved prior to site 1 meizothrombin is the reaction intermediate . Meizothrombin is a two-chain molecule with the same mol. wt as pro' The numbering of amino acids is based on the amino acid sequence of human prothrombin. Human prothrombin consists of 581 residues, whereas bovine prothrombin contains 582 residuea. This difference is due to a deletion in the fourth resddue in the human molecule. Consequently, the numbering of amino acids of human prothrombin differs by one residue from that of the bovine molecule. t The nomenclature used for proteolytically derived products of prothrombin is that rocommended by the International Committee on Thrombosis and Haemostasis (JACKSON, 1977) .
1517
Snake Venom Prothrombin Activators TABIB 1 . TTIB EFFECT OF ACCEY70RY OO~ONEN11 ON THE RATE OF ITt011m01®IN ACTIVATION HY BLOOD OOAOLII.ATION FAChOR X,
Activator
9
RClatiVe rate
Mole prothrombin activated/min/mole X, X, X Ca'+ X Ca~+, PL X Ca2 +, V, ~ ~x+~ pL, V,
0.0046 0.0080 2.8900 10 .6600 1018 .0000
1 .0 1 .7 6.3 x 10' 2 .3 x 103 2.2 x 10 5
The rate of prothrombin activation wen represents the initial (steady-state) rate at 37°C, calculated from the kinetic parameters given in Rosins et at. (1980). thrombin, but which in contrast to prothrombin exhibits catalytic activity. Meizothrombin and thrombin have equal esterolytic and amidolytic activity (KORNALIIC and BLOi~ACIC, 1975 ; F~tANZA et al., 1975; MORITA et al., 197, but meizothrombin has less than 5% of the activity of thrombin towards fibrinogen, factor V and platelets (R061NG et al., 1986 ; DOYLE and MwxN, 1990). Meizothrombin can be further cleaved at site 1 which removes fragment 1.2 and generates a-thrombin . During factor X,~atalysed prothrombin activation both prethrombin 2 and meizothrombin have been shown to accumulate (cf. Rosnvc and TANS, 1988; TANS et al., 1991) and thus, it appears that thrombin can be formed via both pathways. A clear description of product generation during factor X,-catalysed prothrombin activation has always been seriously hampered by the fact that a number of peptide bonds in prothrombin (and in its activation products) are susceptible to proteolytic cleavage by thrombin . The major one is located at Arg15~Ser156 (site 3). Thus, thrombin and meizothrombin will readily cleave prothrombin (or meizothrombin) at site 3, resulting in the removal of fragment 1 and the generation of prethrombin 1 or meizothrombin des fragment 1 . Thrombin can also cleave site 4, which is located between Arg286 and Thr287 . The products of this reaction are a-thrombin (lacking 13 amino acid residues of the A~hain) and fragment 1.2 .3 in the case of meizothrombin, or a polypeptide consisting of 13 amino acids when this cleavage occurs in thrombin . Prothrombin activation shows a typical feature that is common for many other reactions of blood coagulation . The enzyme responsible for prothrombin activation, factor X appears to be a rather poor prothrombin activator. This is shown by the very low rate of prothrombin activation by factor X, alone (Table 1) . In vivo prothrombin activation requires the presence of the so-called accessory components calcium ions, negatively charged phospholipids (in vivo provided by activated platelets) and the nonenzymatic protein cofactor, factor V, (cf. Rosn~ro and TANS, 1988; MANN et al., 1990). These accessory components stimulate prothrombin activation in a multiplicative way and efficient prothrombin activation requires a prothrombinase complex consisting of a factor X.-factor V, complex bound to negatively charged phospholipids in the presence of calcium ions (Table 1) . The stimulatory effect of phospholipids is presumably due to the fact that prothrombin, factor X, and factor V, bind with a high affnity to negatively charged phospholipid surfaces . The coordinate binding of the prothrombinase proteins to the membrane surface is thought to promote strongly the formation of productive
1518
J. ROSING and G . TANS TARIE i. CLA.4sü7CAT10N OP PROTFIItOMBIN AC71VAliNO SNAKE VENOUS
Class
Cofactor dependence
Group I
None
Group II
PhosphoGpids plus CaCl2 plus factor V, Phospholipids plus CaClz
