Identification and characterization of Harobin, a novel fibrino(geno)lytic serine protease from a sea snake (Lapemis hardwickii).

A gene encoding a novel serine protease designated as Harobin is cloned and identified from a sea snake venom gland bacteriophage T7 library. It has 265 amino acids and shares 50-70% similarity to terrestrial snake serine proteases. In addition to the 12 conservative Cys, it has three more Cys residues that may contribute to its higher enzymatic stability. Harobin is expressed in Pichia pastoris and purified. Recombinant Harobin exhibits an amidolytic activity, and specifically degrades Aalpha, Bbeta-chain of fibrinogen. It functions as a defibrase both in vitro and in vivo, and reduces thrombosis. Harobin prolongs the coagulation time and the bleeding time of mice and reduces the fibrinogen levels of rats as well. Meanwhile, intravenous injection of Harobin leads to the reduction of blood pressure in SHR rats. It results from the ability of Harobin that cleaves angiotensin I and release bradykinin from plasma kininogen in vitro and in vivo. These data suggest that Harobin is a novel defibrase and has a potential to be an agent for the therapy of thrombosis and hypertension.

Snake venom thrombin-like enzymes: from reptilase to now.

The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.

Thromboelastograph assay for measuring the mechanical strength of fibrin sealant clots.

In order to provide sustained hemostasis or tissue sealing, fibrin sealants must generate adhesive clots with mechanical properties capable of resisting forces, such as shear, that might break or tear the clot. Commercial preparations of fibrin sealants should generate clots of adequate and consistent mechanical strength. The mechanical strength of fibrin sealants is often measured as bonding strength in in vivo or ex vivo animal wound models. These tests can be useful predictors of clinical efficacy. However, these, as well as many in vitro tensile strength tests for fibrin sealant, tend to be laboratory specific and require extensive reagent preparation time and analyst training. The thromboelastograph has historically been used to screen for plasma protein and platelet disorders that lead to defective clot formation. The authors have developed a simple in vitro test, using a standard thromboelastograph that can provide reliable, reproducible information on the rheology of clots generated by fibrin sealant preparations. Using this method, the shear strength of fibrin sealant clots was measured and shown to correlate with the fibrinogen, but not the thrombin, concentration in the sealant. Shear strength was also shown to correlate with the sealant concentration of the fibrin cross-linking proenzyme, factor XIII. Sealants containing lysine, which can act as an alternate substrate for factor XIII enzyme and prevent efficient fibrin chain cross-linking, were shown by this method to generate clots of substantially reduced shear strength. The method distinguished between thrombin-catalyzed clot formation and other fibrinogen clotting mechanisms as evidenced by the significantly lower shear strength associated with batroxobin-generated fibrin clots.

Effect of dextran on platelet activation by polymerizing fibrin.

Dextran has been shown to alter the mechanical properties and lysability of fibrin. The present study was undertaken to determine whether it would also influence the ability of polymerizing fibrin to activate platelets. Human fibrinogen, with or without the presence of dextran, was incubated with either human thrombin or reptilase at 37 C. Macroscopically evident fibrils first appeared at 7 1/2 to 8 minutes in fibrinogen solution not containing dextran and at 2 1/2 to 3 minutes in solution containing dextran. Both solutions caused aggregation of washed human platelets, but the one containing dextran was less potent (P less than 0.005). Similarly, polymerizing fibrin, under the influence of dextran, was a less potent stimulator of platelet release of labeled serotonin. Scanning electron microscopy showed decreased platelet adhesion to fibrin polymerized in the presence of dextran. Inclusion of dextran in plasminogen-rich bovine fibrin clots facilitated their lysis by urokinase. Apparently, dextran not only increases the lysability of fibrin but also alters the platelet-activating activity of polymerizing fibrin.