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.
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+).
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Rezaie
Alireza R
AR
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D010978:Platelet Factor 4 / Q000378:metabolism*; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection; D014812:Vitamin K / Q000378:metabolism*
D046912:Multiprotein Complexes; D011486:Protein C; D018180:Thrombomodulin; D014812:Vitamin K; D010978:Platelet Factor 4; D002809:Chondroitin Sulfates; D013925:Thromboplastin; D013917:Thrombin; D002118:Calcium