Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets.

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.

Failure of chloramphenicol prophylaxis to reduce the frequency of abscess formation as a complication of envenoming by Bothrops snakes in Brazil: a double-blind randomized controlled trial.

Bites by many species of venomous snake may result in local necrosis at, or extending from, the site of the bite. The use of prophylactic antibiotics to prevent infection as a complication of local necrotic envenoming is controversial. A double-blind randomized controlled trial was carried out to assess whether antibiotic therapy is effective in this situation. Two hundred and fifty-one patients, with proven envenoming by snakes of the genus Bothrops, admitted to two hospitals in Brazil, between 1990 and 1996, were randomized to receive either oral chloramphenicol (500 mg every six hours for five days) or placebo. One hundred and twenty-two of these patients received chloramphenicol (group 1) and 129 were given placebo (group 2). There were no significant differences between the groups at the time of admission. Necrosis developed in seven (5.7%) patients in group 1 and in five (3.9%) patients in group 2 (P>0.05) while abscesses occurred in six patients (4.9%) in group 1 and in six (4.7%) patients in group 2 (P>0.05). In conclusion, the use of orally-administered chloramphenicol for victims of Bothrops snake bite with signs of local envenoming on admission, is not effective for the prevention of local infections.

Age-related biological activity of south american rattlesnake (Crotalus durissus terrificus) venom

An in vitro and in vivo comparative study was performed on the effects of Crotalus durissus terrificus venoms from a mother and its 15 newborns. The venoms were tested for protein content, lethality, proteolytic, myotoxic, hemorrhagic, and phospholipase A2 activity. The minimum coagulant dose in plasma and human fibrinogen, protrhombin, and Factor II activations were analyzed. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE). This showed that despite similar total protein content, the biological effects of the venoms were different. Venom from young snakes exhibited higher enzymatic and coagulant activities and higher myotoxicity compared to the mother's. In addition, the PLA2 content paralleled myotoxicity. However, no difference could be detected in their toxicity (LD50 0.08 mg/Kg). High incidence of blood coagulation disorders and elevated circulating myoglobin may characterize systemic envenoming by young C. d. terrificus.

Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom.

BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 A.

Coagulopathy following lethal and non-lethal envenoming of humans by the South American rattlesnake (Crotalus durissus) in Brazil.

The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for approximately 10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in south-eastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom.

The inhibition of platelet aggregation and blood coagulation by Micropechis ikaheka venom.

Uncoagulable blood and life-threatening bleeding can result from the action of some snake venom toxins on haemostatic components of blood and vessel walls. Although envenoming by Micropechis ikaheka primarily affects neurones and muscle cells causing post-synaptic neuromuscular blockade and rhabdomyolysis, disturbances of haemostasis also occur. Therefore, the present study explored the effects of M. ikaheka venom on platelets and endothelium, which are important components of the haemostatic mechanism. The venom inhibited platelet aggregation in response to ADP and collagen, and also delayed clotting dependent on platelet activation or endothelial cell tissue factor expression. Some of these effects were reduced by the incubation of venom with a phospholipase A2 (PLA2) inhibitor and could be reproduced by a 17 kDa venom fraction containing a PLA2. In addition, an 11 kDa fraction containing a long-chain neurotoxin reduced ADP-induced aggregation. The venom was also found to reduce endothelial cell adherence to vitronectin-, fibronectin- and collagen-coated surfaces. These results suggest that, by inhibiting procoagulant activities of platelets and endothelial cells, a 17 kDa PLA2 plays an important role in the anticoagulant action of M. ikaheka venom.

Characterization of aspercetin, a platelet aggregating component from the venom of the snake Bothrops asper which induces thrombocytopenia and potentiates metalloproteinase-induced hemorrhage.

Thrombocytopenia occurs in a number of patients bitten by Bothrops asper, a species responsible for the majority of snakebites in Central America and southern Mexico. In this work we describe the isolation of a new platelet-aggregating protein, named aspercetin, from the venom of B. asper, which induces thrombocytopenia in mice. Isolation was carried out by a combination of ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on Affi-Gel Blue. Aspercetin is a disulfide-linked heterodimer, with a pI of 4.5 and a molecular mass of 29,759 Da, detemined by MALDI-ESI mass spectrometry. N-terminal sequence shows homology with a number of venom proteins which belong to the C-type lectin family. Aspercetin has functional similarities with botrocetin, from B. jararaca venom, since it induces platelet aggregation only in the presence of plasma or purified von Willebrand factor. Aspercetin-mediated platelet aggregation results from the interaction of von Willebrand factor with platelet receptor GPIb. Aspercetin lacks anticoagulant effect and does not agglutinate erythrocytes, in contrast with other representatives of the C-type lectin family isolated from snake venoms. Moreover, aspercetin is not lethal, nor does it induce myonecrosis, hemorrhage and edema. When injected intravenously or intramuscularly in mice it induces a rapid, dose-dependent drop in platelet counts and prolongs the bleeding time, suggesting that it may play a role in the thrombocytopenia that develops in a number of B. asper envenomations. Moreover, mice injected intravenously with aspercetin and then receiving an intradermal injection of B. asper hemorrhagic metalloproteinase BaP1 develop a larger hemorrhagic lesion than mice receiving only BaP1. This suggests that aspercetin, by reducing platelet numbers, may

