title: Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation.
abstract: Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: Solange M T
authors.initials: SMT
authors.lastname: Serrano
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: Deyu
authors.initials: D
authors.lastname: Wang
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Shannon
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: Antonio F M
authors.initials: AFM
authors.lastname: Pinto
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: Renata K
authors.initials: RK
authors.lastname: Polanowska-Grabowska
authors.affiliation:  Laboratório Especial de Toxinologia Aplicada and CAT-CEPID, Instituto Butantan, Sao Paulo, Brazil Department of Microbiology, University of Virginia, Charlottesville, VA, USA Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Department of Biochemistry and Molecular Genetics University of Virginia, Charlottesville, VA, USA.
authors.forename: Jay W
authors.initials: JW
authors.lastname: Fox
mesh_terms: D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000187:drug effects; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D019610:Cytoprotection / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D045726:Metalloproteases / Q000378:metabolism* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D053657:Platelet-Rich Plasma; D011485:Protein Binding; D012310:Ristocetin / Q000494:pharmacology; D012910:Snake Venoms / Q000201:enzymology*; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D004800:Enzymes, Immobilized; D012910:Snake Venoms; D014841:von Willebrand Factor; D012310:Ristocetin; D045726:Metalloproteases; D003545:Cysteine