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)

[1]  A. Cantor,et al.  Recognition site directing vitamin K-dependent γ-carboxylation resides on the propeptide of factor IX , 1987, Cell.

[2]  C. Esmon,et al.  Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. , 1982, The Journal of biological chemistry.

[3]  D. Rich,et al.  Synthesis of peptide analogues of prothrombin precursor sequence 5-9. Substrate specificity of vitamin K dependent carboxylase. , 1981, Journal of medicinal chemistry.

[4]  C. Walsh,et al.  Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin. , 1990, Journal of Biological Chemistry.

[5]  J. Griffin,et al.  Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[6]  C. Esmon,et al.  Proteolytic formation and properties of gamma-carboxyglutamic acid-domainless protein C. , 1983, The Journal of biological chemistry.

[7]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[8]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[9]  F. Castellino,et al.  The binding of Mn2+ to bovine plasma protein C, des(1-41)-light chain protein C, and activated des(1-41)-light chain activated protein C. , 1987, Archives of biochemistry and biophysics.

[10]  F. Castellino,et al.  A gamma-carboxyglutamic acid (gamma) variant (gamma 6D, gamma 7D) of human activated protein C displays greatly reduced activity as an anticoagulant. , 1990, Biochemistry.

[11]  K. Mann,et al.  Simultaneous purification of bovine prothrombin and factor X. Activation of prothrombin by trypsin-activated factor X. , 1973, The Journal of biological chemistry.

[12]  C. Vermeer,et al.  Isoenzymes of vitamin-K-dependent carboxylase. , 1985, Biochimica et biophysica acta.

[13]  F. Walker.  Regulation of activated protein C by a new protein. A possible function for bovine protein S. , 1980, The Journal of biological chemistry.

[14]  P. Price,et al.  Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[15]  F. Castellino,et al.  Monoclonal antibodies to discrete regions of human Glu1-plasminogen. , 1982, Biochemistry.

[16]  W. Kisiel.  Human Plasma Protein C: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY α-THROMBIN , 1979 .

[17]  K. Kurachi,et al.  Characterization of a cDNA coding for human factor VII. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[18]  L. Patthy,et al.  Common evolutionary origin of the fibrin‐binding structures of fibronectin and tissue‐type plasminogen activator , 1983, FEBS letters.

[19]  K. Kurachi,et al.  Isolation and characterization of a cDNA coding for human factor IX. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[20]  E. Davie,et al.  The nucleotide sequence of the gene for human protein C. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. MacGillivray,et al.  Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. , 1983, Biochemistry.

[23]  B. Lämmle,et al.  Immunoblotting studies of the molecular forms of protein C in plasma. , 1988, Thrombosis research.

[24]  B. Grinnell,et al.  Trans–Activated Expression of Fully Gamma–Carboxylated Recombinant Human Protein C, an Antithrombotic Factor , 1987, Bio/Technology.

[25]  V. V. van Hinsbergh,et al.  Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. , 1985, Blood.

[26]  R. MacGillivray,et al.  Characterization of an almost full-length cDNA coding for human blood coagulation factor X. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A. Tulinsky,et al.  Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain. , 1989, Biochemistry.

[28]  H. Hemker,et al.  Vitamin K-dependent carboxylase: the carboxylation of exogenous substrates in different systems. , 1985, Biochimica et biophysica acta.

[29]  B. Dahlbäck,et al.  Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Walsh,et al.  Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site. , 1989, The Journal of biological chemistry.

[31]  G. Nelsestuen.  Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid. , 1976, The Journal of biological chemistry.

[32]  D. Mcclure,et al.  Characterization and Novel Purification of Recombinant Human Protein C from Three Mammalian Cell Lines , 1990, Bio/Technology.

[33]  P. Fernlund,et al.  beta-Hydroxyaspartic acid in vitamin K-dependent protein C. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[34]  F. Taylor,et al.  A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes--platelet suspensions stimulated by phorbol diester. , 1985, Thrombosis research.

[35]  B. Furie,et al.  Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro. , 1988, The Journal of biological chemistry.

[36]  G. Crabtree,et al.  The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs. , 1985, Nucleic acids research.

[37]  K. Kurachi,et al.  Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). , 1985, Biochemistry.

[38]  H. Ehrlich,et al.  Direct expression of recombinant activated human protein C, a serine protease. , 1989, The Journal of biological chemistry.

[39]  E. Davie,et al.  Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma. , 1979, Biochemistry.

[40]  R. Marlar,et al.  Recombinant human protein C: comparative functional studies with human plasma protein C. , 1990, Thrombosis research.

[41]  W. Kisiel,et al.  Anticoagulant properties of bovine plasma protein C following activation by thrombin. , 1977, Biochemistry.

[42]  E. Davie,et al.  Characterization of a cDNA coding for human protein C. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[43]  E. Davie,et al.  Preparation and properties of bovine factor VIII (antihemophilic factor). , 1980, Biochemistry.

[44]  J. Vieira,et al.  Production of single-stranded plasmid DNA. , 1987, Methods in enzymology.

[45]  W. Kisiel,et al.  Interaction of calcium with bovine plasma protein C. , 1981, Biochemistry.