Role of the hexapeptide disulfide loop present in the gamma-carboxyglutamic acid domain of human protein C in its activation properties and in the in vitro anticoagulant activity of activated protein C.
In order to examine whether the structural integrity of the hexapeptide disulfide loop (residues 17-22), present in the gamma-carboxyglutamic acid (gamma) domain of human protein C (PC), and common to all vitamin K dependent coagulation proteins, is necessary for its anticoagulant properties, we employed recombinant (r) DNA technology to generate two important variants that would address this issue. One such mutein contained aspartic acid for gamma-residue substitutions at sequence positions 19 and 20 ([gamma 19D, gamma 20D]r-PC) in the light chain of the mature protein, and the other possessed a serine for cysteine substitution at position 22 ([C22S]r-PC of the same light chain. A subpopulation of molecules of these mutant proteins, containing the maximum levels of gamma-residues in each, has been purified by fast-protein anion-exchange liquid chromatography and affinity chromatography on an anti-human PC column. A study of the kinetic characteristics of the inhibition by Ca2+ of the thrombin-catalyzed activation rates of these variants, and the corresponding stimulation by Ca2+ of the thrombin/thrombomodulin-catalyzed activation rates of the same recombinant PC molecules, demonstrated that higher concentrations of Ca2+ were required to display these effects, when compared to wild-type (wt) r-PC and human plasma PC. This suggested that the kinetically relevant Ca2+ site responsible for these effects on activation of PC, and known to be present in another domain of PC, was affected by both mutations in the gamma-domain. The recombinant PC variants were converted to their activated forms ([gamma 19D, gamma 20D]r-APC and [C22S]r-APC) and assayed for their Ca(2+)-dependent anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
L
L
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D000925:Anticoagulants / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005169:Factor VIII / Q000037:antagonists & inhibitors; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D011486:Protein C / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D000925:Anticoagulants; D004220:Disulfides; D009842:Oligopeptides; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D005169:Factor VIII; D004247:DNA; D002118:Calcium