Disintegrins are the antagonists of integrin receptors that can be found mostly in snakes’ venom. They can inhibit platelet aggregation, thus preventing the formation of blood clots. By blocking the integrin receptors of cancer cells, disintegrins can inhibit proliferation and metastasis. Thus, the search for new sources of disintegrins and development of methods of their purification is an important task of modern biotechnology. This work was dedicated to the purification and characterization of inhibiting polypeptides from Bitis arietans venom. Crude venom of B. arietans was fractionated using ion-exchange chromatography on Q Sepharose followed by size-exclusion chromatography on Superdex 75 using FPLC method. Analysis of molecular weight of protein components was performed using SDS-PAGE and MALDI-TOF analysis on Voyager-DE. Aggregation of platelet-rich plasma (PRP) in the presence of platelet aggregation inhibitor was investigated using aggregometry on the AR2110. MTT test was used for measuring HeLa cells proliferation and survival in vitro. Two-step chromatography allowed us to obtain fraction that contained polypeptides possessing the dose-dependent inhibitory action on adenosine diphosphate (ADP)-induced platelet aggregation in PRP. SDS-PAGE showed that obtained fraction contained two polypeptides with molecular weight 9.0 and 13.67 kDa according to MALDI-TOF analysis. Purified polypeptides inhibited ADP-induced platelet aggregation with IC50 0.09 mg/ml. However, 0.005 mg/ml of fraction suppressed viability of HeLa cells according to MTT test on 20%. Discovered biological effects of fractions allowed us to conclude the possible use of these polypeptides as anti-aggregatory or anti-proliferative agents. Keywords: antithrombotic action, disintegrins, glycoprotein IIb/IIIa, platelets, snake venom
Aim. In our work, we studied platelet aggregation in blood plasma of pregnant women and estimated the possibility of ex vivo normalization of aggregation rate using a polypeptide from the Echis multisquamatus snake venom. Previous reports demonstrated that it directly interacts with glycoprotein IIb/IIIa receptors on the surface of platelets, preventing their adhesion, thereby affecting the degree of aggregation. Methods. chromatography followed by size-exclusion chromatography on Superdex 75 using the FPLC system (ӒKTA, GE Healthcare, USA). Analysis of molecular weight of protein components was performed using SDS-PAGE. The concentration of protein was measured using spectrophotometer Optizen POP (Korea) at 280 nm. The ability of obtained protein to inhibit platelet aggregation was measured directly by aggregometry. Blood samples of women with placental disfunction during pregnancy (n = 28) were kindly provided by “Perinatal Center of Kyiv”. This study was approved by the Ethics Commission of the Shupyk National Medical Academy of Postgraduate Education and the Ethics Commission of the Kyiv Perinatal Center (# 3 from 05/05/2020). Aggregation of platelet-rich plasma (PRP) induced by ADP was investigated using aggregometry on the AP 2110 (Solar, Belarus). We compared the rate of platelet aggregation in the presence vs absence of platelet aggregation inhibitor. Results. Two-step chromatography protocol allowed us to obtain the polypeptide from the venom of Echis multisquamatus that possessed the anti-aggregatory action. SDS-PAGE analysis confirmed the homogeneity of obtained polypeptide with apparent molecular weight 14 kDa that corresponds to the platelet aggregation inhibitor reported earlier. Initial studies of ADPinduced platelet aggregation allowed selecting active concentration for the effective inhibitory action as 0.02 mg/ml. Conclusions. Platelet aggregation inhibitor from Echis multisquamatis snake venom of can be assumed as the effective agent that reduce the rate of platelet aggregation. We demonstrated it efficacy in platelet rich plasma of pregnant women that had placenta dysfunction. The use of direct antagonist of platelet integrin receptors was assumed as the prospective approach for suppressing of platelet reactivity in particular during complicated pregnancy.
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