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Keywords:

Animals, Bothrops, CHO Cells, Cricetinae, Cricetulus, Crotalid Venoms, Focal Adhesion Kinase 1, Hemostasis, Hemostatics, Humans, Phosphorylation, Platelet Adhesiveness, Platelet Aggregation, Recombinant Proteins, Transfection, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Cell Biology, RGD-disintegrin, platelet aggregation, Bothrops alternatus, dynamic conditions, beta(3) integrin, thrombosis, focal adhesion kinase, phosphorylation, bleeding time, PLATELET-AGGREGATION, FLOW CONDITIONS, INTEGRIN, VENOM, PHOSPHORYLATION, BINDING, ECHISTATIN, FIBRINOGEN, ADHESION, LIGAND, 0601 Biochemistry and Cell Biology, 1112 Oncology and Carcinogenesis, 1116 Medical Physiology, 3102 Bioinformatics and computational biology, 3205 Medical biochemistry and metabolomics, 3207 Medical microbiology

Abstract:

A monomeric RGD-disintegrin was recently identified from a cDNA library from the venom gland of Bothrops alternatus. The corresponding 12 kDa-recombinant protein, DisBa-01, specifically interacted with alpha(v)beta3 integrin and displayed potent anti-metastatic and anti-angiogenic properties. Here, the interaction of DisBa-01 with platelet alphaIIb beta3 integrin and its effects on hemostasis and thrombosis were investigated. DisBa-01 bound to Chinese Hamster Ovary (CHO) cells expressing beta3 or alphaIIb beta3 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 alphaIIb beta3 integrin and potently inhibits thrombosis.