In vivo and in vitro complexes of activated protein C with two inhibitors in baboons

In vivo complex formation of activated protein C with protein C inhibitor (APC-PCI) and with α₁-antitrypsin (APC-α₁AT) following infusion of 0.25 or 1.0 mg APC/kg in 1 hour into baboons was studied using immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA)s. Before APC infusion, detectable plasma levels (about 30 ng/mL) of APC-α₁AT complex were found in the baboon plasma. At the lower APC dose, APC-PCI and APC-α₁AT complex levels were 1.4 ± 0.3 (mean ± SD) and 0.8 ± 0.1 μg/mL after 1 hour of infusion. At the higher APC dose, the APC-PCI level was similar to the APC-α₁AT level during the first 30 minutes, but after 1 hour of infusion the APC-α₁AT level was higher than the APC-PCI level, reaching 4.1 ± 1.2 and 2.9 ± 1.2 μg/mL, respectively. After 24 hours, complex levels had returned to basal conditions. During infusion of protein C (1.0 mg/kg in 1 hour), both complexes were detected in low concentrations. Following bolus injection of APC, half-lives (t_1/2) for APC and APC-PCI and APC-α₁AT complexes of 10, 40, and 140 minutes, respectively, were observed. After 1-hour incubation with 2.5 μg/mL APC, baboon plasma contained 1.0 ± 0.2 and 0.8 ± 0.1 μm/mL of APC-PCI and APC-α₁AT, respectively. Addition of 10 μg/mL APC to baboon plasma yielded 2.5 and 2.4 μg/mL APC-PCI and APC-α₁AT after 1 hour, respectively, Immunoblotting analysis also showed in vivo formation of complexes of APC with an auxiliary inhibitor but not in vitro in citrated plasma. These data show that both PCI and α₁AT are physiologic inhibitors of APC and suggest that when PCI is depleted by a high dose of APC, α₁AT becomes the major inhibitor of APC.