Mechanism of Inhibition of Activated Protein C by Protein C Inhibitor 1

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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 −8 M, 6.7×10 −8 M and 3.1×10 −7 M, 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 125 I-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 1 M ammonia or hydroxyl-amine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hy-droxylamine 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.