Inhibition of platelet aggregation by the recombinant cysteine-rich domain of the hemorrhagic snake venom metalloproteinase, atrolysin A

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins.     

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.     

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.     

Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase

The functionality of the disintegrin-like/cysteine-rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine-rich region, which can interact with VWA-containing proteins. Recently, the hyper-variable region (HVR) of the cysteine-rich domain was suggested to constitute a potential protein-protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P-III SVMP, containing the cysteine-rich domain (disintegrin-like/cysteine-rich and cysteine-rich proteins) but not the disintegrin-like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti-alpha_M/beta₂ antibodies. These results show, for the first time, that the cysteine-rich domain and its HVR play a role in triggering pro-inflammatory effects mediated by integrins.     

Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation

Halysase, a hemorrhagic metalloprotease, has an apparent molecular weight of 66kDa and belongs to the class P-III snake venom metalloprotease. Class P-III snake venom metalloproteases have multifunctional domains including a protease domain and a disintegrin-like domain. Halysase was able to preferentially hydrolyze the alpha-chain of fibrinogen. Proteolytic activity of the enzyme was completely inhibited by metal chelating agents but not by other typical protease inhibitors. The enzyme principally cleaves X-Leu, X-Tyr, X-Phe, and X-Ala peptide bonds of the oxidized insulin B-chain. Halysase strongly suppresses collagen-induced human platelet aggregation in a dose-dependent manner. Apohalysase that is devoid of its metalloprotease activity was also able to inhibit the platelet aggregation to a certain extent. Experimental evidence clearly indicates that each of the two distinct domains of halysase, the metalloprotease and the disintegrin-like domains, plays its characteristic role to inhibit human platelet aggregation.     

Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom

Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.     

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.     

Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.     

Crystal structures of human ADAMTS-1 reveal a conserved catalytic domain and a disintegrin-like domain with a fold homologous to cysteine-rich domains

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type I motifs) family of proteases plays a role in pathological conditions including arthritis, cancer, thrombotic thrombocytopenic purpura and the Ehlers-Danlos type VIIC and Weill-Marchesani genetic syndromes. Here, we report the first crystal structures for a member of the ADAMTS family, ADAMTS-1. Originally cloned as an inflammation-associated gene, ADAMTS-1 has been shown to be involved in tissue remodelling, wound healing and angiogenesis. The crystal structures contain catalytic and disintegrin-like domains, both in the inhibitor-free form and in complex with the inhibitor marimastat. The overall fold of the catalytic domain is similar to related zinc metalloproteinases such as matrix metalloproteinases and ADAMs (a disintegrin and metalloproteinases). The active site contains the expected organisation of residues to coordinate zinc but has a much larger S1' selectivity pocket than ADAM33. The structure also unexpectedly reveals a double calcium-binding site. Also surprisingly, the previously named disintegrin-like domain showed no structural homology to the disintegrin domains of other metalloproteinases such as ADAM10 but is instead very similar in structure to the cysteine-rich domains of other metalloproteinases. Thus, this study suggests that the D (for disintegrin-like) in the nomenclature of ADAMTS enzymes is likely to be a misnomer. The ADAMTS-1 cysteine-rich domain stacks against the active site, suggesting a possible regulatory role.     

The reprolysin jararhagin,a snake venom metalloproteinase,functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling

The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins.     

Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis-inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis-inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino-acid residues. The amino-acid sequence predicted from the cDNA was highly homologous to VAP1. The amino-acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.     

The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.     

Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases

Hemorrhage is one of the most significant effects in envenomings induced by viperid snakebites. Damage to the microvasculature, induced by snake venom metalloproteinases (SVMPs), is the main event responsible for this effect. The precise mechanism by which SVMPs disrupt the microvasculature has remained elusive, although recent developments provide valuable clues to deciphering the details of this pathological effect. The main targets of hemorrhagic SVMPs are components of basement membrane (BM) and surrounding extracellular matrix (ECM), which provide mechanical stability to capillaries. P-III SVMPs, comprising disintegrin-like and cysteine-rich domains in addition to the catalytic domain, are more potent hemorrhagic toxins than P-I SVMPs, constituted only by the metalloproteinase domain. This is likely due to the presence of exosites in the additional domains, which contribute to the binding of SVMPs to relevant targets in the microvasculature. Recent in vivo studies have shown that P-III SVMPs are preferentially located in microvessels. On the other hand, the structural determinants responsible for the different hemorrhagic potential of P-I SVMPs remain largely unknown, although backbone flexibility in a loop located near the active site is likely to play a role. Moreover, hemorrhagic and non-hemorrhagic SVMPs differ in their capacity to hydrolyze in vivo key BM proteins, such as type IV collagen and perlecan, as well as other ECM proteins, like types VI and XV collagens, which play a critical role by connecting BM components to perivascular fibrillar collagens. The evidence gathered support a two-step model for the pathogenesis of SVMP-induced hemorrhage: initially, hemorrhagic SVMPs bind to and hydrolyze components of the BM and associated extracellular matrix proteins that play a key role in the mechanical stability of BM. In conditions of normal blood flow in the tissues, such cleavage results in the weakening, distension and eventual disruption of capillary wall due to the action of biophysical forces operating in vivo.     

Mapping von Willebrand factor A domain binding sites on a snake venom metalloproteinase cysteine-rich domain

The PIII class of the snake venom metalloproteinases (SVMPS) are acknowledged to be one of the major hemorrhage producing toxins in crotalid venoms. This class of SVMPS are structurally distinguished by the presence of disintegrin-like and cysteine-rich domains carboxy to the metalloproteinase domain and thus share structural homology with many of the ADAMs proteins. It has been suggested that the presence of the carboxy domain are the key structural determinants for potent hemorrhagic activity in that they may serve to target the proteinases to specific key extracellular matrix and cell surface substrates for proteolysis leading to hemorrhage production at the capillaries. Following from previous studies in our laboratory in this investigation we scanned the cysteine-rich domain of the PIII hemorrhagic SVMP jararhagin using synthetic peptides in an attempt to identify regions which could bind to von Willebrand factor (vWF), a known binding partner for jararhagin. From these studies we identified two such peptide, Jar6 and Jar7 that could support binding to vWF as well as block the recombinant cysteine-rich domain of jararhagin binding to vWF. Using the coordinates for the recently solved crystal structure of the PIII SVMP VAP1, we modeled the structure of jararhagin and attempted to dock the modeled cysteine-rich structure of that protein to the A1 domain of vWF. These studies indicated that effective protein-protein interaction between the two ligands was possible and supported the data indicating that the Jar6 peptide was involved, whereas the Jar7 peptide was observed to be sterically blocked from interaction. In summary, our studies have identified a region on the cysteine-rich domain of a PIII SVMP that interacts with vWF and based on molecular modeling could be involving in the interaction of the cysteine-rich domain of the SVMP with the A1 domain of vWF thus serving to target the toxin to the protein for subsequent proteolytic degradation.     

cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

Primary structure and autoproteolysis of brevilysin H6 from the venom of Gloydius halys brevicaudus

The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.     

Analysis of fertilin alpha (ADAM1)-mediated sperm-egg cell adhesion during fertilization and identification of an adhesion-mediating sequence in the disintegrin-like domain

Fertilin alpha (also known as ADAM1) is a member of the ADAM (A disintegrin and A metalloprotease domain) family of proteins. In this study, we examine the mechanism of mouse fertilin alpha's in adhesion of sperm to the egg plasma membrane during fertilization. We find that recombinant forms of fertilin alpha corresponding to either the disintegrin-like domain or the cysteine-rich domain and the EGF-like repeat can perturb sperm-egg binding, suggesting that both of these domains can participate in fertilin alpha-mediated adhesion events. In further examination of the fertilin alpha disintegrin-like domain, we find that a subdomain of disintegrin-like domain with the sequence DLEECDCG outside the putative disintegrin loop but with homology to the fertilin beta disintegrin loop can inhibit the binding of both sperm and recombinant fertilin alpha to eggs, suggesting that this is an adhesion-mediating motif of the fertilin alpha disintegrin-like domain. This sequence also inhibits the binding of recombinant fertilin beta to eggs and thus is the first peptide sequence found to block two different sperm ligands. Finally, a monoclonal antibody to the tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant fertilin alpha to eggs in one type of binding assay, suggesting that, under certain conditions, fertilin alpha may interact with a KMC.8-sensitive binding site on the egg plasma membrane.     

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background

Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.

Methodology/Principal Findings

In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.

Conclusions/Significance

These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.