Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus

Recombinant human prothrombin (r-prothrombin) and recombinantmutant prothrombin with active site Asp419 substituted by Asn(D419N-prothrombin) were expressed in recombinant CHO cells,isolated and purified from the fermentation supernatant. Ther-Prothrombin and D419N-prothrombm were digested by both Echiscarinatus venom and Oxyuranus scutellatus venom. Prior to, duringand after activation, generation of thrombin activity and theproteolytic degradation of the prothrombin polypeptide chainwere analysed. Owing to the recombinant preparation and inactivityof D419N-prothrombin and its activation products, the proteolyticaction of E.carinatus and O.scutellatus venoms could be studiedwithout addition of thrombin inhibitor, without interferencefrom autocatalytic digestion of prothrombin and in the absenceof any other blood coagulation protease. The comparison betweenthe activation of r-prothrombin and D419N-prothrombin by snakevenoms permitted differentiation between proteolytic activationand autocatalytic conversion of prothrombin. Incubation of D419N-prothrombinwith E.carinatus venom resulted in the generation of stableD419N-meizothrombin by hydrolysis of the peptide bond Arg320-Ile321.By contrast, O.scutellatus venom exhibited activity towardspeptide bonds Arg320-Ile321 and Arg271-Thr272 and lower activitytowards peptide bond Arg155-Ser156, thus converting D419-prothrombininto D419N-thrombin and also liberating Fragment-1, Fragment-2and Fragment-1/2 activation peptide. Activation of r-prothrombinby E.carintitus and O.scutellatus venoms demonstrated the autocatalyticpotential of prothrombin-derived molecules and indicated thatmeizothrombin hydrolysed the cleavage between Fragment-2 andthrombin A-chain in the meizothrombin molecule, but not in prothrombin,preferentially at position Arg284-Thr285. By contrast, bothmeizothrombin and thrombin exhibited no detectable activitytowards peptide bond Arg320-Ile321 between thrombin A- and B-chain,although this site exhibits the optimum sequence for thrombincleavage.