Calcium-dependent activation of protein C by thrombin/thrombomudulin: role of negatively charged amino acids within the activation peptide of protein C.
In the absence of its cofactor thrombomodulin (TM) thrombin is only a poor activator of the anticoagulant serine protease protein C (PC). The TM-dependence of PC-activation has been restricted to a series of molecular structures of the PC molecule including high-affinity calcium binding sites and single amino acid residues. However, thrombin induced activation of a PC derivative altered in all these critical positions is markedly enhanced by TM indicating that additional structures of the PC molecule are involved in determining the TM specificity. Based on the hypothesis that such an additional regulatory element should be located near the thrombin cleavage site and should include negatively charged amino acids to ascertain calcium binding, we studied whether Glu and Asp in positions P7 and P6 relative to the thrombin cleavage site together with Asp in P3 are involved in formation of such a regulatory element. Three PC derivatives containing the neutral counterpart of the negatively charged amino acids in positions P3; P3 and P6; and P3, P6, and P7, respectively, were generated using site-directed mutagenesis. Compared to rPC-wt the initial rates of PC activation of all three mutants were increased 4.0-fold for thrombin/TM and 4.0-, 5.3-fold for activation by thrombin alone. However, compared to the PC derivative neutralized exclusively in P3, additional changes in P6 and P7 showed no increase in the thrombin activation kinetics and calcium binding properties were identical in all of the three mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Friedrich
U
U
Hemostasis Research Unit, Max-Planck-Institut für physiologische, Kerckhoff-Klinik, Bad Nauheim, Germany.
Pötzsch
B
B
Preissner
K T
KT
Müller-Berghaus
G
G
Ehrlich
H
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000502:physiology*; D001483:Base Sequence; D002118:Calcium / Q000502:physiology*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D018698:Glutamic Acid / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D018180:Thrombomodulin / Q000378:metabolism*
D011486:Protein C; D011993:Recombinant Fusion Proteins; D018180:Thrombomodulin; D001224:Aspartic Acid; D018698:Glutamic Acid; D013917:Thrombin; D002118:Calcium