Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases

Pergamon

Torrwn. Vol. 34, No. 6. pp. 621 642. 1996 Copyright 1996 Elsevw Science Ltd Pnnted I” Great Bntam All rqhts reserved 0044)101!96 $15.00 + 0.00

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PII: S0041-0101(96)00017-7

REVIEW ARTICLE INSIGHTS INTO THE MECHANISM OF HAEMORRHAGE CAUSED BY SNAKE VENOM METALLOPROTEINASES AURA S. KAMIGUTI,‘* CHARLES R. M. HAY,’ R. DAVID G. THEAKSTON2 and MIRKO ZUZEL’ ‘Department of Haematology, Royal Liverpool University Hospital, University of Liverpool. Liverpool. U.K. and ‘Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, U.K. (Received I3 Nooember

1995; accepted

5 Febuarv 1996)

A. S. Kamiguti, C. R. M. Hay, R. D. G. Theakston and M. Zuzel. Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases. Toxicon 34,627-642, 1996.-Local and systemic haemorrhage are common consequences of crotaline and viperine envenoming. Several studies carried out using purified toxins have indicated that local haemorrhage can be attributed to a distinct class of venom metalloproteinases. Analyses of their cDNAs predict multi-domain enzymes, with an N-terminal metalloproteinase domain, a disintegrin-like domain and a Cys-rich C-terminus. Haemorrhagic metalloproteinases are responsible for degrading proteins of the extracellular matrix and they also have cytotoxic effects on endothelial cells. However, to date very few investigations have been carried out on the effects of venom haemorrhagic metalloproteinases on components of the haemostatic system. We describe here the effects of a high molecular weight haemorrhagic metalloproteinase, jararhagin, from the venom of a South American pit viper Bothrops jararaca, on platelet and plasma components involved in haemostasis. Jararhagin, which is not inhibited in plasma, causes the loss of the platelet collagen receptor ~~13,integrin (gpIa/IIa or VLA-2) and degrades the adhesive plasma protein von Willebrand factor. Alterations of these haemostatic components are known to result in bleeding. This suggests that venom haemorrhagic metalloproteinases, in addition to causing local bleeding, may also contribute to systemic haemorrhage. Copyright 0 1996 Elsevier Science Ltd

INTRODUCTION Manifestations of envenoming by viperid or both. Local effects which frequently

and crotalid snakes may be either local, systemic include pain, swelling, ecchymoses and local

* Author for correspondence: Aura S. Kamiguti, Department of Haematology, Royal Liverpool University Hospital, Prescot Street, Liverpool L69 3BX, U.K. Tel. (0151) 706 4311 Fax (0151) 706 5810. 627

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haemorrhage are usually apparent within minutes of the bite. Such signs are sometimes followed by necrosis of the area surrounding the bite. Systemic effects include alterations in blood coagulation or coagulopathy and various types of bleeding distant from the bite site, including gingival haemorrhage, purpura, macrohaematuria, epistaxis, uterine bleeding and haemoptysis. These symptoms occur because snake venoms are rich sources of both enzymatic and non-enzymatic pharmacologically active substances. Viperid and crotalid venoms possess components that can affect haemostasis by causing changes in blood coagulation and platelet function; this subject has been extensively reviewed in the past (Seegers and Ouyang, 1979; Ouyang et al., 1992; Hutton and Warrell, 1993; Kamiguti and Sano-Martins, 1995). Most of these components have coagulant property (e.g. conversion of fibrinogen to fibrin, activation of prothrombin or factor X), causing consumption of clotting factors, thus often rendering blood completely incoagulable. Numerous other venom toxins, enzymes and non-enzymes, have also been extensively reviewed in the past (Iwanaga and Suzuki, 1979; Ohsaka, 1979). Among these, several toxic enzymes lacking coagulant activity but possessing the ability to cause local haemorrhage have been characterized, as recently reviewed by Bjarnason and Fox (1994). This group, responsible for the induction of rapid local bleeding, consists almost entirely of zinc-containing metalloproteinases, varying in size from 20,000 to 100,000 mol. wt. Their effects on the blood vessel wall components have been extensively studied (see reviews by Bjarnason and Fox, 1988, 1989, 1994). The arrest of bleeding from the damaged blood vessels, however, depends on normal function of blood platelets and clotting factors, which are the principal blood components involved in haemostasis. The extent to which these latter components are directly affected by venom haemorrhagic metalloproteinases has not been explored in detail. The present review discusses the mechanisms of action of venom haemorrhagic metalloproteinases, in the light of our recent investigations into the effects of jararhagin, a principal metalloproteinase from the venom of a South American pit viper Bothrops jura~aca (Jararaca), on platelet and plasma proteins involved in haemostasis. MECHANISM

