Antithrombotic effects of combining activated protein C and urokinase in nonhuman primates

Background. We 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 Results. To 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, β-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 lU/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±3.8×10⁹ 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±2.6×10⁹ 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±1.0×10⁹ 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 T₅₀ 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 fibrinolytic system, thrombin activity was released, and the protein C pathway was activated. Conclusions. A combination of intermediate-dose APC and u-PA produce substantial and efficient antithrombotic effects without impairing hemostatic function.