Antiplatelet and anti-proliferative action of disintegrin from Echis multisquamatis snake venom.

AIM: To purify the platelet aggregation inhibitor from Echis multisquamatis snake venom (PAIEM) and characterize its effect 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 for PAIEM identification. Platelet aggregation in the presence of PAIEM was studied on aggregometer Solar-AP2110. The changes of shape and granularity of platelets in the presence of PAIEM were studied on flow cytometer COULTER EPICS XL, and degranulation of platelets was estimated using spectrofluorimetry. Indirect enzyme-linked immunosorbent assay was used for the determination of target of PAIEM on platelet surface. An assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to evaluate the effect of PAIEM on the proliferation of HeLa cells in cell culture. RESULTS: The molecular weight of the protein purified from Echis multisquamatis venom was 14.9 kDa. Half-maximal inhibitory concentration (IC50) of PAIEM needed to inhibit adenosine diphosphate (ADP)-induced platelet aggregation was 7 µM. PAIEM did not affect thrombin- or ADP-induced platelet activation, but it did prevent binding of the anti-IIb antibody to glycoprotein IIb/IIIa (GPIIbIIIa)-receptor of adhered platelets and inhibited the viability of HeLa cells by 54%. CONCLUSION: As a member of the disintegrin family, PAIEM inhibited platelet aggregation and cell proliferation possibly by blocking integrin-mediated interactions. However, it did not impair cellular signaling causing any changes in platelet shape and granularity and did not affect ADP-induced platelet degranulation. This disintegrin was shown to be a potent inhibitor of integrin-mediated cellular interactions including platelet aggregation or cancer cell proliferation.

Non-enzymatic activation of prothrombin induced by interaction with fibrin ß26-42 region.

We have discovered that addition of monomeric desAB fibrin to prothrombin leads to appearance of the thrombin-like activity of prothrombin towards S2238 chromogenic substrate. DesA and desABß(15-42)2 fibrin forms did not cause any activation of prothrombin. From this observation we could suggested that amino acid residues of the 15-42 fragment of BßN-domain presented in desAB fibrin, cleaved in desABß(15-42)2 fibrin and protected in desA fibrin, play a crucial role in the non-enzymatic activation of prothrombin. To identify the Bß amino acid residues involved in the fibrin-prothrombin binding we used monoclonal antibodies 1-5G and 2d2a with epitopes in Bß26-42 and Bß12-26 fibrin fragments respectively. The thrombin-like activity in the mixture of prothrombin and desAB fibrin was monitored in the presence of each of these monoclonal antibodies. It was found that anti-Bß12-26 antibody does not exhibit any inhibitory effect on the thombin-like activity of the mixture. In contrast, adding of Bß26-42 antibody into the mixture of desAB fibrin with prothrombin diminished the thrombin-like activity by 70%. Recombinant dimeric peptides Bß(15-44)2 and Bß(15-66)2 that mimic amino acid residues in fibrin were also tested for their ability to activate prothrombin. It was found that both peptides were able to induce non-enzymatic activation of prothrombin. The activation was more evident in the case of Bß(15-44)2 peptide. From the data obtained we can conclude that desAB fibrin binds to prothrombin through the Bß26-42 amino acid residues and the formation of such a complex caused a non-enzymatic activation of prothrombin.