OF HAEMOSTASIS

The normal state of blood fluidity in the circulation is maintained by the non-thrombogenic properties of the blood vessel walls. Damage to blood vessels triggers a prompt response of haemostatic components to stop the haemorrhage (Bithell, 1993). These components are the vessel wall itself (which contracts due to the action of released vasoactive agents), the circulating platelets (because of their adhesive and aggregating properties) and the blood coagulation factors (which lead to the formation of the fibrin clot). In order to allow full tissue healing, the clots are subsequently removed by the fibrinolytic enzyme, plasmin. In situations where any component of these mechanisms are altered, haemostasis is compromised, resulting in bleeding. In small blood vessels, platelets alone can arrest bleeding. Their first reaction to the vessel damage is adhesion to the exposed subendothelium mediated by adhesive proteins, von Willebrand factor and collagen. The respective platelet receptors for these proteins are glycoprotein (gp) Ib/IX complex (Baumgartner et al., 1978; Weiss et al., 1986) and ~13, integrin, also known as gpIa/IIa complex (Santoro et al., 1988). Engagement of these receptors stimulates platelets to secrete their granular contents and in particular ADP, which promotes activation of platelet a,&, integrin or gpIIb/IIIa. This receptor binds the RGD-containing ligands (fibrinogen and von Willebrand factors) and thereby promotes platelet aggregation,

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resulting in the formation of a platelet plug, which stops bleeding. Thus, the function of platelets clearly depends on their granular content, their surface receptors and the plasma proteins which serve as the ligands of these receptors. INDUCTION

OF LOCAL

HAEMORRHAGE

BY METALLOPROTEINASES

Various studies have demonstrated that purified venom metalloproteinases cause local haemorrhage. Haemorrhagic metalloproteinases from crotalid and viperid venoms produce local bleeding by causing lesions in the walls of small blood vessels (Ohsaka, 1979; Ownby, 1990). It is believed that this is caused by proteolysis of components of the basal lamina of the microvasculature (Ohsaka et al., 1973; Bjarnason and Tu, 1978; Bjarnason rt al., 1988). In animal experiments, such disruption of microvessels becomes evident within minutes of an intradermal injection of venom metalloproteinases. The studies of their substrates have demonstrated that these enzymes degrade all major proteins of the extracellular matrix (ECM) (Bjarnason and Fox, 1988/89; Baramova et al., 1989, 1990). Thus, a positive correlation exists between the proteolytic activity of metalloproteinases and their haemorrhagic potencies; larger enzymes are more potent than the smaller ones in degrading the extracellular matrix (Bjarnason et al., 1988; Bjarnason and Fox, 1988j89). However, the proteolytic attack of these enzymes on ECM proteins is slow, while the in viuo haemorrhagic effect of the venom occurs within minutes of the bite or experimental injection. This indicates that the mechanism of action of these enzymes may be considerably more complex (Ownby, 1990; Lomonte, 1994). Two mechanisms by which erythrocytes and blood components escape from the blood vessels damaged by haemorrhagic toxins and enter the tissue compartments have been described. One is haemorrhage per diapedesis, through widened junctions between endothelial cells, and the other is per rhrxis, through gaps within the damaged endothelial cells (review by Ownby, 1990; Lomonte, 1994). Moreover, a direct cytotoxic effects of haemorrhagic metalloproteinases on endothelial cells in culture has been demonstrated, suggesting that such an effect may play an important role in the development of haemorrhage (Obrig et al., 1993). In conclusion, it has been well documented that haemorrhagic metalloproteinases, because of their broad substrate specificity, cause digestion of the extracellular matrix proteins and damage the integrity of blood vessels. Although this can explain the phenomenon of local bleeding, past studies have shed little light on the possibility that metalloproteinases can have effects on other components of the haemostatic mechanism and thereby contribute to systemic bleeding. SYSTEMIC

HAEMORRHAGE

CAUSED

BY ENVENOMING

As mentioned above, the presence of activators of clotting factors in the venoms frequently causes consumption coagulopathy in envenomed victims, which is often associated with systemic bleeding. In some envenomed patients systemic bleeding can be fatal (Warrell et al., 1975, 1977) and, interestingly, in one case fatal intracranial haemorrhage has been described apparently in the absence of coagulopathy (Kouyoumdjian et al., 1991). Moreover, there have been other reports describing spontaneous systemic bleeding following snake bite in which no coagulation defects have been recorded (Gitter and de Vries, 1968; Warrell et al., 1975; Kamiguti et al., 1992). Systemic bleeding without coagulopathy has also been reported in a case of envenoming

