Immunoradiometric Assay for the Calcium-Stabilized Conformation of Human Protein S

Summary

Rabbit polyclonal anti-protein S serum was fractionated with immobilized human protein S to establish solid-phase immunoradiometric assays recognizing Ca(II)-dependent and NonCa(II)-dependent epitopes of human protein S. The two assays were specific for PS:Ca(II)Ag and PS:NonCa(II)Ag and highly sensitive with a lower limit of detection of about 2.5 ng/ml.

PS:Ca(II)Ag and PS:NonCa(II)Ag levels were measured in immunopurified protein S, thrombin-modified protein S and chy-motrypsin-cleaved protein S. Only in chymotrypsin-cleaved protein S an important discrepancy between the two antigen levels was observed.

Ranges for the concentration of PS:Ca(II)Ag and PS:NonCa(II)Ag and their ratio were established in plasma of healthy individuals (0.92 ± 0.13 U/ml, 0.98 ± 0.21 U/ml, 0.96 ± 0.17, respectively). In a group of patients using oral anticoagulant therapy the ratio PS:Ca(II) Ag/PS : NonCa(II)Ag decreased at increasing intensity of anticoagulation suggesting the presence of sub- and noncarboxylated protein S molecules.

In plasma of patients with a hereditary type I protein S deficiency PS:Ca(II)Ag and PS:NonCa(II) Ag were reduced to the same extent: mean ratio 1.02 ± 0.12 in the group not on oral anticoagulant treatment and 0.94 ± 0.10 in the group on oral anticoagulant therapy. Analysis of patients with a history of unexplained thrombo-embolic disease did not reveal individual patients with a PS:Ca(II)Ag/PS:NonCa(II)Ag ratio below the lower limit of the normal range (mean ratio 1.05 ± 0.17), suggesting that the frequency of genetic protein S variants with defects in the Ca(II)-stabilized conformation is very low.

Keywords

Protein S antibodies - Oral anticoagulation - Calcium - Immunoradiometric assay - Thrombotic disease

