Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin

Summary

Structural data have indicated that the 60-loop of thrombin with 8–9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However, previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved ~2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.

Keywords

Thrombin - specificity - protein C - fibrinogen - antithrombin

• References

• 1 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-56.
• 2 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation, maintenance and regulation. Biochemistry 1991; 30: 10363-70.
• 3 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-18.
• 4 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 1-5.
• 5 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 6: 2561-7.
• 6 Bock SC, Wion KL, Vehar GA. et al. Cloning and expression of the cDNA clone for human antithrombin III. Nucleic Acids Res 1982; 10: 8113-25.
• 7 Damus PS, Hicks M, Rosenberg RD. Anticoagulant action of heparin. Nature 1973; 246: 355-7.
• 8 Olson ST, Björk I. Regulation of thrombin by antithrombin and heparin cofactor II. In Thrombin: Structure and Function. Plenum Press New York 1992; 159-217.
• 9 Bode W, Mayr I, Baumann U. et al. The refined 1.9 Å crystal structure of human a-thrombin: interaction with D-Phe-Pro-Arg chlorometheylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 3467-75.
• 10 Rydel TJ, Ravichandran KG, Tulinsky A. et al. The structure of a complex of recombinant hirudin and human a-thrombin. Science 1990; 249: 277-80.
• 11 Stubbs MT, Bode W. A player of many parts: The spotlight falls on thrombin's structure. Thromb Res 1993; 69: 1-58.
• 12 Mathews II, Padmanabhan KP, Tulinsky A. Structure of a nonadecapeptide of the fifth EGF domain of thrombomodulin complexed with thrombin. Biochemistry 1994; 33: 13547-52.
• 13 Fuentes-Prior P, Iwanaga Y, Huber R. et al. Structural basis for the anticoagulant activity of the thrombin- thrombomodulin complex. Nature 2000; 404: 518-25.
• 14 Pineda AO, Chen ZW, Caccia S. et al. The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket. J Biol Chem 2004; 279: 39824-8.
• 15 Fenton JW II. Thrombin functions and antithrombotic intervention. Thromb Haemost 1995; 74: 493-8.
• 16 Vu TH, Wheaton VI, Hung DT. et al. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674-7.
• 17 Esmon CT, Lollar P. Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 1996; 271: 13882-7.
• 18 Myles T, Yun TH, Leung LLK. Structural requirements for the activation of human factor VIII by thrombin. Blood 2002; 100: 2820-6.
• 19 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci (USA) 1994; 91: 5518-22.
• 20 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: 1301-5.
• 21 He X, Ye J, Esmon CT. et al. Influence of arginine 93, 97, and 101 of thrombin to its functional specificity. Biochemistry 1997; 36: 8969-76.
• 22 Le Bonniec BF, Guinto ER, MacGillivray RTA. et al. The role of thrombin's Tyr-Pro-Pro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III and the Kunitz inhibitors. J Biol Chem 1993; 268: 19055-61.
• 23 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: 349-57.
• 24 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: 26104-9.
• 25 Rezaie AR, Yang L. Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin. Biochem Biophys Acta 2001; 1528: 167-76.
• 26 Yang L, Manithody C, Rezaie AR. Contribution of basic residues of the 70–80-loop to heparin binding and anticoagulant function of activated protein C. Biochemistry 2002; 41: 6149-57.
• 27 Smirnov MD, Esmon CT. Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994; 269: 816-9.
• 28 Rezaie AR. DX-9065a inhibition of factor Xa and the prothrombinase complex: mechanism of inhibition and comparison with therapeutic heparins. Thromb Haemost 2003; 89: 112-21.
• 29 Manithody C, Fay PJ, Rezaie AR. Exosite-dependent regulation of factor VIIIa by activated protein C. Blood 2003; 101: 4802-7.
• 30 Rezaie AR, Yang L. Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin. Proc Natl Acad Sci (USA) 2003; 100: 12051-6.
• 31 Yang L, Manithody C, Rezaie AR. Localization of the heparin binding exosite of factor IXa. J Biol Chem 2002; 277: 50756-60.
• 32 Olson ST, Björk I, Sheffer R. et al. Role of the antithrombin- binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J Biol Chem 1992; 267: 12528-38.
• 33 Guinto ER, Ye J, Le Bonniec BF. et al. Glu192 [to] Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor. J Biol Chem 1994; 269: 18395-400.
• 34 Myles T, Yun TH, Hall SW. et al. An extensive interaction interface between thrombin and factor V is required for factor V activation. J Biol Chem 2001; 276: 25143-9.
• 35 Pineda AO, Carrell CJ, Bush LA. et al. Molecular dissection of Na+ binding to thrombin. J Biol Chem 2004; 279: 31842-53.
• 36 Rezaie AR, Esmon CT. Molecular basis of residue 192 participation in determination of coagulation protease specificity. Eur J Biochem 1996; 242: 477-84.
• 37 Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204: 433-51.
• 38 Li W, Johnson DJD, Esmon CT. et al. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol 2004; 11: 857-62.
• 39 Dementiev A, Petitou M, Herbert J-M et al. The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity. Nat Struct Mol Biol 2004; 11: 863-7.
• 40 O’Reily MS, Pirie-Shepherd S, Lane WS. et al. Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 1999; 285: 1926-8.
• 41 Orcutt SJ, Pietropaolo C, Krishnaswamy S. Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate. Biol Chem 2002; 278: 46191-6.