Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: Possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors

Likui Yang; Alireza R. Rezaie

doi:10.1160/TH06-12-0697

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(06): 899-906
DOI: 10.1160/TH06-12-0697

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: Possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors

Likui Yang

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

,

Alireza R. Rezaie

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

› Author Affiliations

Abstract

Summary

The Ca2⁺-dependence of protein C activation by thrombin in complex with thrombomodulin (TM) containing chondroitin sulfate (CS) exhibits saturation at ~0.5–1 mM Ca2⁺, but withTM lacking CS, it has a distinct optimum at ~0.1 mM Ca2⁺. Since the substrate protein C has multiple Ca2⁺-binding sites, and the cofactor TM also interacts with Ca2⁺, the basis for differences in Ca2⁺ effect on protein C activation by thrombin in complex with TM containing or lacking CS is not known. In this study, by using full-length and Gla-domainless mutants of protein C whose activation by thrombin is independent of either Ca2⁺ or both Ca2⁺ and TM, we demonstrate that i) the Ca2⁺ occupancy of a high-affinity binding site in TM is essential for the high-affinity interaction of the cofactor with thrombin, ii) the Ca2⁺ occupancy of a binding site (KD ~50 μM) in the catalytic domain of protein C is required for the substrate recognition by the thrombin-TM complex, however, at this concentration of Ca2⁺ the Gla domain of protein C is not folded properly and thus interacts with exosite-2 of thrombin in complex with TM that lacks CS but not withTM that contains CS, and finally iii) platelet factor 4 can nonspecifically interact with the Gla domain of protein C and other coagulation factors to influence their activation only at subphysiological concentrations of Ca2⁺.

Keywords

Thrombomodulin - chondroitin sulfate - protein C - thrombin - calcium - platelet factor 4

Full Text

References

References
1 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K dependent clotting factors. Blood 1991; 78: 1637-1651.
2 Esmon CT, Esmon NL. Protein C activation. Sem Thromb Hemost 1984; 10: 122-130.
3 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 6: 2561-2567.
4 Dahlbäck B. New molecular insights into the genetics of thrombophilia. Resistance to activated protein C caused by Arg506 to Gln mutation in factor V as a pathogenic risk factor for venous thrombosis. Thromb Haemost 1995; 74: 139-148.
5 Mather T, Oganessyan V, Hof P. et al. The 2.8 Å crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822-6831.
6 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 1-5.
7 Xu H, Bush LA, Pineda AO. et al. Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C. J Biol Chem 2005; 280: 7956-7961.
8 Bode W, Mayr I, Baumann U. et al. The refined 1.9 Å crystal structure of human α -thrombin: interaction with D-Phe-Pro-Arg chlorometheylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 3467-3475.
9 Rezaie AR, Esmon NL, Esmon CT. The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain. J Biol Chem 1992; 267: 11701-11704.
10 Rezaie AR, Mather T, Sussman F. et al. Mutation of Glu 80 to Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombinthrombomodulin complex. J Biol Chem 1994; 269: 3151-3154.
11 Bode W, Schwager P. The refined crystal structure of bovine beta-trypsin at 1.8 Å resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. J Mol Biol 1975; 98: 693-717.
12 Öhlin A, Landes G, Bourdon P. et al. β -Hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C: Its role in Ca 2+ binding and biological activity. J Biol Chem 1988; 263: 19240-19248.
13 Rezaie AR, Esmon CT. The function of calcium in protein C activation by thrombin and the thrombinthrombomodulin complex can be distinguished by mutational analysis of protein C derivatives. J Biol Chem 1992; 267: 26104-26109.
14 Fukudome K, Esmon CT. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486-26491.
15 Oganesyan V, Oganesyan N, Terzyan S. et al. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem 2002; 277: 24851-24854.
16 Fuentes-Prior P, Iwanaga Y, Huber R. et al. Structural basis for the anticoagulant activity of the thrombin- thrombomodulin complex. Nature 2000; 404: 518-525.
17 Grutter MG, Priestle JP, Rahuel J. et al. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition. EMBO J 1990; 9: 2361-2365.
18 Ye J, Esmon NL, Esmon CT. et al. The active site of thrombin is altered upon binding to thrombomodulin: Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. J Biol Chem 1991; 266: 23016-23021.
19 Zushi M, Gomi K, Yamamoto S. et al. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem 1989; 264: 10351-10353.
20 Stearns DJ, Kurosawa S, Esmon CT. Micro-thrombomodulin: Residues 310–486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation. J Biol Chem 1989; 264: 3352-3356.
21 Meininger DP, Hunter MJ, Komives EA. Synthesis, activity, and preliminary structure of the fourth EGFlike domain of thrombomodulin. Protein Sci 1995; 4: 1683-1695.
22 Yang L, Rezaie AR. The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C. J Biol Chem 2003; 278: 10484-10490.
23 Knobe K, Berntsdotter A, Shen L. et al. Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling. Proteins 1999; 35: 218-234.
24 Gale AJ, Griffin JH. Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site of factor Va. Proteins 2004; 54: 433-441.
25 Liu L, Rezaie AR, Carson CW. et al. Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation. J Biol Chem 1994; 269: 11807-11812.
26 Bourin MC, Lindahl U. Glycosaminoglycans and the regulation of blood coagulation. Biochem J 1993; 289: 313-330.
27 Church FC, Pratt CW, Noyes CM. et al. Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gammaT - thrombin. J Biol Chem 1989; 264: 18419-18425.
28 He X, Ye J, Esmon CT. et al. Influence of arginine 93, 97, and 101 of thrombin to its functional specificity. Biochemistry 1997; 36: 8969-8976.
29 Parkinson JF, Grinnell BW, Moore RE. et al. Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. J Biol Chem 1990; 265: 12602-12610.
30 Slungaard A, Key NS. Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis. J Biol Chem 1994; 269: 25549-25556.
31 Amphlett GW, Kisiel W, Castellino FJ. Interaction of calcium with bovine plasma protein C. Biochemistry 1981; 20: 2156-2161.
32 Light DR, Glaser CB, Betts M. et al. The interaction of thrombomodulin with Ca2+. Eur J Biochem 1999; 262: 522-533.
33 Yang L, Manithody C, Rezaie AR. Activation of protein C by the thrombin-thrombomodulin complex: Cooperative roles of Arg-35 of thrombin and Arg-67 of protein C. Proc Natl Acad Sci USA 2006; 103: 879-884.
34 Smirnov MD, Safa O, Regan LM. et al. A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity. J Biol Chem 1998; 273: 9031-9040.
35 Rezaie AR. Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin. Protein Sci 1998; 7: 349-357.
36 Rezaie AR, Yang L. Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin. Thromb Haemost 2005; 93: 1047-1054.
37 Smirnov MD, Esmon CT. Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994; 269: 816-819.
38 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci USA 1994; 91: 5518-5522.
39 Gan Z-R, Li Y, Chen Z. et al. Identification of basic amino acid residues in thrombin essential for heparincatalyzed inactivation by antithrombin III. J Biol Chem 1994; 269: 1301-1305.
40 Tsiang M, Jain AK, Gibbs CS. Functional requirement for inhibition of thrombin by antithrombin III in the presence and absence of heparin. J Biol Chem 1997; 272: 12024-12029.
41 Slungaard A. Platelet factor 4 modulation of the thrombomodulin-protein C system. Crit Care Med 2004; 32: S331-335.
42 Eslin DE, Zhang C, Samuels KJ. et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood 2004; 104: 3173-3180.