Plasma protein S residues 37–50 mediate its binding to factor Va and inhibition of blood coagulation

 

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Summary

Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilised FVa. Epitope mapping identified N-terminal region residues 37–67 of PS as this antibody’s epitope. A peptide representing PS residues 37–50 inhibited FVa-dependent prothrombinase activity in a noncompetitive manner, with 50% inhibition observed at 11 µM peptide, whereas a peptide with a D-amino acid sequence of 37–50 was ineffective. FVa, but not FXa, bound specifically to the immobilised peptide representing residues 37–50, and the peptide inhibited binding of FVa to immobilised PS. These data implicate PS residues 37–50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS.

• References

• 1 Burstyn-Cohen T, Heeb MJ, Lemke G. Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis. J Clin Invest 2009; 119: 2942-2953.
  CrossrefPubMedGoogle Scholar
• 2 Saller F, Brisset AC, Tchaikovski SN, Azevedo M. et al. Generation and phenotypic analysis of protein S-deficient mice. Blood 2009; 114: 2307-2314.
  CrossrefPubMedGoogle Scholar
• 3 Mahasandana C, Suvatte V, Marlar RA. et al. Neonatal purpura fulminans associated with homozygous protein S deficiency. Lancet 1990; 335: 61-62.
  PubMedGoogle Scholar
• 4 Schwarz HP, Fischer M, Hopmeier P. et al. Plasma protein S deficiency in familial thrombotic disease. Blood 1984; 64: 1297-1300.
  PubMedGoogle Scholar
• 5 Mahmoodi BK, Brouwer JL, Veeger NJ. et al. Hereditary deficiency of protein C or protein S confers increased risk of arterial thromboembolic events at a young age: results from a large family cohort study. Circulation 2008; 118: 1659-1667.
  CrossrefPubMedGoogle Scholar
• 6 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528.
  CrossrefPubMedGoogle Scholar
• 7 Walker FJ. Protein S and the regulation of activated protein C. Semin Thromb Hemost 1984; 10: 131-138.
  Article in Thieme ConnectPubMedGoogle Scholar
• 8 Griffin JH, Zlokovic BV, Mosnier LO. Protein C anticoagulant and cytoprotective pathways. Int J Hematol 2012; 95: 333-345.
  CrossrefPubMedGoogle Scholar
• 9 Heeb MJ, Mesters RM, Tans G. et al. 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.
  PubMedGoogle Scholar
• 10 Heeb MJ, Rosing J, Bakker HM. et al. Protein S binds to and inhibits factor Xa. Proc Natl Acad Sci USA 1994; 91: 2728-2732.
  CrossrefPubMedGoogle Scholar
• 11 Fernandes NL, Mosnier LO, Tonnu L. et al. Zinc-containing protein S inhibits extrinsic factor X activating complex independently of tissue factor pathway inhibitor. J Thromb Haemost 2010; 08: 1976-1985.
  CrossrefPubMedGoogle Scholar
• 12 Hackeng TM, van't Veer C, Meijers JCM. et al. 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.
  PubMedGoogle Scholar
• 13 Schwarz HP, Heeb MJ, Wencel-Drake JD. et al. Identification and characterization of protein S in human platelets. Blood 1985; 66: 1452-1455.
  CrossrefPubMedGoogle Scholar
• 14 Stavenuiter F, Davis NF, Duan E. et al. Platelet protein S directly inhibits procoagulant activity on platelets and microparticles. Thromb Haemost 2013; 109: 229-237.
  Article in Thieme ConnectPubMedGoogle Scholar
• 15 Heeb MJ, Marzec U, Gruber A. et al. Antithrombotic activity of protein S infused without activated protein C in a baboon thrombosis model. Thromb Haemost 2012; 107: 690-698.
  Article in Thieme ConnectPubMedGoogle Scholar
• 16 Hackeng T, Sere KM, Tans G. et al. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
  CrossrefPubMedGoogle Scholar
• 17 Yegneswaran S, Hackeng TM, Dawson PE. et al. The thrombin-sensitive region of protein S mediates phospholipid-dependent interaction with factor Xa. J Biol Chem 2008; 283: 33046-33052.
  CrossrefPubMedGoogle Scholar
• 18 Stenberg Y, Muranyi A, Steen C. et al. EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X. J Mol Biol 1999; 293: 653-665.
  CrossrefPubMedGoogle Scholar
• 19 Heeb MJ, Kojima Y, Tans G. et al. C-terminal residues 621-635 of protein S are essential for binding to factor Va. J Biol Chem 1999; 274: 36187-36192.
  CrossrefPubMedGoogle Scholar
