Functional Significance of Two Kunitz-type Protease Inhibitors Isolated from Daboia russelii russelii Snake Venom

Snake venoms contain a wide variety of biologically active substances that upset the intricate balance of hemostasis. Many of these proteins have been thoroughly characterized in relation to their functions and applications to molecular and clinical research. However, the functional significance of Kunitz-type protease inhibitors remains obscure. In this study, we purified two Kunitz-type protease inhibitors, namely DrKIn-I and DrKIn-II, from the venom of Daboia russelii russelii (Pakistan), with molecular masses of 7550 and 6940 respectively. APTT, PT, TT and ST tests revealed that both DrKIn-I and DrKIn-II inhibit the intrinsic pathway of coagulation, and tests with single factor deficient plasma showed that this inhibition is mainly through the inactivation of FXII. Amidolytic activity assays revealed that DrKIn-I, compared to DrKIn-II, has higher specificities for FXIIa and FXIa, while DrKIn-II has higher specificities for plasma kallikrein, plasmin and activated protein C. Incubation of DrKIn-I with human plasma decreased the generation of bradykinin in a dose-dependent manner and that this decrease correlates well with increased ADP-induced platelet aggregation and decreased vascular permeability. The pro-aggregatory effect on platelets appears to be mediated through a decrease in intracellular cyclic AMP levels. Furthermore, 7 nM of DrKIn-I almost completely abrogated the activation of protein C by the thrombin-thrombomodulin complex, and that co-injection of RVV-X (the factor X activator in the same venom) and DrKIn-I, but not DrKIn-II, significantly decreased the level of fibrinogen in mice compared to RVV-X alone. DrKIn-II, on the other hand, has less pronounced effect on bradykinin generation and protein C activation. However, treatment of DrKIn-II with the euglobulin fraction significantly prolonged the clot lysis time, compared to DrKIn-I, and that co-administration of RVV-X and DrKIn-II reduced the generation of fibrinogen/fibrin degradation products in mice, confirming the anti-fibrinolytic role of DrKIn-II. We therefore propose that both DrKIn-I and DrKIn-II are prothrombotic agents that help to synergize the procoagulating effects of RVV-X in snakebite symptoms.