Journal
MOLECULAR PHARMACOLOGY
Volume 72, Issue 4, Pages 984-992Publisher
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.107.038018
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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 alpha and vWF. Cleavage of platelet GPIb alpha by kistomin resulted in release of 45- and 130kDa soluble fragments, indicating that kistomin cleaves GPIb alpha 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 alpha in whole blood. Moreover, GPIb alpha 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 alpha and vWF and its proteolytic activity on GPIb alpha-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.
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