Abstract: Studies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. 125 I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxo-bin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of 125 I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batrox-Correspondence obin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radio-labeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs.When the dose of TAFIa was 218 µg/kg, giving a peak plasma level of TAFIa 0.9 ±0.05 µg/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circu-lating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.

Abstract: Platelet contractile force (PCF), which is absent in blood obtained during cardiopulmonary bypass, significantly recovers after protamine sulfate administration. In vitro studies reveal this effect to be primarily caused by heparin. Because many of heparin's effects are mediated by suppression of thrombin generation and activity, this study assessed the influence of thrombin inhibition on PCF. The effects of natural and synthetic antithrombins were measured. Clots were formed by the addition of batroxobin (0.21 μg/mL) to whole blood (platelet count 200,000/μL). Force development was measured from the moment of batroxobin addition. After 1200 s of clotting, purified antithrombin III (22 μM) reduced PCF by 74%. Thrombomodulin (0.014 μM) reduced PCF by 60%. At 0.040 μM, PCF was reduced by 82% (6.5–1.2 Kdynes). Hirudin decreased PCF in a dose-dependent fashion, with complete suppression at concentrations ≥0.30 μM. At concentrations between 0.04 and 0.29 μM, Lepirudin (Refludan, a recombinant therapeutic hirudin) produced dose-dependent delay and suppression of PCF. Above 0.29 μM Lepirudin, PCF was totally suppressed. At 1.60 μM, bivalirudin (a synthetic, 20 amino acid peptide) delayed and reduced PCF by 50%. At 6.40 μM, PCF was completely suppressed. Although 20 μM of P-PACK II (d-Phenylalanyl-L-Phenylalanylarginine-chloro-methyl ketone 2 HCl) had little effect, 40 μM delayed onset of force development from 300 to 600 s and reduced PCF at 1200 s from 5.2 to 3.3 Kdynes. At 120 μM, force development was totally suppressed. Four micromol Thromstop® (BNas-Gly-(pAM)Phe-Pip) delayed force development of greater than 800 s and PCF at 1200 s was reduced by 70%. At 0.20 μM, Argatroban (a synthetic polypeptide direct thrombin antagonist) delayed onset of PCF from 300 to 540 s and decreased PCF by 40%. At a concentration of 0.40 μM and above, Argatroban totally suppressed PCF. These results indicate that some of the antiplatelet effects of heparin are the result of thrombin inhibition and that low-level thrombin generation is essential for clot retraction. The sensitivity of PCF to the presence of thrombin may permit monitoring of antithrombin agents via this assay.

Abstract: A recombinant thrombin-like enzyme batroxobin expressed by yeast, a production method thereof, and use of the batroxobin as a hemostatic agent or antithrombotic agent is disclosed. In order to express recombinant thrombin-like enzyme, an expression vector is prepared by inserting cDNA encoding the enzyme, the transformed cell is prepared by introducing the expression vector into the cell, the transformed cell then is incubated and the recombinant thrombin-like enzyme is obtained from the cell. The recombinant thrombin-like enzyme may be usefully used as a hemostatic agent because the enzyme effectively decrease bleeding time and blood coagulation time and does not affect various blood coagulation factors. The recombinant thrombin-like enzyme is also useful for a hemostatic agent or antithrombotic agent comprising the recombinant thrombin-like enzyme as an active component