Group III
Best-known example Echis carinatus (saw-scaled viper) Notechis scutatus scutatus (tiger snake) Oxyuramcs scutellatus scutelJatus (Taipan snake)
enzyme-substrate complexes. This is reflected in a considerable decrease of the 11~, for prothrombin and a corresponding increase in the reaction rate (RoslrrG et al., 1980). The CaZ+-dependent binding of prothrombin and factor Xo to phospholipids requires so-called y-carboxyglutamic acid residues (or Gla-residues) which are introduced in the amino terminal domain of these proteins after post-ribosomal carboxylation of specific glutamic acid residues (cf. $LTITIE and JwcxsoN, 1977). The major function of the protein cofactor, factor Va, is to ensure a high V,o ~ of thrombin formation (Rosnvc et al., 1980 ; Klusi-nvwswwa~r et al., 1986) . This appears to be caused by at least three different effects: (1) stimulation of the binding of factor Xe to the lipid surface which increases the amount of factor X. participating in the reaction; (2) a factor VQinduced shift in the pathway of prothrombin activation from a reaction sequence producing the catalytically inactive intermediate, prothrombin 2, into one producing thrombin ; and (3) a factor VQdependent increase of a forward rate constant of the prothrombin activation pathway. SNAKE VENOM PROTHROMBIN ACTIVATORS
Within the group of snake venom prothrombin activators one encounters a rather large variety of enzymes with different structural and functional properties . In view of the mechanism of factor Xs-catalysed prothrombin activation described above it will not be surprising that a meaningful classification of prothrombin activators can be based on the possible stimulation of venom prothrombin-converting activity by the accessory components of the prothrombinase complex, i.e. Cap+, negatively charged phospholipids or factor V, (cf.1JENSON, 1976). By these criteria snake venom prothrombin activators can be divided into throe different classes, the properties of which are summarized in Table 2. Activators belonging to the first group do not require added metal ions nor is their activity affected by phospholipids or by factor V, . The well-known activator from the venom of Echis carinatus is representative for this group. The second group, the best-known example of which is the activator obtained from Notechis scutatus (tiger snake) venom, contains activators whose prothrombin~onverting activities are strongly stimulated by negatively charged phospholipids and by factor V, in the presence of calcium ions. The third group consists of venom activators that are stimulated by negatively charged phospholipids in the presence of calcium ions but not by factor Ve . The prothrombin activator present in the venom from Oxyuranus scutellatus (Taipan snake) is characteristic for this last group. The actual effects of the accessory components on rates of prothrombin activation by the crude venoms of the snakes mentioned above are summarized in Table 3. These data illustrate the lack of effect of accessory components on the prothrombin activator from E.
Snake Venom Prothrombin Activators
151 9
TAHIE 3. THE EFPEC,T OP CALCIUM IONS, NEGATIVELY CHARGED PH08PHOLIPIDB AND FACTOR V, ON THE RATE OF PROTHROMBIN ACTIVATION HY THE CRUDE VENOMS OF Echis Cali7latY.r, NOteChis BCUtatfL4 sCUlates AND OXyYlatlIL4
scuteltatur scuteltatus
Activator Venom Venom, Caz+ Venom, Caz+, PL Venom, CaZ+, PL, V,
Echis carinatur 3 nnrole prothrombin activated/min/mg venom
Notechis scutatus seutatus 9 nmole prothrombin activated/min/mg venom
Oxyurarae scvte!latus scutellatus S nmole prothrombin activated/min/mg venom
34 .2 54 .3 42 .3 48 .2
O .000l8 0 .00027 2 .04000 9234 .00000
0 .27 2 .13 968 .00 985 .00
The rate of prothrombin activation given represents the initial (steady-state) rate determined at 37°C. Final concentrations of reactants were: 0.5 PM prothrombin, 5 mM CaQ=, 50 ltM phospholipid vesicles (20% phosphatidylserine/80% phosphatidylcholine ; mole/mole), 5 nM factor V, and appropriate amounts of purified venom activator. Data based on $PEIJER et al. (1986) and YULE[SON et at. (1991) .
