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Antithrombotic Effect of a Protein-Type I Class Snake Venom Metalloproteinase, Kistomin, Is Mediated by Affecting Glycoprotein Ib-von Willebrand Factor Interaction

Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (C.-C.H., Y.-H.C., H.-L.K., T.-F.H); and School of Medicine, Fu-Jen Catholic University, Taipei County, Taiwan (W.-B.W.)

Binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib-IX-V mediates platelet activation in the early stage of thrombus formation. Kistomin, a snake venom metalloproteinase (SVMP) purified from venom of Calloselasma rhodostoma, has been shown to inhibit vWF-induced platelet aggregation. However, its action mechanism, structure-function relationship, and in vivo antithrombotic effects are still largely unknown. In the present study, cDNA encoding kistomin precursor was cloned and revealed that kistomin is a P-I class SVMP with only a proteinase domain. Further analysis indicated that kistomin specifically inhibited vWF-induced platelet aggregation through binding and cleavage of platelet GPIb and vWF. Cleavage of platelet GPIb by kistomin resulted in release of 45- and 130-kDa soluble fragments, indicating that kistomin cleaves GPIb at two distinct sites. In parallel, cleavage of vWF by kistomin also resulted in the formation of low-molecular-mass multimers of vWF. In ex vivo and in vivo studies, kistomin cleaved platelet GPIb in whole blood. Moreover, GPIb agonist-induced platelet aggregation ex vivo was inhibited, and tail-bleeding time was prolonged in mice administered kistomin intravenously. Kistomin's in vivo antithrombotic effect was also evidenced by prolonging the occlusion time in mesenteric microvessels of mice. In conclusion, kistomin, a P-I class metalloproteinase, has a relative specificity for GPIb and vWF and its proteolytic activity on GPIb-vWF is responsible for its antithrombotic activity both in vitro and in vivo. Kistomin can be useful as a tool for studying metalloproteinase-substrate interactions and has a potential being developed as an antithrombotic agent.

Address correspondence to: Tur-Fu Huang, Department of Pharmacology, College of Medicine, National Taiwan University, No1, Sec1, Jen-Ai Rd, Taipei, Taiwan. E-mail: turfu{at}ntu.edu.tw