FUNCTIONAL CHARACTERIZATION OF PROTEIN C INHIBITORS (PCI) FROM PLASMA AND URINE

Immunoblotting studies showed that human plasma and urine contain components that form heparin-dependent complexes with activated protein C (APC) and urokinase (UPA) and that the presence of either active enzyme decreases complex formation by the other (Geiger et al. 1986, Circ. 74:11-234). To investigate whether these findings are due to competition of APC and UPA for one or more heparin-dependent inhibitors, a functional assay for PCI was developed to study the time course of inhibition of protein C (PC) in purified or crude systems in the presence or absence of stimulating or interfering substances. Microtiter plates coated with monoclonal anti-protein C antibodies were incubated with diluted plasma supplemented with ¹²⁵I-pc. After activation of bound PC with Protac C, the wells were washed and then incubated with samples containing PCI in the presence or absence of heparin for varying times. The remaining amidolytic activity on the substrate S-2366 was determined as Δ A₄₀₅/time/cpm bound. Using plasma as a source of PCI, APC inhibition followed pseudo first order kinetics and a linear relation between the pseudo first order rate constant, k, and plasma concentration was observed from 6% to 40% plasma with k = 0.012/min at 20% plasma. A dose dependent increase in k was observed in the presence of heparin, with half maximal stimulation at 2 to 4 U/ml and maximal stimulation (k = 0.05/min at 20% plasma at heparin concentration > 10 U/ml. Urinary PCI was partially purified vising heparin Sepharose chromatography from which it coeluted at 0.35 M NaCl with UPA inhibitory activity. Partially purified urinary PCI inhibited APC and UPA, and competitions similar to those obtained with plasma PCI were observed. The effect of UPA on the inhibition of APC by PCI in plasminogen-depleted plasma and by urinary PCI was studied. In both systems, UPA caused a significant decrease in the inhibition of APC in the absence or presence of heparin. These results and our previous immunobloting data demonstrate competition of APC and UPA for common heparin-dependent PCIs in plasma and urine.