carinates . The venom activator from N. scutatus is stimulated by both phospholipids plus CaZ+ and by factor V,. A maximal stimulation of 3 x 106-fold was observed when all accessory components were present at the same time. Prothrombin activation by the crude venom from O. scutellatus is stimulated approximately 3600-fold by phospholipids plus CaZ+ and is not affected by the presence of factor V,. Finally, it should be mentioned that there are several snake venoms which convert prothrombin into the precursor forms of thrombin that do not exhibit catalytic activity (i.e. prothrombin 1 and prethrombin 2). These venoms are discussed in the last paragraph of this review. Group I activators Group I activators (Table 2) constitute a group of snake venoms that contain an activating principle that activates prothrombin efficiently without requiring the presence of phospholipids or the protein cofactor V,. Studies on prothrombin activation by the crude venoms belonging to this group are complicated by the fact that many of these venoms also contain thrombin-like enzymes or factor X activators (DErrsox, 1976; BRADLOW et al., 1980; HEMKER et al., 1984; HOFMANN and Box, 1987; Govl?RS-RIEMSLAG et al., 1987). Several group I prothrombin activators have, however, been purified to homogeneity and they appear to share many structural and functional properties (Table 4). The bestknown example of a group I activator is the one present in Echis carinates venom. This activator, which has been purified by several groups, is a single~hain enzyme with an estimated mol . wt of 55,0003,000 (MORITA aIId IWANAGA, 1978, 1980; R.i~e et al., 1982; FORTOVw et al., 1983). Other venom activators from this group which have been purified to homogeneity are those present in the venoms from Dispholidus typos (Gtra.Lnv et al., 1978; BRADLOW et al., 1980), Thelotornis kirtlandi (KORNALIR and TAHORSIU, 1978; ATRINSON et al., 1980), Bothrops atrox (HOFMANN and Box, 1987) and Bothrops t:euwiedi (Govtnes-R11?MSLA(i et al., 1987) . With the possible exception of Rhabdophis tigrinus tigrirtus (MoRrrw et al., 1988) group I prothrombin activators appear to be single-chain enzymes with mol . wts ranging between 50,000 and 70,000. They are not inhibited by serine protease inhibitors and most likely belong to the metalloproteases since chelating
1520
J . ROSING and G. TANS TAHIL 4 . STRUCTURAL AND FUNCTIONAL PROPF1tTIP3 OF GROUP I PROTFDtOMBIN ACTTVATORS
Source Echis carinates
Disphotides typos Rhabdophis tigrirea tigrimrs
M,
Subunits
Inhibitors
Cofactors
Product
55,000 56,000 63,000
Single chain
Chelating and reducing agents
None
MT
EDTA
None
MT
85,000 50,000 58,000
170,000
84,000 58,000 52,000
-
-
MT
Thetotornis kirttartdi capensis
56,000
Roducing agents
None
-
Bothrops atrox
70,000
Single chain Single chain
-
MT
Bothrops neewiedi
60,000
Chelating and reducing agents TPCK Chelating and reducing agents
None
MT
Single chain
The list given in this table contains activators for which the purification to homo~neity has been reported. Similar properties have been reported for several other F.chis and Bothrops species. Abbreviations: EDTA, ethylenediaminetetraacetic acid; TPCK, tosyl-phenylalanine chloromethyl~etone ; MT, meizothrombin .
agents such as EDTA or o-phenantroline readily destroy the prothrombin-activating capacity of these enzymes. One of the most striking features of group I activators, at least as judged from their mode of action in prothrombin activation, is that these enzymes appear only capable of cleaving the Arg322-rie323 bond (site 2, Fig. 1) in prothrombin (MORITw et al., 1976; Guu,I,nv et al., 1978 ; ATKINSON et al., 1980; HOFMANN and Box, 1987; GOVERS-R>EMSLAG et al., 1987). So the product of prothrombin activation by these activators is meizothrombin. Due to autocatalytic removal of the fragment 1 region, prothrombin 1 and fragment 1 are also readily observed during activation . BRISI' et al. (1982) reported that the prothrombin activator from Echis carirtatus can also remove fragment 1 by cleaving at G1y158-Ser159, a peptide bond just adjacent to the one cleaved by thrombin . In some cases it has been reported that prolonged incubation of prothrombin with group I venom activators finally results in thrombin formation. Such thrombin formation has been mainly observed with human prothrombin (FRwNZw et al ., 1975 ; GUII.LIN et al., 1978 ; RIB et al., 1982) and not with bovine prothrombin (KORNALIK and BE.OMHACR, 1975 ; MORrrw et al., 1976; GovExs-RIEM3LAG et al., 1987). These differences probably resulted from sutocatalytic cleavage of bond 4 (cf. Fig. 1) by meizothrombin itself, which is a rather efficient reaction in the case of human prothrombin (RISE et al., 1982). Group II activators The prothrombin-converting activities of group II activators (Table 2), the best-known examples of which are the activators present in the venoms of several Notechis species, are strongly stimulated both by factor V, and by negatively charged phospholipids plus calcium ions (Josnv and EsxouF, 1966 ; HERRMANN et al., 1972; CI~sTER and CRAWFORD, 1982 ; MwucuAr .L and HERRMANN, 1983). Two group II activators (both from the Notechis
