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

Abstract

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.

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

“…Indeed, the Mascot hits found where procoagulant activity was observed revealed the presence of both these toxin types, along with C-type lectins and an L-amino acid oxidase (Table 2). The SVSP identified (“Thrombin-like enzyme ancrod-2”; SC, 53%) has previously been shown to be fibrinogenolytic (51), while the literature relating to the SVMPs detected (“Snake venom metalloproteinase kistomin” and “Zinc metalloproteinase/disintegrin” SC, 33% and 27%, respectively) suggests they predominately exert anticoagulant activities, including interfering with platelet aggregation and promoting haemorrhage (52, 53). It is therefore possible that these toxins are co-eluting with procoagulant components, although given that many SVMPs are multi-functional proteins that can exert procoagulant bioactivities (54), they may be contributing to the functional effect observed here despite these activities not previously being reported.…”

Metalloproteases Affecting Blood Coagulation, Fibrinolysis and Platelet Aggregation from Snake Venoms: Definition and Nomenclature of Interaction Sites

“…Intact GPIb plays critical role in the extent and rate of platelet aggregation stimulated by low concentrations of thrombin [80]. Kistomin cleaves platelet GPIbα at two distinct sites releasing 45- and 130-kDa soluble fragments and specifically inhibits von Willebrand factor- (vWF-) induced platelet aggregation [81]. Kistomin also cleaves vWF resulting in the formation of low-molecular-mass multimers.…”

“…Kistomin also cleaves vWF resulting in the formation of low-molecular-mass multimers. It inhibits GPIbα agonist-induced platelet aggregation, and prolongs the occlusion time in mesenteric microvessels and tail-bleeding time in mice [81]. Kistomin also inhibits platelet aggregation induced by collagen and convulxin (Glycoprotein VI (GPVI) [82].…”

Caracterização funcional e estudo dos mecanismos de resposta ao dano celular causado por uma L-aminoácido oxidase de Calloselasma rhodostoma em linhagens celulares humanas

“…A maioria dos estudos com a peçonha de C. rhodostoma, está relacionado com a purificação e caracterização de proteínas que interferem na hemostasia sanguínea, como por exemplo, a rhodosthoxin que é uma hemorragina com alto potencial proteolítico, hemorrágico e indutor de edema e a kistomin, que é uma metaloprotease da classe P-I, com efeito antitrombolítico in vivo (PONNUDURAI et al, 1993;HSU et al, 2007).…”