Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.
Snake venoms contain various molecules that can be used as tools in the diagnosis and in the treatment of hemostatic disorders. This study reports the isolation and functional characterization of a new thrombin-like enzyme and its role in the modulation of platelet aggregation and coagulation. The molecule was purified by gel filtration, anion exchange chromatography and reverse-phase-HPLC on C8 column; its molecular weight was determined. Natural and synthetic substrates were used to evaluate its enzymatic activities. The fibrinogenolytic activity was tested electrophoretically and by reverse-phase-HPLC on C18 column. Otherwise, the effect on blood coagulation and deficient plasma factors were also evaluated. The mechanism by which a thrombin-like enzyme VLCV (thrombin-like enzyme)-induced platelet aggregation was explored in presence of ticlopidin, clopidogrel and aspirin. VLCV (45 kDa) isolated from Vipera lebetina as a thrombin-like enzyme seems to be able to modulate platelet function. This enzyme showed an amidolytic activity by hydrolyzing the chromogenic-specific substrate of thrombin and the α-chain of fibrinogen. It is also able to clot human plasma and the deficient human plasma in factor X, suggesting that it is involved in the intrinsic and common pathways. The aggregating effect of VLCV is more sensitive to ticlopidine than to the clopidogrel suggesting the involvement of ADP/P2Y12/PI3K pathway. VLCV seems to be able to promote human platelet aggregation suggesting an interaction between P2Y12 and PAR1. Due to its ability to replace the missing factor X and its proaggregating activity, VLCV could be used as molecular tool to better understand the hemostasis mechanism.
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