Snake Venom Prothrombin Activators
152 1
family) have thus far been purified to homogeneity (TANS et al., 1985 ; WILLIAMS and WIC, 1989 ; Table ~. The activator from the venom of Notechis scutatus was structurally and functionally characterized by TArrs et al. (1985) . This activator is a two-chain enzyme with a mol. wt of 54,000 that consists of a heavy chain and a light chain with mol. wts of 32,000 and 23,000, respectively, held together by one or more disulphide bridges. The active site is located on the heavy chain of the molecule, as evidenced by the incorporation of the fluorescent active site-directed chloromethylketone dansyl~lu~rly-Arg-CH ZCI. The enzyme appears to belong to the class of serine professes since it is inhibited by wellknown serine protease inhibitors . Surprisingly, the venom activator was found to contain 8 Gla-residues which by analogy with factor X, will most likely be located in the aminoterminal portion of the light chain of the molecule. The prothrombin~onverting activity of the Notechis activator is stimulated in a multiplicative way by the prothrombinase accessory components, negatively charged phospholipids, factor V, and calcium ions (Table 3). The presence of all accessory components results in an overall stimulation of more than a million-fold . The mechanism by which the accessory components stimulate prothrombin activation by the venom protease is actually quite similar to that observed in factor X,~atalysed prothrombin activation . Like factor X, the purified venom activator appears to be a very poor activator of prothrombin with very unfavourable kinetic parameters . The 1~ for prothrombin is very high (105 ~M) and V~ is only 0.13 enzyme turnovers per min. The rate enhancement observed in the presence of negatively charged phospholipids is explained by a dramatic decrease in the 1~ for prothrombin to result in a ~ of approximately 0.2 ~M with little change in the V~ of the reaction . The major effect of factor V, is a 3000-fold increase of the V~ of the reaction . The venom activator appears capable of randomly cleaving both peptide bonds necessary to be split in order to convert prothrombin into thrombin . This can be concluded from the fact that in reaction mixtures in which prothrombin is activated by the venom activator appreciable amounts of prethrombin 2, meizothrombin and thrombin accumulate . In Table 6 we have compared a number of structural and functional properties of the venom activator from N. scutatus with those of the physiological prothrombin activator, factor X,. These data illustrate that the activator present in N. scutatrts venom is an enzyme which is remarkably similar to factor X,. Tt siB 5 . S~rxucrvre~r. ~ Fuivctmrui. Paor~erms oF anour II M,
Subunita
Notechis scutatus scutatur
54,000
32,000 23,000
Nottclw ater niger
58,000
37,000 23,000
Source
PaOTrfltOèIBIN
Inhibitors
ACr1VA10RS
Cofactors
roduct
BenTSmidine PL, V Ca~+ T, MT, PT2 DFP, SBTI dansylGGACK PL, V Ca=+ -
Although no other purißcation data are as yet available, activators with similar properties have been reported to be preaeat in the venom of several other Noteclria species and of Cryptophis nigrracena, HoplooepkaGrs atepMrrü, Paerrdechis porpbyriacus and Tropid~eclris carirtotus. Abbreviations : DFP, düsopropylfluorophosphate; SBTI, soybean trypsin inhibitor, dansylGGACK, dansyl(ilu-0ly-Arg~hloromethylketo~ ; PL, phospholipids V blood coagulation factor V,; T, thrombin ; MT, meizothrombin ; P'I'2, prethrombin 2.
1522
J. ROSING aad G. TANS TABLE
6.
X, AND l'H8 ACI7VATOR Notcchis scutatus scutatus vBNOt~
Snt1LARrr~s OF BOVINB FACIOR
PURIF~D FROLi
Noteckis scutatus scalants
Factor X,
54,000 32,000 23,000 2 .00
44,000 27,000 17,000 2.40
0.16 25 .00
0.21 46.00
Mol. wt Heavy chain" Light chain GLA~ontent (%) Prothrombin activationt tip (pM) kq, (sec -')
'Both enzymes have the active site located on the heavy chain. tKinetic parameters determined in the presence of CaCh, phospholipid and factor V, . Data from RosnvG et aJ. (1980), TANS et at. (1985) and Sum and JACKSON (1977) .