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by a colubrid snake (Silva and Buononato, 1983/84), which led to the discovery of one haemorrhagic metalloproteinase in this type of venom (Assakura et al., 1992). Experimental studies have shown that venoms of various other snakes can also cause pronounced local haemorrhage in the absence of coagulant activity (Gitter and de Vries, 1968; Theakston and Reid, 1983). In addition, in one such study using a purified non-coagulant haemorrhagic toxin systemic bleeding was observed (Kamiguti et al., 1991a), whereas in another study with a different haemorrhagic toxin such an effect could not be demonstrated (Kornalik, 1992). Therefore, different metalloproteinases appear to differ in their ability to cause systemic effects; this clearly requires further in depth studies of the effects of these enzymes on the haemostatic mechanism.

METALLOPROTEINASEAND

DISINTEGRINS:STRUCTURE/FUNCTION RELATIONSHIPS

Recently, the elucidation of the primary structure of different members of the venom haemorrhagic metalloproteinasedisintegrin family (Takeya et al., 1990; Paine et al., 1992; Hite et al., 1992; Kini and Evans, 1992) has generated new interest in the function of these enzymes. Comparison of amino acid sequences have indicated that, despite differences in their molecular sizes, all these enzymes may be related through a common ancestral gene (Hite et al., 1992; Paine et al., 1992). A multi-domain structure characterizes the large haemorrhagic metalloproteinases; the N-terminal region possessing a zinc-containing metalloproteinase domain is followed by a disintegrin-like and a C-terminal cysteine-rich domain. Moreover, disintegrin-like domains of high molecular weight metalloproteinases possess a high sequence homology to the venom disintegrins. Disintegrins are low molecular weight non-enzymatic venom components containing RGD or KGD sequences by which they bind to platelet surface integrins and thereby potently inhibit platelet aggregation (Huang et al., 1987; Gan et al., 1988; Scarborough et al., 1993). However, the disintegrin-like domains of the metalloproteinases do not have an RGD sequence. The haemorrhagic metalloproteinase (from B. jararaca venom), jararhagin (Paine et al., 1992) like most other venom haemorrhagic and non-haemorrhagic metalloproteinases, has a conserved ECD sequence near the region where an RGD sequence is found in the ‘true’ disintegrins (Fig 1). However, in ecarin, a prothrombin-activator metalloproteinase, a DCD sequence was found instead of ECD (Nishida et al., 1995). While it is known that

Jararhagin

CRASMSECDPAEHC

HRlB

CRAAESECDIPESC CRGIRSECDLAEHC

ECH-

II

RW/X PH-308 Trigramin Echistatin Fig.

CRRARDECDVPEHC CRESTDECDLPEYC CRIARGD-DMDDYC CKRARGD-DMDDYC

I. Comparison of the predicted amino acid sequences of a limited region of the metalloproteinase disintegrin-like domain and venom disintegrins. This region comprises residues 464478 of the following haemorrhagic metalloproteinases: Jararhagin from Bothrops jararaca (Paine et al., 1992) HRIB from Trimeresurus _/?avoviridis (Takeya et al., 1990) and ECH-II from Echis pyramidum leakeyi venoms (Paine et al., 1994). RVV/X is a factor X-activator metalloproteinase from Russell’s viper venom (Takeya et al., 1992). PH-30 13 is a sperm protein from guinea pig (Blobel et al., 1992). Trigramin and Echistatin are disintegrins from Trimeresurus gramineus (Neeper and Jacobson, 1990) and Echis carinatus venoms (Gan et a/., 1988), respectively. Note the presence of a conserved ECD sequence in all metalloproteinases near the RGD sequence of the disintegrins.

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the RGD sequence in true disintegrins has cell-binding function, the function of the disintegrin-like domain in the metalloproteinases is still unknown. Sequence homology to the disintegrin-like domain of venom haemorrhagic metalloproteinases has also been found in a sperm protein, considered to be a candidate for egg binding in sperm-egg fusion (Blobel et al., 1992). Recently, it was shown that the disintegrin-like region of this sperm protein recognizes a&, integrin on the egg membrane (Almeida et al., 1995). It is possible that similar domain found in venom haemorrhagic metalloproteinase has a function in cell recognition. Since the large haemorrhagic metalloproteinases possess a disintegrin-like domain, it is thought that some disintegrins may originate from autoproteolysis of these enzymes (Takeya et al., 1993). A recent demonstration that a venom component, jararhagin C, which is identical to the disintegrin-like domain of jararhagin, causes inhibition of platelet aggregation (Usami et al., 1994) supports this proposal. Purified jararhagin interferes with platelet function, in particular with collagen- (Kamiguti et al., 199la, 1995a) and ristocetin-induced platelet aggregation (Kamiguti et al., 1995b). This implies that jararhagin could be affecting either the platelet itself, the different ligands involved in platelet function, or both.