Coagulant and anticoagulant activities of Bothrops lanceolatus (Fer de lance) venom.

Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.

The use of snake venom toxins as tools to study platelet receptors for collagen and von Willebrand factor.

A large proportion of the biologically active proteins and peptides present within snake venoms interact with components of the haemostatic system to promote or inhibit the normal sequence of events that lead to clot formation. The venom proteins achieve their effects through interaction with various components of the coagulation cascade, endothelial matrix and platelets. Within the latter group, a number of venom proteins target the interaction of platelets with the major adhesive proteins, von Willebrand factor and collagen. The venom proteins bind either the adhesive protein itself or their receptors on the platelet surface, notably GP-Ib-IX-V and GPVI. This review discusses the substantial contribution that venom proteins have made to our understanding of the role of these two adhesive proteins and their receptors (excluding GPIIb-IIIa) in platelet regulation.

Activation-dependent proteolytic degradation of polymorphonuclear CD11b.

CD11b/CD18 is the principal integrin of polymorphonuclear (PMN) leucocytes and is involved in their adhesion, migration and phagocytosis. In quiescent cells, the receptor is stored in intracellular granules from where it is translocated to the cell surface in response to a variety of stimuli. In this study, we demonstrated that strong stimulation of PMNs not only leads to the upregulation of CD11b surface expression, but also to the subsequent time-dependent apparent loss of this receptor, as detected by fluorescence-activated cell sorting (FACS) using a monoclonal antibody (mAb) against an N-terminal CD11b epitope. This epitope loss was observed following either direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or after multiple receptor stimulation using a combination of the agonist N-formylmethionyl-leucyl-phenylalanine (FMLP) and the priming agents granulocyte macrophage-colony stimulating factor (GM-CSF) and platelet factor (PF) 4. However, upregulation following weak stimulation with FMLP alone was not followed by subsequent epitope loss of the receptor. The increases and subsequent decreases in CD11b expression induced by PMA were paralleled by an increase and a decrease in PMN adhesion to CD11b-specific ligands, fibrinogen and intercellular adhesion molecule (ICAM)-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that this epitope loss of PMN CD11b was the result of proteolytic degradation of the N-terminal region of the molecule. The use of a range of proteinase inhibitors indicated that this CD11b degradation involves a cell-associated serine proteinase. This is the first demonstration of the proteolytic alteration of CD11b in response to strong PMN stimulation. Given the central role of CD11b/CD18 in all aspects of PMN function, this alteration of the CD11b molecule and its effect on PMN adhesion are probably of considerable pathophysiological importance.

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus).

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.

Evidence against a direct role of the integrin alpha2beta1 in collagen-induced tyrosine phosphorylation in human platelets.

In the present study we have investigated whether the collagen receptor alpha2beta1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with alpha2beta1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the beta1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of alpha2beta1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of alpha2beta1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than alpha2beta1 as previously thought. These observations show that the integrin alpha2beta1 is not required for regulation of tyrosine phosphorylation by collagen.

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation.

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).

Snake venom metalloproteinases and disintegrins: interactions with cells.

Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin.

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

The platelet antigens CD9, CD42 and integrin alpha IIb beta IIIa can be topographically associated and transduce functionally similar signals.