A number of other venoms (all from Australian elapidae) appear to contain a group II activator (Ci-~sTeR and CxAwIroRD, 1982; M ARCr-tzt .L and HExRMANN, 1983; SPaIJaIt et al., 198 . However, none of these activators has as yet been purified or has been characterized in more detail (cf. legend to Table 5). Group III activators Group III (Table 7) consists of venom activators whose action on prothrombin is strongly stimulated by the presence of negatively charged phospholipids plus calcium ions but not by factor V,. Like group II activators, the activators belonging to group III are only fotmd in the venoms from Australian elapidae . The prothrombin activator from the venom of Oxyuranus scutellatus has been extensively characterized (WALx>;It et al., 1980; $P13IJER et al., 1986) and the data from SPI~JER et al. (1986) will be used as a guide in the further discussion of the properties of this activator . The enzyme responsible for prothrombin activation in O. scutellatus venom likely belongs to the class of serine professes since its activity is inhibited by a large number of well-known serine protease inhibitors (Table ~. It appears that the venom activators classified in group III are rather unusual in that they appear to be multiple subunit enzymes (WALKI~t et al., 1980; SPEIJER et al., 1986; MASCI et al., 1988; NAKAGAKI et al., 1992). SDS-gel electrophoretic analysis of non-reduced samples of the purified venom activator from O. scutellatus venom (SPEUER et al., 1986) shows four major bands with TARIE
7.
ST UCTURAL AND FUNCIiONAL PAOPERI'~4 OF GROUP
Source Oxyuraxus scutettatus PsetrdonaJa textitir
M, 300,000 (380,000) ~ 200,000
III
PROTIÜtOQ~IN ACfIVATOR3
Subunits
Inhibitors
Cofactors
Product
Multiple subunits Multiple subunits
Hen7amidine SBTI, dansylGGACK -
PL, Ca2 +
T, MT
PL, Ca~+
Activators with similar properties have been reported to be present in the venom of Oxyurarttts microlepittotus, Psettdonaja r~îreis (Dugite) and Pseudonaja rruchalis (Crwardar) . Abbreviations: SBTI, soybean trypsin inhibitor; dansyl-GGACK, dansyl--Glu~rly-Arg~hloromethylketone; PL, phospholipid ; T, thrombin ; MT, meizothrombin .
Snake Venom 1?rothrombin Activators
1523
220,000 Mr uMt
c E
0 r 0
4 2
a 0 0.00 0 .05 0 .10 0 .15 0 .20 0.25 0 .30 0 .35
FIG .
2.
nM factor Va or ml 220,000 Mr unit EFFF.CI' OP FACTOR V, AND THE VITiO!! COFACTOR SIJBIJNIT ON YROTIütOMBIN AC'T'IVATION HY TIC CATALYTTC SUBUNIT OF TIC OJCyUrIDJtl3 Scutellatus ACTIVATOR .
The initial rate of prothrombin activation by the catalytic subunit (mol . wt 57,000) of the venom activator was determined at 37°C in the presence of increasing amounts of factor V, or purified venom cofactor subunit (mol . wt 220,000) . The rate of prothrombin activation was determined at 0.5 pM prothrombin, 5 mM CaC h and 50 pM phospholipid vesicles (25% phoaphatidylserine/ 75% phosphatidylcholine; mole/mole). Reprinted with permisaion from $PEIJER et ol. (1986) .
mol. wts of 220,000, 120,000, 80,000 and 60,000, respectively, whereas bands with mol. wts of 110,000, 80,000 and 30,000 were observed after reduction. The catalytic site is located on the 60,000 polypeptide as evidenced by the incorporation of active site-directed fluorescent chloromethylketone, dansyl-Glu-Gly-Arg~H 2C 1 . After reduction, the fluorescent label migrated with a mol. wt of 30,000, which indicates that the subunit with mol. wt of 60,000 probably consists of two 30,000 mol. wt subunits held together by one or more disulphide bonds. Table 3 shows that prothrombin activation by the purified venom activator is strongly stimulated by negatively charged phospholipids plus calcium ions, whereas factor V, has little effect. The reason for this apparent lack of stimulation by factor V° is due to the fact that the large subunits of the activator actually act as a factor Vélike cofactor in venom-catalysed prothrombin activation . This was concluded from experiments in which the cofactor and the enzymatic unit of the venom activator were separated during gradient gel electrophoresis . The cofactor unit appeared to have a mol. wt of 220,000 and the enzymatic unit migrated at 57,000 . Compared with the native activator the 57,000 enzymatic unit exhibited a very low prothrombin-converting activity. Prothrombin activa-
1524
J. ROSING and G. TANS
Factor V,-like part
Factor X,-like part active site FIG.
3.
HYPOTHETICAL
MODEL
FOR
TfIE
VENOM
PROTHROMHIN
scutellates. For further details see text .
