A new fibrinogenase was purified and characterized. The purified protease preferably cleaved the R42-A43 bond of fibrinogen Bbeta-chain and with much lower activity hydrolysed C-terminus of Aalpha-chain. As to the polymerized fibrin, the enzyme hydrolyzed C-terminal part of alpha-chain and N-terminus of beta-chain of fibrin at similar kinetics to the respective values for the fibrinogen, but both reactions were much slower. Brief incubation of fibrinogen with protease elongated thrombin clotting time by 60%. The clot formed from a mixture of native fibrinogen and desВbeta(1-42)2 (truncated fibrinogen) was much more sensitive to plasmin digestion than the native fibrin clot. The protease did not activate platelets nor cause changes in their shape and granularity, but it reduced platelet aggregation induced by ADP. An in vitro study of the influence of fibrinogenase on haemostatical potential allowed us to conclude that the fibrinogenase might serve as a fibrinogen-depletive agent. To evaluate the accessibility of BbetaR42-A43 to fibrinogenase hydrolytic action, we compared the kinetics of Fg, monomeric and polymeric fibrin desA and desABbeta15-42 peptide cleavage. Rate of peptide cleavage was determined using electrophoretic and densitometric analyses of hydrolized substrates. It was found that kinetic parameters of the hydrolysis of fibrinogen, monomeric fibrin desA and desAB (in presence of 1 mM GPRP peptide) were 8.0; 9.6; 10.4 mkМ for Km, 0.13; 0.08; 0.019 s-1 for kcat and 0.016, 0.008 and 0.0018 mkМ-1·s-1 for kef (Km/kcat), respectively. The narrow range of Km values showed that fibrinogenase has similar affinity to the monomeric substrates. Decreased values of kef indicated a possible change within polypeptide structure, surrounding BbetaR42-A43 peptide bond. Computer modeling of fibrinogen (Bbeta1-60) and desAB fibrin (beta15-60) using server I-TASSER predicted some models of their tertiary structures. One model for fibrinogen and another for desAB fibrin were selected according to the location of the split peptide bond within the loop of peptide chain. Meanwhile Km, kcat and kef values varied significantly for fibrinogenase reaction for the polymeric fibrin desA (60 mkМ, 0,041 s-1 and 0,00007 mkМ-1·s-1, respectively). Taking into account the concentration of the fibrin molecules in the surface layer of a fibril (~18 % of total), Km and kef, normalized to the available concentration of polymerized fibrin desA, were 10.8 mkМ and 0.004 mkМ-1·s-1. Cleavage of fibrin desAB betaN-domain from fibrin polymer was not observed, probably due to its involvement into formation of protofibrils. The data indicated that the BbetaR42-A43 peptide bond was available for cleavage in all forms of fibrin(ogen) except polymerized fibrin desAB. It was found that fibrin desAB but no fibrin desA and desABbeta(15-42)2 induced appearance of thrombin activity in the mixture with prothrombin. Thus we could predict the crucial role in this process of 15-42 sequence of betaN-domain cleaved off in fibrin desABbeta(15-42)2 and protected in fibrin desA. To map the beta15-42 residue we studied the influence of specific monoclonal IgG antibody to beta26-42 on thrombin-like activity in the mixture of prothrombin and fibrin desAB. It was shown that blocking of Вbeta26-42 residue by antibody significantly diminished the induction of thrombin-like activity in the mixture. Thus we have found that prothrombin was activated after forming a complex with fibrin. The non-enzymatic activation of prothrombin could be an important process during thrombin over-formation on fibrin matrix and fibrin degradation products. ADP- and collagen-induced platelet aggregation of washed platelets in the presence of native fibrinogen or the partly hydrolyzed form - desBbeta(1-42)2 was studied. In both cases the rate and level of platelet aggregation did not statistically different from control. However, desBbeta(1-42)2 fibrinogen caused further disaggregation of platelets. Washed platelets were activated by ADP in the presence of native or desBbeta(1-42)2 fibrinogen to quantify PF-4 and PDGF (markers of alpha-granules) release. The levels of PF-4 and PDGF were measured using ELISA. Neither rates differed from the controls. From the data we can speculate that the abnormalities in platelet aggregation in the presence of desBbeta(1-42)2 fibrinogen were related rather to lesser stability of fibrinogen-platelets interaction mediated by the GPIIbIIIa receptor or other platelet integrins, than to platelet activation. Analytical size-exclusion chromatography of fibrin desAB with D and DD fragments of fibrin(ogen) revealed a ternary fibrin-DD-D complex. In the mixture of D and DD with desABbeta(15-42)2 or desA fibrin only binary complex of fibrin and DD was formed. Also, the complex of DD with fibrin desABbeta(15-42)2 was much less stable than that with fibrin desA. Thus we can speculate about the role of ВbetaN-domain in the DDE-triad formation as well as in lateral association of protofibrils. In conclusion, fibrinogenase from the Echis multisquamatis snake venom cleaved N-terminal peptide 1-42 of fibrinogen Вbeta-chain with high specificity, hydrolysed C-terminal part of Aalpha chain in both fibrinogen and fibrin. Truncated fibrinogen showed slow response to clotting by thrombin but the formed clot hydrolysed much faster than clot from the native fibrinogen. fibrinogenase did not activate platelets but diminished aggregation of ADP-activated platelets in platelet rich plasma or washed platelets with added fibronogen. Therefore we can conclude that the action of fibrinogenase from the venom of Echis multisquamatis had an anticoagulant, thrombolytic and antiaggregatory action on blood plasma and could be a potential antithrombotic an thrombolytic agent.
