Mechanism of Inhibition of Activated Protein C by Protein C Inhibitor

Abstract

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K₁) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6×10^-8M, 6.7×10^-8M and 3.1×10^-7M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (M_r=62.000) was incubated with protein C inhibitor (M_r=57, 000), enzyme-inhibitor complexes with apparent M_r=102, 000 and 88, 000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with M_r=54, 000 were detected. When ¹²⁵I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent M_r=102, 000 and 54, 000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with M_r=54, 000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor. Thus, the results indicate that the protein C inhibitor inhibits activated protein C by forming an enzyme-inhibitor complex accompanied with the proteolytic modification of the inhibitor by the enzyme.