EFFECTS

OF HAEMORRHAGIC

METALLOPROTEINASES

ON BLOOD

COMPONENTS

Of all venom components known to interfere with haemostasis, the haemorrhagic metalloproteinases have been the least investigated. Little attention has been paid to the effects of these enzymes on platelets which, as mentioned before, play the central role in the haemostatic mechanism. Earlier studies of venom metalloproteinases showed that they can inhibit platelet aggregation in vitro (Ouyang et al., 1979). Since the majority of these enzymes are also known to hydrolyse fibrinogen, and since fibrinogen is an important cofactor in platelet aggregation (Hawiger et al., 1982; Plow et al., 1984), it has been proposed that the inhibition of platelet aggregation caused by these enzymes is due to degradation of fibrinogen (Teng and Huang, 1991; Ouyang et al., 1992). We have investigated the effects of envenoming by B. jararaca, because this species constitutes the major problem in southeastern Brazil, and local and systemic haemorrhage is one of the most prominent results of envenoming by this species (Rosenfeld, 1971; Maruyama et al., 1990; Cardoso et al., 1993). The main venom component responsible for this effect is thought to be jararhagin, which causes intense local haemorrhage, inhibits platelet aggregation in vitro and, as shown in animal experiments, also contributes to systemic bleeding (Kamiguti et al., 1991a). This haemorrhagic metalloproteinase has a disintegrin-like domain whose function in intact jararhagin is not precisely known; however, here we present evidence suggesting that this domain may play an important role in the substrate specificity of the enzyme. Jararhagin could interfere with platelet function in two ways: first, by degrading different platelet receptors and adhesive proteins involved in haemostasis, and, second, by a non-enzymatic (disintegrin-mediated) interference with the function of platelet adhesion receptors. The effects of jararhagin on platelet and plasma proteins involved in haemostasis are described below. We demonstrate that jararhagin can contribute to the systemic bleeding of envenoming because this enzyme is not effectively inhibited by plasma proteinase inhibitors and proteolytically degrades the main platelet collagen receptor, a,IJ, integrin, and the adhesive ligand, von Willebrand factor.

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Interaction of jararhagin with plasma proteinase inhibitors During platelet aggregation studies, jararhagin was always more effective in washed platelet suspensions than in platelet-rich plasma. Thus, in plasma, either competitive substrates or enzyme inhibitors interfere with the effects of jararhagin on platelets. The inhibitors are particularly important, since the survival of jararhagin in the circulation would critically depend on their efficacy. Excessive proteolysis in plasma is physiologically controlled by various proteinase inhibitors, notably a,-proteinase inhibitor (Arnaud and Chapuis-Cellier, 1988) a,-macroglobulin (Q-M), Cl-esterase inhibitor (Shapira et al., 1988), antithrombin III and a,-antiplasmin (Wilman and Collen, 1978). Previous studies of several crotalid venoms have shown that plasma ccz-M effectively inhibits the proteolytic effects of some of these venoms (Kress and Catanese, 1981). Some purified low molecular weight haemorrhagic metalloproteinases from Crotalus atrox (Western diamondback rattlesnake) venom are also effectively inhibited by a,-M (Baramova et al., 1990). In contrast, a,-M failed to inhibit Ht-a, a large metalloproteinase from the same venom (Baramova et al., 1990). a,-M is a large (720,000 mol. wt) glycoprotein consisting of two noncovalently linked pairs of identical disulphide-linked subunits. This protein can form 1:1 or 2:1 stoichiometric complexes with various enzymes including metalloproteinases (Barrett and Starkey, 1973; Sottrup-Jensen et al., 1980). Following attack by proteinases, the middle portions of a,-M subunits (bait regions) are cleaved; this causes a conformational change which results in the enzyme becoming physically entrapped by the inhibitor. During this conformational change, the internal Cys-Glu thioester bonds of a,-M become transiently available for nucleophilic attack by the lysyl t-amino groups of the enzyme, followed by covalent enzyme-inhibitor binding via y-glutamyl(a,-M)-c-lysyl(proteinase) cross-links (Sottrup-Jensen et al., 1981). The overall result of these reactions is that the activity of the bound enzyme is restricted to low molecular weight substrates only (Barrett et al., 1979; Sottrup-Jensen et al., 1990). When radiolabelled jararhagin was incubated with whole plasma and its binding to plasma proteins examined by immunoelectrophoresis, it was demonstrated that only a,-M was capable of binding jararhagin. However, jararhagin retained considerable proteolytic activity towards high molecular weight substrates, such as fibrinogen and hide powder azure, in the presence of a large molar excess of a,-M (Kamiguti et al., 1994a). Others have also shown that some venom metalloproteinases are not completely inhibited by equimolar amounts of a,-M (Pandya and Budzynski, 1984; Baramova et al., 1990) but the exact reason why the inhibitor-enzyme reaction failed to proceed to completion was not clarified. We demonstrated that jararhagin cleaved the inhibitor subunits, but failed to become covalently linked and entrapped in a manner which would prevent its interaction with high molecular weight substrates. Also, when the interaction of jararhagin with a,-M was studied using SDS-PAGE (Laemmli, 1970) in the presence of whole venom, it was found that other venom components can compete with jararhagin for a,-M (Kamiguti et al., 1994a). As such competition could also take place with enzymes intrinsically generated during Bothrops envenoming (i.e. thrombin and plasmin), it is likely that the inhibition of jararhagin in the circulation is ineffective. This is supported by earlier studies, using an experimental rodent model, in which jararhagin alone caused systemic bleeding (Kamiguti et al., 199la). Systemic bleeding without coagulopathy has also been recorded in cases of human envenoming by B. jararaca (Kamiguti et al., 199lb, 1992). The results of our more

