A qualitative and quantitative analysis of the activation and inactivation of protein C in vivo in a primate model

Abstract

Abstract A model of Protein C (PC) activation in vivo was used to investigate the complexing of activated PC (APC) with its plasma inhibitors, PC inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT). Chimpanzees were infused with a bolus of activated factor X (F.Xa) together with vesicles of phosphatidylcholine and phosphatidylserine (PCPS). Pre- and post-infusion plasma samples were analyzed using enzyme linked immunosorbent based assays (ELISA) for PC and APC complexes, and immunoblotting of PC from nondenaturing polyacrylamide gel electrophoresis. Within 2 minutes of infusion, a 60% decrease in nonactivated PC zymogen (PCz) levels was observed. This coincided with a precipitous drop in plasma activities of cofactors VIIIa and Va. In contrast, total PC antigen (PCt) levels decreased by only 1%, indicating APC generation. Complexes of APC with both PCI and alpha 1AT were observed on immunoblots, and further identified and quantified using a sandwich ELISA employing antibodies to both PC and these inhibitors. The distribution of APC between these two inhibitors varied with the dose of F.Xa/PCPS infused. At a dose of F.Xa/PCPS of 24.05 pmol and 37.70 nmol/kg, respectively, an initial spike of APC generation, associated with decreases in the levels of factors VIIIa and Va, was noted but dissipated over the next 30 minutes. During this period, APC/inhibitor complexes appeared with the levels of APC-PCI and APC-alpha 1AT reaching 8.5 nmol/L and 2.2 nmol/L by 30 minutes, respectively. In contrast, at a higher dose of F.Xa/PCPS of 36.60 pmol and 56.30 nmol/Kg respectively, complexes of APC-alpha 1AT appeared rapidly and reached a level of 6 nmol/L by 30 minutes postinfusion, whereas APC-PCI complexes were only present at a concentration of 3.4 nmol/L by this time. Additional experiments with lower doses of F.Xa/PCPS suggest that PCI is the preferred inhibitor of APC, but as the availability of this inhibitor becomes limiting, alpha 1AT plays an increasingly crucial role as a secondary inhibitor of endogenously generated APC. Moreover, evidence is presented suggesting the existence of additional inhibitor(s) of APC that may have a role similar to alpha 1AT.