title: Crotalin, a vWF and GP Ib cleaving metalloproteinase from venom of Crotalus atrox.
abstract: Binding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP lb antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP lb as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP lb antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin autoproteolytically degraded to approximately 14 and approximately 10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP lb interaction.
authors.affiliation: Department of Pharmacology, College of Medicine, National Taiwan University, Taipei.
authors.forename: W B
authors.initials: WB
authors.lastname: Wu
authors.forename: H C
authors.initials: HC
authors.lastname: Peng
authors.forename: T F
authors.initials: TF
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D015153:Blotting, Western; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D017839:Crotalus / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008666:Metalloendopeptidases / Q000145:classification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D012310:Ristocetin / Q000494:pharmacology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D012310:Ristocetin; D008666:Metalloendopeptidases