The Interaction of Protein S with the Phospholipid Surface Is Essential for the Activated Protein C-independent Activity of Protein S

Summary

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces.

During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-inde-pendent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S.

We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

• References

• 1 Mahasandana C, Suvatte V, Marlar RA, Manco JohnsonMJ, Jacobson LJ, Hathaway WE. Neonatal purpura fulminans associated with homozygous protein S deficiency [letter]. Lancet 1990; 335: 61-62
• 2 Schwarz HP, Heeb MJ, Lottenberg R, Roberts H, Griffin JH. Familial protein S deficiency with a variant protein S molecule in plasma and platelets. Blood 1989; 74: 213-221
• 3 Engesser L, Broekmans AW, Briet E, Brommer EJ, Bertina RM. Hereditary protein S deficiency: clinical manifestations. Ann Intern Med 1987; 106: 677-682
• 4 Bertina RM. Hereditary protein S deficiency. Haemostasis 1985; 15: 241-246
• 5 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528
• 6 Hackeng TM, Hessing M, van’t Veer C, Meijer HuizingaF, Meijers JC, de Groot PG, van Mourik JA, Bouma BN. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C. J Biol Chem 1993; 268: 3993-4000
• 7 Bakker HM, Tans G, Janssen ClaessenT, Thomassen MC, Hemker HC, Griffin JH, Rosing J. The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C. Eur J Biochem 1992; 208: 171-178
• 8 Koedam JA, Meijers JC, Sixma JJ, Bouma BN. Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 1988; 82: 1236-1243
• 9 Solymoss S, Tucker MM, Tracy PB. Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection. J Biol Chem 1988; 263: 14884-14890
• 10 Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RF, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-2648
• 11 Rosing J, Hoekema L, Nicolaes GAF, Christella M, Thomassen LGD, Hemker HC, Varadi K, Schwarz HP, Tans G. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-27858
• 12 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor Villa. J Biol Chem 1994; 269: 18735-18738
• 13 Hackeng TM, van’t Veer C, Meijers JC, Bouma BN. Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa. J Biol Chem 1994; 269: 21051-21058
• 14 Heeb MJ, Rosing J, Bakker HM, Fernandez JA, Tans G, Griffin JH. Protein S binds to and inhibits factor Xa. Proc Natl Acad Sci USA 1994; 91: 2728-2732
• 15 Heeb MJ, Mesters RM, Tans G, Rosing J, Griffin JH. Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-2877
• 16 Koppelman SJ, Hackeng TM, Sixma JJ, Bouma BN. Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII. Blood 1995; 86: 1062-1071
• 17 van ’t Veer C, Hackeng TM, Biesbroeck D, Sixma JJ, Bouma BN. Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma. Blood 1995; 85: 1815-1821
• 18 Dahlbäck B. Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846
• 19 Chang GT, Ploos van Amstel HK, Hessing M, Reitsma PH, Bertina RM, Bouma BN. Expression and characterization of recombinant human protein S in heterologous cells - studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein. Thromb Haemost 1992; 67: 526-532
• 20 Meijers JC, Davie EW, Chung DW. Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency. Blood 1992; 79: 1435-1440
• 21 Grinnell BW, Walls JD, Marks C, Glasebrook AL, Berg DT, Yan SB, Bang NU. Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties. Blood 1990; 76: 2546-2554
• 22 DiScipio RG, Davie EW. Characterization of protein S, a gamma-carboxy-glutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904
• 23 Klein JD, Walker FJ. Purification of a protein C activator from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Biochemistry 1986; 25: 4175-4179
• 24 Bock PE, Craig PA, Olson ST, Singh P. Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-p-guanidinoben-zoate. Arch Biochem Biophys 1989; 273: 375-388
• 25 Mertens K, Cupers R, van Wijngaarden A, Bertina RM. Binding of human blood-coagulation Factors IXa and X to phospholipid membranes. Biochem J 1984; 223: 599-605
• 26 Kane WH, Majerus PW. Purification and characterization of human coagulation factor V. J Biol Chem 1981; 256: 1002-1007
• 27 Kisiel W. Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J Clin Invest 1979; 64: 761-769
• 28 Di Scipio RG, Hermodson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 1977; 16: 698-706
• 29 Bajaj SP, Rapaport SI, Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem 1981; 11: 397-412
• 30 Nesheim ME, Myrmel KH, Hibbard L, Mann KG. Isolation and characterization of single chain bovine factor V. J Biol Chem 1979; 254: 508-517
• 31 Brunner J, Skrabal P, Hauser H. Single bilayer vesicles prepared without sonication. Physico-chemical properties. Biochim Biophys Acta 1976; 455: 322-331
• 32 Rouser G, Fkeischer S, Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970; 5: 494-496
• 33 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16
• 34 Walker FJ. Regulation of vitamin K-dependent protein S. Inactivation by thrombin. J Biol Chem 1984; 259: 10335-10339
• 35 Dahlbäck B, Wiedmer T, Sims PJ. Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles. Biochemistry 1992; 31: 12769-12777
• 36 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-1651
• 37 Sugo T, Dahlbäck B, Holmgren A, Stenflo J. Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxy-glutamic acid-containing region. J Biol Chem 1986; 261: 5116-5120