Insights into the Mechanism of Haemorrhage

633

recent studies suggest mechanisms by which jararhagin can participate in the aetiology of such bleeding.

Effects of jararhagin on platelet surface proteins The most pronounced effects of jararhagin on platelets in platelet-rich plasma in aitro were the inhibitions of both collagen- and ristocetin-induced cell aggregation. This suggested either the proteolysis by jararhagin of collagen and von Willebrand factor (vWF), or an interference with their respective platelet receptors, ccJ3, integrin (gpIa/IIa or VLA-2) and gpIb. We describe here first the effects of jararhagin on platelet receptors and later its effects on adhesive protein ligands, fibrinogen and vWF. The collagenase activity of jararhagin is excluded as a mechanism of collagen-induced platelet aggregation inhibition, because jararhagin selectively degrades non-fibrillar type IV collagen (Maruyama et al., 1992) which is not used in platelet aggregation studies. The major platelet receptors for macromolecular ligands involved in adhesion and aggregation are gpIb (von Willebrand Factor and thrombin receptor), cr,,& integrin or gpIIb/IIIa (receptor for fibrinogen and several other RGD-containing ligands), ~(~13, integrin (collagen receptor) and gpIV (thrombospondin receptor). Of these receptors, gpIb is highly susceptible to proteolysis by both endogenous and exogenous proteinases (Cooper et al., 1982; Wicki and Clemetson, 1985). This is because the exposed extracellular region of gpIb, known as glycocalicin, is a good substrate for different enzymes. The proteolysis of CX,,$,integrin has also been extensively studied (Calvete et al., 1992) but little is known about the proteolysis of other platelet receptors. In jararhagin-treated platelets, no alterations of gpIb could be detected either by flow cytometry or by immunoprecipitation (Kamiguti et af., 1995a). This indicated that jararhagin inhibition of platelet responses to ristocetin/vWF complex may have been due to some effect on vWF. There are several proposed platelet collagen receptors including a,13,and a,,,& integrins and gpIV. a,&, integrin binds collagen only after platelet activation. crJ3, integrin is the primary receptor which, upon collagen binding, generates the signal for activation of the secondary receptor c(,,,&. The role of gpIV in the interaction of collagen with platelets has been questioned recently, because platelet responses to collagen are normal in subjects deficient in this receptor (McKeown et al., 1994). When platelets were treated with jararhagin, gpIV appeared unchanged judged by a fully preserved cell response to 0KM5, a mAb raised against gpIV (Talle et al., 1983). Moreover, our observation that gpIV remained intact in jararhagin-treated platelets, which had lost their ability to interact with collagen, supports the lack of involvement of gpIV in the platelet/collagen interaction. a,,& Integrin was also found to be both structurally and functionally unaltered in jararhagin-treated platelets (Kamiguti et al., 1995a). This was expected from the preserved fibrinogen-dependent platelet aggregation, which is mediated by cr,,,B, integrin. In contrast, the expression of platelet LQ, integrin in jararhagin-treated (Fig. 2) and platelets was markedly reduced, as shown by how cytometry immunoprecipitation (not shown). This change in cc,D,integrin not only explains the altered responses of platelet to collagen, but also supports the hypothesis that txJ3, integrin plays a vital role in the platelet/collagen interaction (Santoro, 1986; Staatz et al., 1989). Moreover, since congenital deficiency of this receptor leads to bleeding manifestations (Nieuwenhuis et al., 1985), it is to be expected that jararhagin-treated platelets would not be fully haemostatically competent.

