Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Identification of a region in protein C involved in thrombomodulin-stimulated activation by thrombin: potential repulsion at anion-binding site I in thrombin. 798 Apr 64

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.
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PMID:Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin. 879 83