Antithrombotic Effects of Combining Activated Protein C and Urokinase in Nonhuman Primates

BackgroundWe have determined in vivo the relative antithrombotic efficacy and hemostatic safety of combining low-dose activated protein C (APC) and urokinase (urinary plasminogen activator, u-PA), two natural proteins that regulate thrombogenesis. Methods and ResultsTo model acute thrombotic responses of native blood under conditions of arterial flow, thrombogenic segments of Dacron vascular graft (VG) were incorporated into chronic exteriorized femoral arteriovenous (AV) access shunts in baboons. Thrombus formation on VG was determined by measuring 1) the deposition of autologous 111In platelets using real-time scintillation camera imaging, 2) the accumulation of 125I fibrin, 3) segment patency by Doppler flow analysis, and 4) blood tests for thrombosis, including plasma concentrations of platelet factor 4, 8-thromboglobulin, fibrinopeptide A (FPA), and D-dimer. Treatments consisting of low-dose and intermediate-dose APC (0.07 or 0.25 mg/kg hr), u-PA (25,000 or 50,000 IU/kg hr), or the combination were administered for 1 hour by continuous intravenous infusion. In untreated controls, platelets and fibrin accumulated rapidly, reaching plateau values at 1 hour of 15.1 % 3.8x i09 platelets and 7.8 % 2.2 mg fibrin. Although the low-dose APC or u-PA alone did not decrease either platelet or fibrin deposition significantly, this combination moderately reduced both platelet and fibrin accumulation (7.3 ± 2.6x 109 platelets, p < 0.05; 3.9 ± 0.6 mg fibrin, p < 0.05). Furthermore, intermediate-dose APC or u-PA reduced thrombus formation by half when administered alone (p < 0.001 for both platelet and fibrin deposition), and the combination markedly interrupted the accumulation of platelets (3.0 % 1.0X 109 platelets, p < 0.001) and fibrin (13 % 0.6 mg fibrin, p < 0.001). During active treatments, all VG segments remained patent. Hemostatic plug forming capability, as measured by template bleeding times, remained normal during all experiments (p > 0.05). The T;% clearance time for APC activity was not affected by the concurrent administration of u-PA. u-PA alone increased the plasma levels of D-dimer, FPA, and, interestingly, APC, implying that during pharmacological activation of the fibnrnolytic system, thrombin activity was released, and the protein C pathway was activated. ConclusionsA combination of intermediate-dose APC and u-PA produce substantial and efficient antithrombotic effects without impairing hemostatic function.