ACTIVATOR FROM
Oxyuranes
tion by the 57,000 unit was, however, greatly stimulated by incubation either with the 220,000 cofactor unit from the venom activator or with bovine factor V, (Fig. 2). Thus, factor V, can substitute for the 220,000 subunit present in the venom activator. These data led to a hypothetical model (Fig. 3) for the venom prothrombin activator from O. scutellatus. In this model it is proposed that the activator is comprised of a factor X,-like catalytic unit of 60,000 mol. wt and of a 220,000 factor V~like subunit. The data obtained after SDS-gel electrophoretic analysis of the purified activator (see above) suggest that the cofactor unit may actually consist of subunits with mol. wts of 110,000 and 80,000 and that the catalytic unit is composed of two subunits with a mol. wt of 30,000. TABLE
8.
VENOMS rHAr wNVIasT PROrtoeoMHnv Ihro
Source
Ineonucls wITTTOUr
cArALYrTC ACrMTY
M,
Inhibitors
CofactoTS
Product
Agkistrodort acutes AgkistrodorT rhodostoma Agkistrodhn covTtortrix Bothrops atrox
33,500 25,600 36,000
Ca2* Ca~*
PT1, PT2 PTI, PT2 PTI, PT2
Crotales odmnantees
32,700
DFP, PMSF PMSF, al-M, AT-III DFP, SBTI, AT-III, chloromethylketonea DFP, 2-ME, chloromethylketo~s
PL, CA~*
PTl PTI, PT2
Abbrcviations: DFP, düsopropylfluôrophosphate; PMSF, phenyhnethylsulfonylchlolide; arM, az-macroglobulin ; AT-III, antithrombin III; SBTI, soybean trypsin inhibitor, 2-ME, 2-mercaptoetbanol; PL, phospholipid; PTI, prethrombin 1; P'I'2, Prethtnmbin 2.
Snake Venom Prothrombin Activators
152 5
Like the activator from the venom of N. scutatus, the O. scutellatus prothrombin activator was found to contain Gla residues ($PEIISR et al., 198 . By analogy with the function of Gla residues in factor X,-catalysed prothrombin activation, it is likely that these residues play an important role in the calcium-dependent binding of the venom activator to the negatively charged phospholipid membranes. The prothrombin activation products observed with the venom activator of O. scutellatus venom are meizothrombin and thrombin (Spit et al., 1986). This indicates that group III activators can also cleave the two peptide bonds necessary for the conversion of prothrombin into thrombin . Venoms that convert prothrombin into products without enrymatic activity There are several snake venoms that can cleave peptide bonds in prothrombin without converting it into products with catalytic activity (i.e. thrombin or meizothrombin) . These venoms contain enzymes that are capable of limited proteolysis of prothrombin at or near bond 1 or 3 (Fig. 1). This results in the formation of products which are similar or identical to prethrombin 1 and prethrombin 2, the enzymatically inactive intermediary products observed during factor X,-catalysed prothrombin activation . The enzymes thus far reported for this group are actually thrombin-like enzymes. There are, however, relatively few data in literature on the action of these enzymes on prothrombin, since prothrombin is not considered to be their target substrate. Moreover, some caution should be exerted as to which enzyme in the venom is actually responsible for the conversion of prothrombin, since the crude venom may contain more than one enzyme with thrombin-like activity . For example, the prothrombin-converting enzyme in Agkistrodon rhodostoma venom, ARHy (Lor i.wit et al., 1987) is not the same enzyme as the well-known fibrinogenolytic enzyme snood, an enzyme that has no activity towards prothrombin (hIRICLB et al., 1976). In Table 8 we have only summarized those venoms for which the prothrombinconverting activity has been well defined. These include enzymes present in the venoms of A. acutus (OUYANG et al., 1971 ; SEeoets et al., 1980), A . contortrix (PIRKLE et al., 197, A. rhodostoma (LotLwit et al., 1987), Bothrops atrox (NIEVVIAROWSKI et al., 1979) and Crotales adamanteus (PIRRLE et al., 1976). As far as the mechanism of prothrombin activation is concerned these enzymes may be less interesting. They may, however, serve as useful tools in the production and purification of the non-enzymatic prothrombin derivatives prethrombin 1 and prethrombin 2. REFERENCES Almvsox, P. M ., Hnwntaw, B. A ., Wnrre, J . A . M ., Gn~a, H . H. W. sad (}wrr r " Qn, M. C. (1980) Clinical features of twig snake (Thelotorrtis cgpacrir) eaveaomation. S. Afr. med. 1. SB, 1008-1011 . Bnwnt.ow, B. A ., A~t~sox, P. M ., Croup~tl3, E . D . and Gwn .r wQn , M . C. (1980) Studies on the coagulant effect of boomslang (Displroltdus types) venom. Cllr. lab . Haemat. 2, 317-331 . Bnmr, E ., NOYZD, C. M., Roa~rta, H. R and fviau+lrnx, M . J. (1982) Cleavage and activation of human prothrombin by Echir carbratrrs venom. Tlrrornb . Res . 27, 591-b00. C,~~st~t, A . and Ctuwt~n, ß . P. M . (1982) In vitro coagulant properties of venoms from Australian snakes. Toxlcarr 20, SOI-504. Dwvm, E. W ., Ftr~awww, K. and Kts~., W . (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10,363-10,370 . Dwsot+, K. W. E . (1976) Clot-inducing substances present in snake venoms with particular reference to Echis carirwtur venom. 77tmmb . Res . 8, 351-360 .
