Identification of a region in protein C involved in thrombomodulin-stimulated activation by thrombin: potential repulsion at anion-binding site I in thrombin.
During coagulation human protein C is activated by thrombin; however, this cleavage reaction is slow unless thrombin is complexed with a cofactor, thrombomodulin. Near the thrombin cleavage site in protein C is a cluster of basic residues, at positions P5' (Lys-174), P8' (Arg-177) and P9' (Arg-178). We have explored the role of this basic cluster in the activation of protein C by thrombin, and by thrombin-thrombomodulin complex, by substitution of glutamic acid at each position to generate the acidic protein C derivative P'-EEE. The activation rate of P'-EEE by free alpha-thrombin was approx. 12-fold faster than that observed for wild-type (wt) human protein C zymogen (HPC) in the presence of calcium, but unchanged in the absence of calcium. While the thrombin-catalysed activation of wt-HPC was stimulated approx. 300-fold by thrombomodulin, we observed no effect of thrombomodulin on thrombin-catalysed activation of the P'-EEE derivative. Using synthetic peptides that bind to anion-binding site I of thrombin (thrombin-receptor sequence 52-66 and hirudin sequence 54-65 SO4 Tyr), we found that the rate of thrombin-catalysed activation of wt-HPC in the presence of calcium could be increased severalfold in a dose-dependent manner. However, the enhanced rate of thrombin-catalysed activation of P'-EEE could be progressively reduced to wt-HPC levels with increasing concentrations of both synthetic peptides. Our data suggest that the P' basic cluster in protein C reduces interaction with free alpha-thrombin through electrostatic repulsion with anion-binding site I, a site that is masked when thrombomodulin binds thrombin. Further, the lack of thrombomodulin cofactor activity with thrombin-catalysed activation of P'-EEE suggests that the basic cluster in protein C forms a contact site with thrombomodulin.
Department of Cardiovascular Research, Lilly Research Laboratories, Indianapolis, IN 46285-0434.
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Grinnell
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Gerlitz
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D000595:Amino Acid Sequence; D000838:Anions; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000502:physiology*
D000838:Anions; D017931:DNA Primers; D010455:Peptides; D011486:Protein C; D018180:Thrombomodulin; D013917:Thrombin