Antithrombotic effect of a protein-type I class snake venom metalloproteinase, kistomin, is mediated by affecting glycoprotein Ib-von Willebrand factor interaction.
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 GPIbalpha and vWF. Cleavage of platelet GPIbalpha by kistomin resulted in release of 45- and 130-kDa soluble fragments, indicating that kistomin cleaves GPIbalpha 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 GPIbalpha in whole blood. Moreover, GPIbalpha 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 GPIbalpha and vWF and its proteolytic activity on GPIbalpha-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.
Hsu
Chun-Chieh
CC
Department of Pharmacology, College of Medicine, National Taiwan University, No1, Sec1, Jen-Ai Rd, Taipei, Taiwan.
Wu
Wen-Bin
WB
Chang
Ya-Hui
YH
Kuo
Heng-Lan
HL
Huang
Tur-Fu
TF
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000494:pharmacology*; D001483:Base Sequence; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000494:pharmacology; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D008297:Male; D045726:Metalloproteases / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000187:drug effects* / Q000378:metabolism; D014841:von Willebrand Factor / Q000187:drug effects* / Q000378:metabolism
D000991:Antithrombins; D003435:Crotalid Venoms; D017931:DNA Primers; D018076:DNA, Complementary; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D045726:Metalloproteases; C524409:kistomin protein, Calloselasma rhodostoma