1526
J . ROSING and G. TANS
DoYt.a, M . F. and Mwrtx, K. G . (1990) Multiple active forms of thrombin . IV . Relative activities of meizothrombins. J. biol. Chem . 266, 10,693-10,701 . Fexrox, J . W., Fwsoo, M . J ., STwctcxow, A. B., Axoxsox, D . L., Youra, A. M . and Ftxr.wYSOx, J . S. (1977) Human thrombins . Production, evaluation and properties of a-thrombin. J. biol. Chem. 252, 3587-3598 . FORTOVA, H., Dvx, J . E ., Voutuztcw, Z . and Kottxwt.tx, F . (1983) Isolation of the prothrombin-converting enzyme from fibrinogenolytic enzymes of Echù carirtater venom by chromatographic and electrophoretic methods . J. Chromat. 259, 47379 . Fxwxzw, B . R ., Ateortsox, D. L. and Fnvuvsox, J. S . (1975) Activation of human prothrombin by a procoagulant fraction from the venom of Echù carinates. J. biol. Chem. 250, 7057-7068 . Govetess-Rtt~rst.wa, J . W . P., Kxwrt=.x, M . J. H ., Twxs, G., ZWML, R . F . A . and Rostra, J . 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M. (1977) 77tromb . Haemost. 38, 567-577 . Joetx, F. and EsrouF, M . P. (1966) Coagulant activity of tiger snake (Notechù scutates scetates) venom. Nature 211, 873,875. Kotexwt.tte, F . and Br-o~xcx, B. (1975) Prothrombin activation induced by Ecarin-a prothrombin converting enzyme from Echù carinates venom . Thromb. Res. 6, 53 3 . Kotsrtwt nc, F . and Twuottsttw, E . (1978) Procoagulant and defibrinating potency of the venom gland extract of Thelotornù kirtlandi. Thromb . Res . 12, 991-1000. Kxtstn~twswwrnr, S ., Mwxx, K. G. and NFSt~tM, M . E . (1986) The prothrombinase~atalysed activation of prothrombin procceds through the intermediate meizothrombin in an ordered, sequential reaction. J. biol. Chem . 261, 8977984. Lot.t-wx, P., Pww~e, C . G., Kwssrtstet, P. J., Ltzwtt.t.px, R . D. and Fwss, D . N . (1987) Degradation of coagulation proteins by an enzyme from Malayan pit viper (Agkùtrodon rhodostoma) venom. Biochemùtry 26, 7627-7636. Mwxx, K . G ., Nlst~tur, M . E., Cttuxcty W . R ., Hwt.t:v, P . and Ktetst~nvwswwt~rsr, S . (1990) Surface-dependent reactions of the vitamin K-dependent enzyme complexes . Blood 76, 1-16. Mw4VHAi .L, L . R. and Ht~xt~twrx, R. P . (1983) Coagulant and anticoagulant actions of Australian snake venoma. Thromb . Kaemost. 50, 707-711 . Mwsct, P . P., WEa'rwxt:x, A. N. and Dtirt:ast=.u, J . (1988) Purification and characterization of a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilù textilù). Biochem . Int . 17, 825-835. Moxrrw, T. and Iwwxwaw, S . (1978) Purification and properties of prothrombin activator from the venom of Echù carinates . J. Biochem . 83, 559-570. Moterrw, T . and Iwwxwaw, S. (1980) Prothrombin activator from Echù carinates venom . Meth . Enzym . 80, 303-311 . Moxrrw, T., IWANAGw, S . and Suzutu, T . (1976) The mechanism of activation of bovine prothrombin by an activator isolated from Echù carinates venom and characterization of the new active intermediates. J. Biochem . 79, 1089-1108 . Moxrrw, T ., Mwrsuttoro, H ., IWANAGA, S . and Swtw, A . (1988) A prothrombin activator found in Rhabdophù tigrim~s tigrirets (Yamakagashi snake) venom. In: Hemostasù and Animal Venoms, pp. 556 (Pmtci .t:, H. and Mwx~r-wxn, F . S., Jte Eds). New York : Marcel Dekker . Nwxwawta, T., Ltx, P. and Ktst~ ., W . (1992) Activation of human factor VII by the prothrombin activator from the venom of Oxyeranes scetcllates (Taipan snake) . Thromb. Res . 