• References

• 1 DiScipio RG, Hermodson MA, Yates SG, Davies EW. A comparison of human prothombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 1977; 16: 698-706
• 2 Dahlback B. Interaction between vitamin K-dependent protein S and the complement protein C4b-binding protein. A link between coagulation and the complement system. Semin Thromb Hemostas 1984; 10: 139-148
• 3 Walker FJ. Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. J Biol Chem 1986; 261: 10941-10944
• 4 Walker FJ. Protein S and the regulation of activated protein C. Semin Thromb Hemostas 1984; 10: 131-138
• 5 Suzuki K, Stenflo J, Dahlback B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
• 6 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 7 Gardiner JE, McGann MA, Berridge CW, Fulcher CA, Zimmerman TS, Griffin JH. Protein S as a cofactor for activated protein C in plasma and in the inactivation of purified factor VIII: C. Circulation 1984; 70 suppl. 11-205
• 8 Fay PJ, Chavin SI, Schroeder D, Young FE, Marder VJ. Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain. Proc Natl A cad Sci USA 1982; 79: 7200-7204
• 9 Harris KW, Esmon CT. Protein S is required for bovine platelets to support activated protein C binding and activity. J Biol Chem 1985; 260: 2007-2010
• 10 Walker FJ. Regulation of vitamin K-dependent protein S. Inactivation by thrombin. J Bio Chem 1984; 259: 10335-10339
• 11 Lundwall Å, Dackowski W, Cohen E, Shaffer M, Mahr A, Dahlbäck B, Stenflo J, Wydro R. Isolation and sequence of the cDNA for human protein s, a regulator of blood eoagulation. Proc Nat Acad Sci USA 1986; 83: 6716-6720
• 12 Doolittle RF, Feng Df, Johnsson MS. Computer-based charactetiiation of epidermal growth factor precursor. Nature 1984; 307: 558-560
• 13 Prendergast FG, Mann KG. Differentiation of metal ion-induced transitions of prothrombin fragment: l. J Biol Chem 1977; 252: 840-850
• 14 Bloom JW, Mann KG. Metal ion-induced cohfotmational.transitions of prothrombin and prothrombin fragment 1. J Biochemistry 1978; 17: 4430-4438
• 15 Amphlett GW, Byrne G, Castellino FJ. The binding of metal ions to bovine factor IX. J Biol Chem 1978; 253: 6774-6779
• 16 Jackson CM, Nemerson Y. Blood coagulation. Ann Rev Biochem 1980; 49: 765-811
• 17 Johnson AE, Esmon NL, Laue TM, Esmon CT. Structural changes required for activation of protein C are induced by Ca²⁺-binding to a high affinity site that does not contain y-carboxyglutamic acid. J Biol Chem 1983; 258: 5554-5560
• 18 Wakabayashi K, Sakata Y, Aoki N. Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. J Biol Chem 1986; 261: 11097-11105
• 19 Sugo T, Dahlback B, Holmgren A, Stenflo J. Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the y-carboxyglutamic acid-containing region. J Biol Chem 1986; 261: 5116-5120
• 20 Brody T, Suttie JW. Glutamate carboxylase: assays, occurrence and specificity. Methods Enzymol 1984; 107: 552-563
• 21 Bertina RM, van der Marel-van Nieuwkoop W, Loeliger EA. Spectrophotometric assays of prothrombin in plasma of patients using oral anticoagulants. Thromb Haemostas 1979; 42: 1296-1305
• 22 Blanchard RA, Furie BC, Jorgensen M, Kruger SF, Furie B. Acquired vitamin K-dependent carboxylation deficiency in liver disease. New Engl J Med 1981; 305: 242-248
• 23 Furie B, Blanchard RA, Robison DJ, Tai MM, Fuire BC. Conformation-specific antibodies: approach to the study of the vitamin K-dependent blood coagulation proteins. Meth Enzymol 1982; 84: 60-83
• 24 Broze jr GJ, Hickman S, Miletich JP. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII. J Clin Invest 1985; 76: 937-946
• 25 Bom V JJ, van Tilburg NH, Krommenhoek-van EsC, Bertina RM. Immunoradiometric assays for human coagulation factor VII using polyclonal antibodies against the Ca(II)-dependent and Ca(II)-inde-pendent conformation. Thromb Haemostas 1986; 56: 343-348
• 26 Bertina RM, Veltkamp JJ. A genetic variant of factor IX with decreased capacity for Ca²⁺ binding. Brit J Haematol 1979; 42: 632-635
• 27 Poort SR, van der Linden IK, Krommenhoek-van EsC, Briet E, Bertina RM. Rabbit polyclonal antibodies against the calcium-dependent conformation of factor IX and their application in solid phase immunoradiometric assays. Thromb Haemostas 1986; 55: 122-128
• 28 Bray GL, Weinmann F, Thompson AR. Calcium-specific immunoassays for factor IX: reduced levels of antigen in patients with vitamin K disorders. J Lab Clin Med 1986; 107: 269-278
• 29 Mikami S, O’Brien DP, Mellars G, Goodall AH, Tuddenham E GD. Studies on immunological assay of vitamin K-dependent factors III. A double monoclonal immunoradiometric assay for factor IX antigen. Brit J Haematol 1986; 62: 513-524
• 30 Tai MM, Furie BC, Furie B. Conformation-specific antibodies directed against the bovine prothrombin-calcium complex. J Biol Chem 1980; 255: 2790-2795
• 31 Bertina RM, van Wijngaarden A, Reinalda-Poot J, Poort SR, Bom V JJ. Determination of plasma protein S, the protein cofactor of activated protein C. Thromb Haemostas 1985; 53: 268-272
• 32 Loeliger EA, Lewis SM. Progress in laboratory control of oral anticoagulants. Lancet 1982; 2: 318-320
• 33 Bertina RM. Hereditary protein S deficiency. Haemostasis 1985; 15: 241-246
• 34 Dahlback B, Lundwall A, Stenflo J. Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S. J Biol Chem 1986; 261: 5111-5115
• 35 Suzuki K, Nishioka J, Hashimoto S. Regulation of activated protein C by thrombin-modified protein S. J Biochem 1983; 94: 699-705
• 36 Eisenhart C, Hastay MW, Wallis WA. Techniques of Statistical Analysis. pp. 102-107 McGraw-Hill Book Company; New York: 1947
• 37 Morita T, Mizuguchi J, Kawabata S, Iwanaga S. Proteolytic cleavage of vitamin K-dependent bovine plasma protein S by a-thrombin and plasma serine protease. J Biochem 1986; 99: 561-568
• 38 Fair DS, Revak DJ. Quantitation of human protein S in the plasma of normal and warfarin-treated individuals by radioimmunoassay. Thromb Res 1984; 36: 527-535
• 39 Qrstavik KH, Laake K. Factor IX in warfarin treated patients. Thromb Res 1978; 13: 207-218
• 40 Fair DS, Plow EF, Edgington TS. Combined functional and immunochemical analysis of normal and abnormal human factor X. J Clin Invest 1979; 64: 884-894
• 41 Fair DS. Quantitation of factor VII in the plasma of normal and warfarin-treated individuals by radioimmunoassay. Blood 1983; 62: 784-791
• 42 Stern D, Brett J, Harris K, Nawroth P. Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S. J Cell Biol 1986; 102: 1971-1978
• 43 Morita T, Jackson CM. Preparation and properties of derivatives of bovine factor X and factor Xa from which the y-carboxyglutamic acid containing domain has been removed. J Biol Chem 1986; 261: 4015-4023
• 44 Esmon NL, De Bault LE, Esmon CT. Proteolytic formation and properties of y-carboxyglutamic acid-domainless protein C. J Biol Chem 1983; 258: 5548-5553
• 45 Morita T, Isaacs BS, Esmon CT, Johnson AE. Derivatives of blood coagulation factor IX contain a high affinity Ca²⁺-binding site that lacks y-carboxyglutamic acid. J Biol Chem 1984; 259: 5698-5704
• 46 Morita T, Mizuguchi J, Iwanaga S. Proteolytic cleavage of bovine vitamin K-dependent factors, protein C, protein S and protein Z for preparation of their Gla-domainless derivatives. Thromb Haemostas 1983; 50: 81 (Abstr.)
• 47 Bertina RM, Broekmans AW, Krommenhoek-van EsC, van Wijngaarden A. The use of a functional and immunologic assay for plasma protein C in the study of the heterogeneity of congenital protein C deficiency. Thromb Haemostas 1984; 51: 1-5