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Thromb Haemost. 2007 Jun;97(6):899-906.

Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors.

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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.


The Ca(2+)-dependence of protein C activation by thrombin in complex with thrombomodulin (TM) containing chondroitin sulfate (CS) exhibits saturation at approximately 0.5-1 mM Ca(2+), but with TM lacking CS, it has a distinct optimum at approximately 0.1 mM Ca(2+). Since the substrate protein C has multiple Ca(2+)-binding sites, and the cofactor TM also interacts with Ca(2+), the basis for differences in Ca(2+) effect on protein C activation by thrombin in complex with TM containing or lacking CS is not known. In this study, by using full-length and Gla-domainless mutants of protein C whose activation by thrombin is independent of either Ca(2+) or both Ca(2+) and TM, we demonstrate that i) the Ca(2+) occupancy of a high-affinity binding site in TM is essential for the high-affinity interaction of the cofactor with thrombin, ii) the Ca(2+) occupancy of a binding site (K(D) approximately 50 microM) in the catalytic domain of protein C is required for the substrate recognition by the thrombin-TM complex, however, at this concentration of Ca(2+) the Gla domain of protein C is not folded properly and thus interacts with exosite-2 of thrombin in complex with TM that lacks CS but not with TM that contains CS, and finally iii) platelet factor 4 can nonspecifically interact with the Gla domain of protein C and other coagulation factors to influence their activation only at subphysiological concentrations of Ca(2+).

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