The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.
Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.
Rezaie
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Howard Hughes Medical Institute, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Esmon
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D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011486:Protein C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013725:Terbium / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011486:Protein C; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013725:Terbium; D004815:Epidermal Growth Factor; D013917:Thrombin; D015951:Factor Xa; D002118:Calcium