• 20 Heeb MJ, Prashun D, Griffin JH. et al. Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor. FASEB J 2009; 23: 2244-2253.
  CrossrefPubMedGoogle Scholar
• 21 Tans G, Rosing J, Thomassen MC. et al. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-2648.
  PubMedGoogle Scholar
• 22 Fernandez JA, Heeb MJ, Xu X. et al. Species-specific anticoagulant and mitogenic activities of murine protein S. Haematologica 2009; 94: 1721-1731.
  CrossrefPubMedGoogle Scholar
• 23 Heeb MJ, Koenen RR, Fernandez JA. et al. Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals. J Thromb Haemost 2004; 02: 1766-1773.
  CrossrefPubMedGoogle Scholar
• 24 Heeb MJ, Griffin JH. Activated Protein C-dependent and -independent Anticoagulant Activities of Protein S have Different Structural Requirements. Blood Cells Mol Dis 2002; 29: 190-199.
  CrossrefPubMedGoogle Scholar
• 25 Rosing J, Tans G, Govers-Riemslag JWP. et al. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283.
  PubMedGoogle Scholar
• 26 Saller F, Villoutreix BO, Amelot A. et al. The gamma-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding. Blood 2005; 105: 122-130.
  CrossrefPubMedGoogle Scholar
• 27 Stavenuiter F, Gale AJ, Heeb MJ. Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity. FASEB J. 2013 27. in press.
  PubMedGoogle Scholar
• 28 Rezende SM, Lane DA, Mille-Baker B. et al. Protein S Gla-domain mutations causing impaired Ca(2+)-induced phospholipid binding and severe functional protein S deficiency. Blood 2002; 100: 2812-2819.
  CrossrefPubMedGoogle Scholar
• 29 Ikejiri M, Tsuji A, Wada H. et al. Analysis three abnormal Protein S genes in a patient with pulmonary embolism. Thromb Res 2010; 125: 529-532.
  CrossrefPubMedGoogle Scholar
• 30 Neki R, Fujita T, Kokame K. et al. Genetic analysis of patients with deep vein thrombosis during pregnancy and postpartum. Int J Hematol 2011; 94: 150-155.
  CrossrefPubMedGoogle Scholar
• 31 Ahnstrom J, Andersson HM, Canis K. et al. Activated protein C cofactor function of protein S: a novel role for a gamma-carboxyglutamic acid residue. Blood 2011; 117: 6685-6693.
  CrossrefPubMedGoogle Scholar
• 32 Garcia de FP, Fuentes-Prior P, Hurtado B. et al. Molecular basis of protein S deficiency. Thromb Haemost 2007; 98: 543-556.
  Article in Thieme ConnectPubMedGoogle Scholar
• 33 Villoutreix BO, Teleman O, Dahlback B. A theoretical model for hte Gla-TSREGF-1 region of the anticoagulant cofactor protein S: from biostructural pathology to species-specific cofactor activity. J Comput Aided Mol Des 1997; 11: 293-304.
  CrossrefPubMedGoogle Scholar
• 34 He X, Shen L, Villoutreix BO. et al. Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function. J Biol Chem 1998; 273: 27449-27458.
  CrossrefPubMedGoogle Scholar
• 35 Giri TK, de Frutos PG, Yamazaki T. et al. In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S. Thromb Haemost 1999; 82: 1627-1633.
  Article in Thieme ConnectPubMedGoogle Scholar
• 36 Gandrille S, Borgel D, Ireland H. et al. Protein S deficiency: a database of mutations. For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1997; 77: 1201-1214.
  Article in Thieme ConnectPubMedGoogle Scholar
• 37 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-18738.
  PubMedGoogle Scholar
• 38 Cramer TJ, Griffin JH, Gale AJ. Factor V is an anticoagulant cofactor for activated protein C during inactivation of factor Va. Pathophysiol Haemost Thromb 2010; 37: 17-23.
  Google Scholar
• 39 Hayashi T, Nishioka J, Suzuki K. Molecular mechanism of the dysfunction of protein S (Tokushima) (Lys155-- >Glu) for the regulation of the blood coagulation system. Biochim Biophys Acta 1995; 1272: 159-167.
  CrossrefPubMedGoogle Scholar
• 40 Heeb MJ, Kojima Y, Hackeng TM. et al. Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va. Protein Sci 1996; 05: 1883-1889.
  CrossrefPubMedGoogle Scholar
• 41 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.
  PubMedGoogle Scholar
• 42 Heeb MJ, Radtke K-P, Fernandez JA. et al. Plasma contains protein S monomers and multimers with similar direct anticoagulant activity. J Thromb Haemost 2006; 04: 2215-2222.
  CrossrefPubMedGoogle Scholar