Antiplatelet and antiproliferative action of disintegrin from Echis multisquamatis snake venom

摘要

Aim To purify the platelet aggregation inhibitor from Echismultisquamatis snake venom (PAIEM) and characterize itseffect on platelet aggregation and HeLa cell proliferation.Methods Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used forPAIEM identification. Platelet aggregation in the presenceof PAIEM was studied on aggregometer Solar-AP2110. Thechanges of shape and granularity of platelets in the presenceof PAIEM were studied on flow cytometer COULTEREPICS XL, and degranulation of platelets was estimatedusing spectrofluorimetry. Indirect enzyme-linked immunosorbentassay was used for the determination of targetof PAIEM on platelet surface. An assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide wasused to evaluate the effect of PAIEM on the proliferation ofHeLa cells in cell culture.Results The molecular weight of the protein purified fromEchis multisquamatis venom was 14.9 kDa. Half-maximal inhibitoryconcentration (IC50) of PAIEM needed to inhibit adenosinediphosphate (ADP)-induced platelet aggregationwas 7 μM. PAIEM did not affect thrombin- or ADP-inducedplatelet activation, but it did prevent binding of the anti-IIb antibody to glycoprotein IIb/IIIa (GPIIbIIIa)-receptor ofadhered platelets and inhibited the viability of HeLa cellsby 54%.Conclusion As a member of the disintegrin family, PAIEMinhibited platelet aggregation and cell proliferation possiblyby blocking integrin-mediated interactions. However,it did not impair cellular signaling causing any changes inplatelet shape and granularity and did not affect ADP-inducedplatelet degranulation. This disintegrin was shownto be a potent inhibitor of integrin-mediated cellular interactionsincluding platelet aggregation or cancer cell proliferation.