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The importance of thrombin in arterial and venous thrombosis renders thrombin inhibition an important therapeutic target. Identification of novel inhibitors requires an appropriate animal model. We modified a previously reported rat arterial thrombosis model to allow simultaneous assessment of the arterial and venous antithrombotic efficacies of heparin, hirudin, hirulog, a novel thrombin inhibitor H-(N-Me-D-Phe)-Pro-L-trans-4-aminocyclohexyl-Gly-[CO-CO]-NHCH3+ ++ (L-370,518) and the factor Xa inhibitor tick anticoagulant peptide in rabbits. Thrombosis was induced through application of 70% ferric chloride to the femoral artery and jugular vein. Incidence of occlusion, thrombus weight, aPTT and plasma inhibitor concentrations were determined. Heparin was efficacious in preventing arterial and venous occlusive thrombosis but at a dose that profoundly elevated aPTT. On a molar dosing basis, the approximate order of potency of the thrombin and factor Xa inhibitors was similar in artery and vein: hirudin>tick anticoagulant peptide>hirulog> or =L-370,518. Data suggested that compounds tended to be more potent in preventing venous thrombosis than arterial. This thrombin-dependent model is an economical and efficient approach to arterial and venous antithrombotic efficacy screening that eliminates variabilities encountered when multiple model/multiple animal strategies are employed.

Cuticle bleeding time (CBT) measurements in anesthetized rabbits were performed to assess the potential bleeding risks which may accompany the administration of tissue-type plasminogen activator (tPA) or vampire bat salivary plasminogen activator (BatPA). The dose of BatPA or tPA used in this study, 42 nmol/kg, was previously shown to be efficacious using a rabbit femoral artery thrombosis model (Gardell et al, Circulation 84:244, 1991). CBT was determined by severing the apex of the nail cuticle and monitoring the time to cessation of blood flow. CBT was minimally elevated (1.6-fold, p = NS) following bolus intravenous administration of BatPA; in contrast, bolus intravenous administration of tPA dramatically elevated CBT (6.2-fold, p < 0.05). Rabbits treated with tPA, but not BatPA, displayed profound activation of systemic plasminogen and consequent degradation of Factor VIII and fibrinogen. Elevations in CBT after the administration of tPA were reversed by the replenishment of plasma Factor VIII activity to 40% of control, but were unaffected by complete replenishment of plasma fibrinogen. The results of this study suggest that the administration of BatPA, at a dose that promotes thrombolysis, may evoke a minimal bleeding risk, relative to an equi-efficacious dose of tPA. In addition, the tPA-provoked proteolytic consumption of Factor VIII may be a key contributor to the heightened bleeding risk.

Vampire bat salivary plasminogen activator (BatPA), human tissue-type plasminogen activator (tPA) or streptokinase (SK) were incubated in human citrated plasma containing a plasma clot that was radiolabelled with iodine-125 fibrin(ogen). Complete clot dissolution by BatPA (30 nM) was associated with slight activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by only 8 and 19%, respectively. Addition of SK (3,600 IU/ml) to the clot-containing plasma caused complete clot lysis and massive activation of the "fluid phase" plasminogen, leading to greater than 60 and 96% decreases of the functional levels of fibrinogen and alpha 2-antiplasmin, respectively. Incubation of tPA (30 nM) in clot-containing plasma caused complete clot lysis as well as substantial activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by 45 and 79%, respectively. The profound degradation of fibrinogen in the SK and tPA but not BatPA-containing samples was confirmed by immunoblot analysis. Additional experiments showed that the presence of soluble clot lysate in plasma containing tPA enhanced the extent of fibrinogen degradation from 25% to greater than 60%; the addition of soluble clot lysate to the plasma containing BatPA did not prompt further fibrinogen degradation. Finally, studies using exogenous alpha 2-antiplasmin suggested that plasmin generated via tPA-mediated activation of "fluid phase" plasminogen does not play an important role in clot dissolution.