634

A. S. KAMIGUTI

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Fig. 2. Flow cytometry of jararhagin-treated platelets with anti-a, mAb(Gi 9). Platelets in PRP (top panels) or washed suspensions (bottom panels) containing 3 x lO*/ml platelets were treated with 10 ,ug/ml jarahagin for 10 min. Paraformaldehyde-fixed platelets were then washed and reacted with anti-gpIa mAb (2 pg/lO’ cells), followed by a FITC-conjugated anti-mouse IgG. Panel a = control PRP; panel b = jararhagin-treated PRP; panel c = control washed platelets; panel d = jararhagin-treated platelets.

Efects on plasma proteins involved in haemostasis Fibrinogen degradation by jararhagin: functional consequences. Venom metalloproteinases are also known to digest fibrinogen and are therefore referred to as either LYor B-fibrinogenases, depending on whether the enzyme preferentially degrades the Acr or the BD chains of the fibrinogen molecule (Markland, 1991). The majority of haemorrhagic metalloproteinases are a-fibrinogenases. Because some venom a-fibrinogenases have also been shown to inhibit platelet aggregation, their inhibitory activity on platelets was attributed to the fibrinogen degradation (Ouyang et al., 1987; Ouyang et al., 1992; Teng and Huang, 1991). However, snake venom a-fibrinogenases inhibit platelet function even in defibrinogenated blood, suggesting that other mechanisms may also play a part (Kini and Evans, 1991). Whether D-fibrinogenases of this group of enzymes have any effects on platelet function has never been investigated. The fibrinogen molecule (330,000 mol. wt) is a dimer composed of three pairs of disulphide-linked chains termed Aa, BB and y chains (Doolittle, 1984). Fibrinogen functions both as a ligand in platelet aggregation and as a substrate for thrombin in blood coagulation. Stimulation of platelets with ADP, adrenalin or thrombin permits fibrinogen binding and platelet aggregation by altering the conformation of the integrin receptor, a,,&. Fibrinogen binding to activated tx,& is mediated through two types of specific site. One contains an Arg-Gly-Asp (RGD) sequence including Aa 95-97 and Aa 572-574

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of Haemorrhage

(Hawiger et al., 1989). The other is the dodecapeptide 400-411 (HHLGGAKQAGDV) at the carboxy-terminal end of the y chain (Hawiger et al., 1982; Kloczewiak et al., 1984). The fact that fibrinogen-dependent platelet aggregation in response to ADP is unaffected by jararhagin, led us to question the previous hypothesis that the aggregation inhibition by venom metalloproteinases is the result of fibrinogen CYchain degradation (Teng and Huang, 1991; Ouyang et al., 1992). We therefore reexamined the function of jararhagin-treated fibrinogen in platelet aggregation. We found that jararhagin cleaves fibrinogen in the C-terminal portion of the Acr chains, resulting in the removal of a 23,000 mol. wt fragment containing one RGD sequence thought to interact with platelet ~l,,,,l3~ integrin. There were no changes in either the I3 or 7 chains (Fig. 3). The purified remnant fibrinogen molecule (290,000 mol. wt) was still fully functional in platelet aggregation responses to stimulation with ADP or adrenalin (Kamiguti et al., 1994b). The results shown in Fig. 4 are fully compatible with a previous observation that the loss of this C-terminal RGD binding site due to a limited proteolysis of fibrinogen by plasmin also did not alter the fibrinogen-platelet interaction (Peerschke and Galakanis, 1983). The finding that the y chains of fibrinogen are resistant to proteolysis by jararhagin not only

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Fig. 3. SDS-PAGE of jararhagin-treated fibrinogen Following the treatment of human fibrinogen with 12 pg/ml jararhagin for IO min, the reaction WZIS stopped by 5 mM EDTA. Proteins separated under non-reducing conditions (5-15% gel): lane 1 = intact fibrinogen; lane 2 = jararhagin-treated fibrinogen; lane 3 = Sephacryl S-300 gel-filtered jararhagin-treated fibrinogen. Same materials under reducing conditions (10% gel) are shown, respectively, in lanes 4-6 (Kamiguti e/ al., 1994b).

