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

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.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C‐deficient family

“…All assays were performed in the investigators' laboratories either by ELISA: FV antigen [coefficient of variation (CV) 5.8%][21], FVIII antigen (CV 7.8%), FIX antigen (CV 10%), VWF (CV 3%) [22], protein Z (CV 6.5%) [24], ZPI (CV 7.2%) [24], APC–α1AT complex (CV 12.4%) [25] and APC–PCI complex (CV 11.7%) [25], or radioimmunoassay: prothrombin (CV 8%) [26], antithrombin (CV 5%) [27,28], protein S (CV 9.8%) [29], F1.2 (CV 8%) [28], PCP (CV 14%) [28] and FPA (CV 8%) [28], the Clauss method for fibrinogen (CV 1.7%) [30,31] using the ST4 instrument (Diagnostica Stago, Parsipanny, NJ, USA), a clot‐based functional assay for protein C: (CV 5.5%) [20,23] or micro latex bead agglutination for d ‐dimer (CV 9.2%) (Biomerieux, Durham, NC, USA) [32,33].…”

The multi‐functional serpin, protein C inhibitor: beyond thrombosis and hemostasis

“…Because both APC–PCI and APC–α l AT complexes are observed in plasma of patients with intravascular coagulation, α l AT is also considered a secondary physiological APC inhibitor [55,62,63]. In experiments using primate models, soon after injection of APC, the APC–PCI complex appeared first, and then, when free PCI was decreased by consumption, APC‐α l AT and APC‐α 2 macroglobulin complexes appeared [64,65]. In thrombotic patients treated with heparin, the APC–PCI complex was predominantly observed [66].…”

Characterization of a novel human protein C inhibitor (PCI) gene transgenic mouse useful for studying the role of PCI in physiological and pathological conditions

“…reported that urinary PCI prevents LPS‐induced disseminated intravascular coagulation in rats by inhibiting plasma thrombin and kallikrein [44]. On the other hand, several studies have reported that PCI physiologically inhibits plasma APC in vivo [45–48]. However, it is still unknown how PCI acts on the blood coagulation pathway in vivo .…”