66, 105-116 . NtEwtwxowstu, S ., KtxsY, E. P., Bxwzvtvs~t, T. M . and Sam, K . (1979) Thrombocytin, a serine protease from Bothrops atrox venom . 2 . Interaction with platelets and plasma-clotting factors. Biochemùtry 18, 3570-3577 . OuYwxa, C ., HONG, J . S . and Ttaxa, C: M . (1971) Purification and properties of the thrombin-like principle of Agkùtrodort sector venom and its comparison with bovine thrombin. 77womb. Diath . Haemorrh . 26, 224-234 . PrRr~ H ., Mwnt~uxn, F . S . and Tt~monott, I . (1976) Thrombin-like enzymes of snake venoms : actions on prothrombin . Thromb. Res. 8, 61927 . Rtma, M-J ., Monxts, S. and Kasow, D. P. (1982) Role of meizothrombin and meizothrombin-(des Fl) in the conversion of prothrombin to thrombin by the Echù carbates venom coagulant . Blochemùtry 21, 3437-3443. Rostra, J . and Twxs, G. (1988) Meizothrombin, a major product of factor Xa~atalysed prothrombin activation. Thromb. Haemost. 60, 355-360 .
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1527
Rasata, J . and Teas, G . (1991) Inventory of exogenous prothrombin activators. Report for the subcommittee on nomenclature of exogenous hemostatic factors of the scientific and standardization committee of the international society on thrombosis and haemoatasis. Thromb . Nacmost . 66, 62730 . ROBING, J ., Teas, G., Goveßs-R~etsuo, J . W . P ., Zweet ., R. F . A. and Hnricm, H . C. (1980) The role of phospholipids and factor V, in the prothrombinase complex . J. biol. Chem . 256, 274-283 . Roan~o, J ., ZwMI., R . F . A . and TwNS, G. (1986) Formation of meizothrombin as intermediate in factor X,~atalysed prothrombin activation . J. biol. Chem . 261, 4224-4228 . S»aizes, W ., H., TEtao, C: M . and Novoe, E . (1980) Pt+eparation of bovine prethrombin 2 : use of Awtin and activation with prothrombinase or Eearin . Thromb. Res. 19, 11-20. Bream, H ., GovEas-Ru~tsLeo, J. W . P., Zww,+L, R . F . A. and RosINa, J . (1986) Prothrombin activation by an activator from the venom of Oxyuramv scutellatus (Taipan snake) . J . biol. Chem . 261, 13,258-13,267 . Soma:, J . W . and JecICSON, C. M . (1977) Prothrombin structure, activation and biosynthesis. Physiol . Rev . 67, 1-65 . Teas, G., GovIIts-R~uG, J. W. P., v~u~t RuN, J. L . M . L. and Rasnva, J . (1985) Purification and properties of a prothrombin activator from the venom of Nottchis scutarus scutattu . J. biol. Chem . 2611, 9366-9372. Teas, G ., J~xs.~x-C~.w~x, T ., HER, H . C ., Zwwwt, R. F . A . and Rosnvc, J . (1991) Meizothrombin formation during factor X,~atalysed prothrombin activation. Formation in a purified system and in plasma . J. biol. Chem. 266, 21,864-21,873 . Ww. .s~a, F . J ., Owslv, W . G. and ESMON, C. T . (1980) Characterization of the prothrombin activator from the venom of Oxyurarrus scutcllatus scutellatus (Taipan venom) . Biochemistry 19, 1020-1023 . Wn.r ut~ts, V . and W~, J . (1989) Purification and properties of a pracoagulant from peninsula tiger snake (Notechis attr niger) venom. Toxicon 27, 773-779. YuxsISON, L. Ye., TANS, G ., TIIOI~sssN, M . L . C . G . D., Hrttxea, H . C . and Rosnvc, J . (1991) Procoagulant activities in venoms from central Asian snakes. Toxicon 29, 491-502.
