Proteolytic formation and properties of gamma-carboxyglutamic acid-domainless protein C.

Protein C activation is catalyzed by a complex between thrombin and an endothelial cell surface receptor, thrombomodulin.To investigate the function of the y-carboxyglutamic acid (Gla) residues in the calciumdependent activation of protein C, residues 1-41 were removed from the protein C light chain by selective proteolysis with chymotrypsin, and the Gla-domainless protein C was purified to homogeneity.Activated Gladomainless protein C and activated protein C hydrolyzed H-D-Phe-pipecolyl-Arg-p-nitroanilide at the same rate.Unlike activated protein C, Gla-domainless activated protein C did not function as an anticoagulant.Calcium ions were required for Gla-domainless protein C activation by the thrombin-thrombomodulin complex.At 2 p~ substrate, half-maximal rates of activation were achieved at 250 2 50 p~ Ca2+ for protein C and 60 k 5 p~ Ca2+ for Gla-domainless protein C. At saturating levels of Ca2+, the activation properties of these two substrates were identical; Vmax = 250 mol/ min/mol and K,,, = 8 PM.In the absence of thrombomodulin, calcium inhibits thrombin-catalyzed activation of both Gla-domainless protein C and protein C. Half-maximal inhibition of protein C activation occurs at 250 p~ Ca2+, while half-maximal inhibition of Gladomainless protein c activation occurs at 60 CM Ca".Over the surface of rabbit endothelial cells, Gla-domainless protein C, at physiological concentrations, was activated at less than 5% the rate of protein C. Endothelial cell-mediated protein C activation exhibited sites with both high and low affinity for protein C, while only low affinity sites were observed for Gladomainless protein C.These results indicate that protein C possesses a Glaindependent high affinity calcium-binding site that is required for recognition by the thrombin-thrombomodulin complex and that metal ion interaction with this same site is responsible for Ca2+ inhibition of protein C activation by thrombin alone.These studies further show that the Gla domain of protein C is essential for optimal protein C activation at the endothelial cell surface.