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 recombinant mutant p rothrombin with active site Asp419 substituted by Asn (D419N-prothromb in) mere expressed in recombinant CHO cells, isolated and purified fro m the fermentation supernatant. The r-Prothrombin and D419N-prothrombi n mere digested by both Echis carinatus venom and Oxyuranus scutellatu s venom, Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the prothrombin polypepti de chain were analysed, Owing to the recombinant preparation and inact ivity of D419N-protbrombin and its activation products, the proteolyti c action of E.carinatus and O.scutellatus venoms could be studied with out addition of thrombin inhibitor, without interference from autocata lytic digestion of prothrombin and in the absence of any other blood c oagulation protease, The comparison between the activation of r-prothr ombin and D419N-prothrombin by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of prothro mbin, Incubation of D419N-prothrombin with E.carinatus venom resulted in the generation of stable D419N-meizothrombin by hydrolysis of the p eptide bond Arg320-Ile321. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg320-Ile321 and Arg271-Thr272 and low er activity towards peptide bond Arg155-Ser156, thus converting D419-p rothrombin into D419N-thrombin and also liberating Fragment-1, Fragmen t-2 and Fragment-1/2. activation peptide, Activation of r-prothrombin By E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of prothrombin-derived molecules and indicated that meizoth rombin hydrolysed the cleavage between Fragment-2; and thrombin A-chai n in the meizothrombin molecule, but not in prothrombin, preferentiall y at position Arg284-Thr285. By contrast, both meizothrombin and throm bin exhibited no detectable activity towards peptide bond Arg320-Ile32 1 between thrombin A- and B-chain, although this site exhibits the opt imum sequence for thrombin cleavage.