Structural changes required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain gamma-carboxyglutamic acid.

W e have examined the calcium-binding properties and metal ion-dependent conformational changes of a proteolytically modified derivative of bovine protein C that lacks y-carboxyglutamic acid (Gla) residues.Equilibrium dialysis experiments demonstrated that each Gla-domainless protein C molecule retains one high affinity calcium ion-binding site (Kd = 61 k 6 p ~) .Ca2+ binding to this site is accompanied by a 14% decrease in intrinsic fluorescence emission intensity (Kd = 44 k 5 p ~) and a substantial change in circular dichroism.The spectral changes were reversible upon the addition of EDTA, and M&' caused little change in fluorescence intensity below 400 p ~. Titration with Mn2+ or T b 3 + resulted in fluorescence changes similar to those obtained with Ca".Tb3+ and Ca2+ appear to bind to the s a m e site because tryptophan-dependent terbium fluorescence was reduced by the addition of Ca2+.The metal ion-dependent changes in the fluorescence of unmodified protein C were equivalent to those of Gla-domainless protein C at low concentrations of metal ions.However, higher concentrations of Ca2+ induced an additional fluorescence change in protein C with an apparent Kd in excess of 200 p ~.The close correlation of the Kd value inferred from kinetic studies and the Kd values obtained from equilibrium dialysis and fluorescence data indicate that the conformation of Gla-domainless protein C that is recognized and activated b y the soluble thrombin-thrombomodulin complex is nucleated and/or stabilized by calcium binding to a high affinity site which does not include Gla residues.Additional Gla domain-dependent calcium binding and conformational changes are then required for the activation of intact protein C b y the thrombin-thrombomodulin complex.