Hemostatic effects of recombinant DisBa-01, a disintegrin from Bothrops alternatus

Abstract

A monomeric RGD-disintegrin was recently identified from a cDNA library from the venom gland of Bothrops alternatus. The corresponding 12 kDarecombinant protein, DisBa-01, specifically interacted with alphavbeta3 integrin and displayed potent anti-metastatic and anti-angiogenic properties. Here, the interaction of DisBa-01 with platelet alphaIIbbeta3 integrin and its effects on hemostasis and thrombosis were investigated. DisBa-01 bound to Chinese Hamster Ovary (CHO) cells expressing beta3 or alphaIIbbeta3 and promoted their adhesion and the adhesion of resting platelets onto glass coverslips. The disintegrin inhibited the binding of FITC-fibrinogen and FITC-PAC-1 to ADP-stimulated platelets and inhibited ADP-, TRAP- and collagen-induced aggregation of murine, rabbit or human platelets. In a flow chamber assay, DisBa- 01 inhibited and reverted platelet adhesion to immobilized fibrinogen. DisBa-01 inhibited the phosphorylation of FAK following platelet activation. The intravenous injection of DisBa-01 in C57Bl6/j mice, prolonged tail bleeding time as well as thrombotic occlusion time in mesenteric venules and arterioles following vessel injury with FeCl3. In conclusion, DisBa-01 antagonizes the platelet alphaIIbbeta3 integrin and potently inhibits thrombosis.