Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor

Abstract

The inhibition of kallikrein and factor XIa by protein C inhibitor (PCI) was studied. The method of Suzuki et al. [Suzuki, K., Nishioka, J., and Hashimoto, S. (1983) J. Biol. Chem. 258, 163-168] for the purification of PCI was modified in order to avoid the generation of proteolytic activity and subsequent inactivation of PCI. With the use of soybean trypsin inhibitor, an efficient inhibitor of kallikrein and factor XIa, the generation of proteolytic activity was avoided. The kinetics for the inactivation of activated protein C (APC), kallikrein, and factor XIa by PCI were determined. In the absence of heparin, no inactivation of APC was observed, in contrast to kallikrein and factor XIA, which are inhibited with second-order rate constants of (11 .+-. 4) .times. 104 and (0.94 .+-. 0.07) .times. 104 M-1 s-1, respectively. Addition of heparin potentiated the inhibition of APC [(1.2 .+-. 0.2) .times. 104 M-1 s-1] and factor XIa [(9.1 .+-. 0.7) .times. 104 M-1 s-1] by PCI, whereas the inhibition of kallikrein by PCI was unchanged [(10 .+-. 1) .times. 104 M-1 s-1]. The second-order rate constants for the inhibition of kallikrein or factor XIa by PCI were similar to the second-order rate constants for the inhibition of their isolated light chains by PCI, indicating a minor role for the heavy chains of both molecules in the inactivation reactions. With sodium dodecyl sulfate-polyacrylamide slabe gel electrophoresis and immunoblotting, complex formation of APC, kallikrein, and factor XIa with PCI could be demonstrated. APC and kallikrein formed 1:1 molar complexes with PCI. Factor XIa formed 1:1 and 1:2 molar complexes with PCI, indicating that both active sites on the factor XIa molecule can be inactivated by PCI. On the basis of second-order rate constant calculations, PCI would account for 7% of the inactivation of kallikrein and 5% of the inactivation of factor XIa in plasma, indicating that PCI can be physiologically important in the inhibition of both proteinases. Moreover, because of the high second-order rate constants for the inactivation of kallikrein and factor XIa by PCI and the low concentration of PCI in plasma, the PCI activity in plasma could be regulated by these proteinases of the contact system.