Investigation of the specific effects of different mAb known to stimulate platelets (agonist mAb) is complicated by interaction of the Fc portion of these mAb with the platelet Fc gamma RII. This has led to the conclusion that nearly all agonist-mAb-induced activation of platelets is mediated by this receptor. However, the target antigen-mediated signal can be analysed provided that the effects of Fc gamma RII engagement can either be reduced or eliminated. We have therefore blocked platelet Fc gamma RII with IV.3 Fab fragments (an anti-Fc gamma RII mAb), and stimulated the platelets by cross-linking intact agonist mAb with F(ab')2 fragments of an Fc-specific anti-mouse antibody. By analysing functional platelet responses and protein-tyrosine phosphorylation, we found that such non-Fc gamma RII-mediated cross-linking of CD9, CD42 and glycoprotein (gp) IIb/IIIa generates closely similar signals. Since this may indicate molecular associations, we analyzed the surface topography of platelets using the chemical cross-linking agent dithiobis(sulfosuccinimidyl propionate). We found that a proportion of CD9, gpIIb/IIIa and CD42 molecules associate with each other on the platelet surface membrane. Thus, our results suggest that these antigens are able to form a larger molecular complex and induce similar signals. Furthermore, cross-linking of CD9 and CD42 stimulated thrombasthenic platelets completely lacking gpIIb/IIIa. These data therefore indicate that CD9 and CD42 can signal independently of gpIIb/IIIa, and that signals generated by all these molecules may converge on a common pathway.

Proteolytic cleavage of the beta1 subunit of platelet alpha2beta1 integrin by the metalloproteinase jararhagin compromises collagen-stimulated phosphorylation of pp72.

Early signaling events in the stimulation of platelets by collagen include the tyrosine phosphorylations of FcR gamma-chain, pp72(syk) and phospholipase Cgamma2. These events are dependent on the main platelet collagen receptor, alpha2beta1 integrin (glycoprotein Ia-IIa complex). We recently found that jararhagin, a 52-kDa snake venom metalloproteinase, selectively inhibits collagen-induced platelet secretion and aggregation in parallel with the cleavage of the beta1 subunit of the alpha2beta1 integrin. The present study demonstrates that jararhagin also interferes with collagen-induced phosphorylation of the protein-tyrosine kinase pp72(syk). This effect is not observed when the platelet aggregation response to collagen is inhibited by two venom RGD-containing disintegrins, contortrostatin and echistatin. These disintegrins inhibit platelet aggregation through their high affinity binding to the platelet alphaIIbbeta3 integrin (glycoprotein IIb-IIIa complex). We also show that mild stimulation by ADP of jararhagin-treated platelets, but not of platelets treated with the RGD-containing disintegrins, restores the collagen-induced platelet aggregation. ADP also restored both pp72(syk) and pleckstrin phosphorylation of jararhagin-treated platelets in response to collagen, presumably via interaction of collagen with ADP-activated alphaIIbbeta3 integrin. Thus, RGD-containing disintegrins do not interfere with agonist-induced pp72(syk) phosphorylation but inhibit aggregation through occupancy of the alphaIIbbeta3 integrin. Conversely, jararhagin affects early platelet signaling events in response to collagen through its effects on the alpha2beta1 integrin without interfering with the function of the alphaIIbbeta3 integrin. Our demonstration that the degradation of the beta1 subunit of alpha2beta1 by jararhagin results in the loss of pp72(syk) phosphorylation, suggests that this subunit is critically involved in collagen-induced platelet signaling.

Inhibition of collagen-induced platelet aggregation as the result of cleavage of alpha 2 beta 1-integrin by the snake venom metalloproteinase jararhagin.

Jararhagin is a high-molecular-mass (52 kDa) haemorrhagic metalloproteinase from Bothrops jararaca venom and a member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of jararhagin has been implicated in the inhibition of platelet responses to collagen by a mechanism that is not entirely known. The present investigation demonstrates that both active and 1,10-phenanthroline-inactivated jararhagin inhibit platelet aggregation by collagen with an IC50 of 40 and 140 nM respectively. The apparently higher inhibitory effect of the active enzyme clearly indicates that, in addition to the disintegrin region, the metalloproteinase domain of jararhagin also participates in this inhibition. As collagen interacts with platelets via alpha 2 beta 1-integrin, we investigated the effects of jararhagin on this integrin using selected function-blocking monoclonal antibodies against both of its subunits. Flow cytometry of platelets treated with native jararhagin and immunoprecipitation of platelet surface glycoproteins from lysates after jararhagin treatment showed an apparently selective reduction of alpha 2 beta 1-integrin immunoreactivity with both anti-alpha 2 and anti-beta 1 monoclonal antibodies. The loss of immunoreactivity was not due to integrin internalization, since it also took place in cytochalasin D-treated platelets. Here we show that jararhagin cleaved isolated alpha 2 beta 1-integrin resulting in the generation of a 115 kDa beta 1 fragment. We therefore propose that the inhibition by jararhagin of platelet response to collagen is mediated through the binding of jararhagin to platelet alpha 2-subunit via the disintegrin domain, followed by proteolysis of the beta 1-subunit with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands.

Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases.

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 alpha 2 beta 1 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.