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Concentration of fibrinogen (pglml) Fig. 4. Binding of ‘ZSI-labelledfibrinogen to ADP-stimulated platelets. Human washed platelets (6 x 108/ml) were stimulated with 80 PM ADP in the presence of unlabelled intact fibrinogen containing 0.5% by weight of either ?-labelled untreated fibrinogen (0) and jararhagin-treated fibrinogen (m) for 30 min, without stirring. Radioactivity of the pellets was measured. Specific binding was obtained by subtracting non-specific binding in similar mixtures without ADP. Results are means f SD (n = 3) (Kamiguti et al., 1994b).

explains the preserved aggregation, but also confirms that the C-terminal dodecapeptide of y chains is indeed the principal platelet-binding site of fibrinogen (Kloczewiak et al., 1984). Thrombin proteolysis of Acr (Arg’6Gly’7) and Bl3 (Arg’4-Gly’5) chains cleaves fibrinopeptides A and B from the amino-terminal ends of these chains (Blomback and Vestermark, 1958). This results in the formation of fibrin monomers which contain polymerization sites ‘A’ and ‘B’ in the new N-termini of c( and I3 chains, respectively (Blomback et al., 1978; Olexa and Budzynski, 1980). These polymerization sites have complementary sites ‘a’ and ‘b’ located in the carboxy-terminal region of the fibrinogen molecule (Kudryk et al., 1973, 1974). The fibrin monomers first polymerize by end-to-end association into double-stranded protofibrils which then associate side-by-side into fibrin fibres forming a solid clot (Blomback et al., 1978). Although fibrinogen was clottable by thrombin following proteolysis of Aa chains by jararhagin, the polymerization of the fibrin formed from jararhagin-treated fibrinogen was defective (Kamiguti et al., 1994b). This indicated the loss of a polymerization site located in the C-terminal region of the Aa chains. This site was recently identified in a congenital dysfibrinogenemia (Fibrinogen Marburg) which shows a polymerization abnormality caused by absence of 149 amino acids from the C-terminal end of fibrinogen Aa chains 1992). Although our studies showed that jararhagin is an (Koopman et al., a-fibrinogenase, in contrast to previous opinion they clearly demonstrate that the a-fibrinogenase activity of venom haemorrhagic metalloproteinases is not responsible for the platelet inhibitory effects of these enzymes. The only consequence of such fibrinogenolytic activity which could contribute to defective haemostasis is the fibrin polymerization abnormality observed when jararhagin-treated fibrinogen was converted to fibrin by thrombin.

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Proteolysis of von Willebrand factor (v Wf> by jararhagin: functional consequences. In addition to inhibiting collagen-induced aggregation, jararhagin also interfered with the platelet response to ristocetin. Since, as described above, jararhagin did not attack the receptor for von Willebrand factor (gpIb), the most likely explanation for the inhibition of ristocetin-induced aggregation by jararhagin was a direct effect on vWF itself. vWF plays an important role in haemostasis following vascular injury by first binding to the subendothelium and then to platelet gpIb (Baumgartner et al., 1978; Weiss et al., 1986). This reaction is the first event in the formation of haemostatic plugs. In plasma, vWF is found in the form of disulphide-linked multimers of different sizes ranging from 500,000 to > 20,000,OOOmol. wt (Ruggeri and Ware, 1992; Zimmerman et al., 1986). The high molecular weight multimers are principal haemostatically functional species. Reduced vWF is a 225,000 mol. wt single-chain protein of 2050 amino acids (Titani et al., 1986). However, when vWF from normal plasma is reduced, fragments of 189,000, 176,000 and 140,000 mol. wt can be found together with the intact 225,000 mol. wt subunit (Berkowitz et al., 1987). Each native vWF subunit contains the Va1449-Lys72* domain responsible for ristocetin-induced vWF binding to platelet gpIb, and one binding site for platelet a,,& integrin containing the RGDS’7M-‘747 sequence (Titani et al., 1986). We have studied the effect of jararhagin on vWF structure and function using both purified vWF and vWF present in plasma. With purified reactants, we demonstrated that jararhagin cleaves vWF subunits in the N-terminal half of the molecule generating a number of fragments (Kamiguti et al., 1995b). SDS-PAGE of non-reduced protein revealed that the cleavage of vWF by jararhagin results in the appearance of two main products of 270,000 and 230,000 mol. wt, and two minor fragments of 160,000 and 115,000 mol. wt. Western blot analysis showed that the cleavage occurs mainly in the N-terminal portion of the molecule, giving rise to a small fragment of 58,000 mol. wt (Fig 5). Some of these fragments are similar to those generated during physiological catabolism of circulating vWF and, as shown in previous and present experiments, with plasmin. However, at least one major N-terminal jararhagin-specific fragment (58,000 mol. wt) was also observed when vWF was reduced to its constituent monomers. These internal cleavages of vWF caused the disappearance of high molecular weight multimers. This was explained by changes in protein conformation upon proteolysis which lead to the rupture of labile disulphide bonds which hold subunits together in their N-terminal ends within the structure of the multimer (Dong et al., 1994). Since small multimers are not haemostatically effective molecular species, the disappearance of the large multimers upon vWF treatment with jararhagin could seriously impair haemostasis. Although we did not thoroughly investigate the proteolysis of vWF in plasma, our work shows that the treatment of platelet-rich plasma with jararhagin also inhibited the response of platelets to ristocetin. It is possible that jararhagin cleaves a part of the vWF molecule (Al domain) which contains the site responsible for vWF binding to platelet gplb. In the integrin a,I3, (collagen receptor), a homologous domain (I domain) has been identified in the CQchain as the putative binding site for collagen (Kamata et al., 1994). Since jararhagin has been shown here to degrade both vWF and platelet a&3,integrin, this strongly suggests the I domain as the preferential cleavage site of these proteins by jararhagin. A cleavage of vWF within Al domain could explain the inhibitory effect of this enzyme on the ristocetin-induced platelet aggregation in platelet-rich plasma. It can thus be assumed that the disruption of vWF structure caused by jararhagin, and possibly other enzymes contained in B. jararaca venom, contributes to the bleeding process which is often observed in envenomed patients. Jararhagin-induced proteolysis of vWF in

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'4225.

189

4176~

145 130

140 84 58

5678

1234

Fig. 5. Immunoblots of N- and C-termini fragments of reduced vWF: degradation by jararhagin, whole B. jararaca venom and plasmin. The purified human vWF preparation (kindly provided by Dr C. Mazurier from the Centre de Transfusion Sanguine, Lille, France) was incubated for 60 min with IO pg/ml of either jararhagin or venom, or with 0.1 IU/ml plasmin. Blots were developed using M7 (lanes 14) and M31 (lanes

5-8) mAbs (kindly provided by Dr 2. M. Ruggeri, Scripps Clinic and Research Foundation, California, U.S.A.) against epitopes LyP-Met**” and Leu”‘‘-Met’@‘,respectively. Lanes 1 and 5 = control vWF; lanes 2 and 6 = jararhagin-treated vWF; lanes 3 and 7 = B. ,jararaca venom-treated vWF; lanes 4 and 8 = plasm&treated vWF. The 225,000 mol. wt band, vWF subunit,

conjunction haemostatic components

is recognized

by both mAbs.

with its effects on platelet-collagen interaction can impair the first step of the process and this, together with the consumption coagulopathy due to other of B.juraraca venom, can be life-threatening.

CONCLUDING REMARKS 1. Here, we introduce the haemorrhagic metalloproteinases with a disintegrin-like domain (high molecular weight metalloproteinases) as venom components with a new role in the field of toxinology. Apart for being responsible for local bleeding in envenoming, they may also play a role in the systemic bleeding. Platelets and principal plasma proteins involved in haemostasis are targeted by these enzymes, as demonstrated using jararhagin. It is clear that more work on the structure/function relationships of the metalloproteinases needs to be carried out to fully understand how they interfere with haemostasis. For example, the role of the disintegrin-like domain in the inhibition of platelet function requires further clarification.

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2. Venom haemorrhagic metalloproteinases are excellent tools for investigating mechanisms of ligand/receptor and cellkell interactions. This area of cell biology still needs to be explored to fully understand the role of these interactions in normal cell function. 3. With regard to systemic bleeding, it is clear that haemorrhagic metalloproteinases can contribute to some such bleeding manifestations of envenoming, although it is appreciated that these enzymes are not the sole cause of bleeding.

Acknowledgements-We wish to thank Dr J. R. Slupsky from the Haemostasis Research Unit, Max Planck Institute, Germany, who formerly worked in our department, and Drs H. P. Desmond and G. Laing from the Venom Research Unit, Liverpool School of Tropical Medicine, U.K., for their help and advice during the course of this study. We also acknowledge the Wellcome Trust for funds which enabled this work to be presented by A. S. K. at the XIIIth Meeting of the International Society of Haematology in Istanbul (3-8 September, 1995).

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