The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of α₁-proteinase inhibitor M358R for thrombin

Summary

Natural inhibitors of coagulation or inflammation such as the serpins antithrombin (AT), heparin cofactor II (HCII), and 1-proteinase inhibitor (α₁-PI) can be overwhelmed in thrombosis and/or sepsis. The reactive centre (P1-P1) variant α₁-PI M358R inhibits not only procoagulant thrombin but also anticoagulant activated protein C (APC). We previously described HAPI M358R, comprising a fusion of HCII residues 1–75 to the N-terminus of a1-PI M358R that yielded increased anti-thrombin, but not anti-APC activity. We hypothesized that further alterations to the HAPI M358R reactive centre loop would yield additional refinements in specificity. The reactions with thrombin or APC of recombinant α₁-PI M358R variants with or without the HCII extension were characterized electrophoretically and kinetically. Their extension of clotting times and inhibition of fibrin-bound thrombin were measured, and the survival of HAPI M358R in mice was determined. Replacing the P7-P3 and P2’ residues of HAPI M358R with AT residues reduced APC inhibition rates by 140-fold, but those of thrombin less than two-fold;substituting the P16-P2 and P2’-P3’ residues of HAPI M358R with HCII residues reduced APC inhibition rates by 180-fold, but those of thrombin 10.5-fold. Fused variants extended thrombin clotting times more effectively than unfused inhibitors, were at least as effective at inhibiting clot-bound thrombin, and remained intact in the murine circulation. The combination of modifications inside and outside the RCL resulted in a 1,360-fold increase in selectivity of HAPI M358R (AT P7-P3/P2’) for thrombin versus APC relative to α₁-PI M358R. Our results predict that this protein may be effective in limiting thrombosis in vivo.

Keywords

Serpins - thrombin - APC - recombinant proteins - kinetics

• References

• 1 Colman RW. Are hemostasis and thrombosis two sides of the same coin?. J Exp Med 2006; 203: 493-495.
• 2 Bates SM, Weitz JI. The status of new anticoagulants. Br J Haematol 2006; 134: 3-19.
• 3 Owen MC, Brennan SO, Lewis JH. et al. Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder. N Engl J Med 1983; 309: 694-698.
• 4 Beatty K, Bieth J, Travis J. Kinetics of association of serine proteinases with native and oxidized alpha- 1-proteinase inhibitor and alpha-1-antichymotrypsin. J Biol Chem 1980; 255: 3931-3934.
• 5 Silverman GA, Bird PI, Carrell RW. et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 2001; 276: 33293-33296.
• 6 Gettins PG. Serpin structure, mechanism, and function. Chem Rev 2002; 102: 4751-4804.
• 7 Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 157-162.
• 8 Stratikos E, Gettins PG. Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A. Proc Natl Acad Sci U.S.A 1999; 96: 4808-4813.
• 9 Travis J, Matheson NR, George PM. et al. Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases. Biol Chem Hoppe Seyler 1986; 367: 853-859.
• 10 Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood 2005; 106: 2605-2612.
• 11 Travis J, Owen M, George P. et al. Isolation and properties of recombinant DNA produced variants of human alpha 1-proteinase inhibitor. J Biol Chem 1985; 260: 4384-4389.
• 12 Luisetti M, Travis J. Bioengineering: alpha 1-proteinase inhibitor site-specific mutagenesis. The prospect for improving the inhibitor. Chest 1996; 110: 278S-283S.
• 13 Scott CF, Carrell RW, Glaser CB. et al. Alpha- 1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf. J Clin Invest 1986; 77: 631-634.
• 14 Heeb MJ, Bischoff R, Courtney M. et al. Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358. J Biol Chem 1990; 265: 2365-2369.
• 15 Harper PL, Taylor FB, DeLa Cadena RA. et al. Recombinant antitrypsin Pittsburgh undergoes proteolytic cleavage during E. coli sepsis and fails to prevent the associated coagulopathy in a primate model. Thromb Haemost 1998; 80: 816-821.
• 16 Bernard GR, Vincent JL, Laterre PF. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 17 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505.
• 18 Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 1982; 257: 2162-2169.
• 19 Hopkins PC, Pike RN, Stone SR. Evolution of serpin specificity: cooperative interactions in the reactivesite loop sequence of antithrombin specifically restrict the inhibition of activated protein C. J Mol Evol 2000; 51: 507-515.
• 20 Filion ML, Bhakta V, Nguyen LH. et al. Full or partial substitution of the reactive center loop of alpha- 1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition. Biochemistry 2004; 43: 14864-14872.
• 21 Van Deerlin VM, Tollefsen DM. The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans. J Biol Chem 1991; 266: 20223-20231.
• 22 Ragg H, Ulshofer T, Gerewitz J. On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans. J Biol Chem 1990; 265: 5211-5218.
• 23 Sheehan JP, Wu Q, Tollefsen DM. et al. Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines Myles T. Church FC. Whinna HC. et al. Role of thrombin anionbinding exosite-I in the formation of thrombin-serpin complexes. J Biol Chem 1998; 273: 31203-31208.
• 24 Sutherland JS, Bhakta V, Filion ML. et al. The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition. Biochemistry 2006; 45: 11444-11452.
• 25 Pace CN, Vajdos F, Fee L. et al. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995; 4: 2411-2423.
• 26 Sutherland JS, Bhakta V, Sheffield WP. Investigating serpin-enzyme complex formation and stability via single and multiple residue reactive centre loop substitutions in heparin cofactor II. Thromb Res 2006; 117: 447-461.
• 27 Cunningham MA, Bhakta V, Sheffield WP. Altering heparin cofactor II at VAL439 (P6) either impairs inhibition of thrombin or confers elastase resistance. Thromb Haemost 2002; 88: 89-97.
• 28 Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-559.
• 29 Sheffield WP, Smith IJ, Syed S. et al. Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris. Blood Coagul Fibrinolysis 2001; 12: 433-443.
• 30 Francischetti IM, Valenzuela JG, Ribeiro JM. Anophelin: kinetics and mechanism of thrombin inhibition. Biochemistry 1999; 38: 16678-16685.
• 31 Sheffield WP, Mamdani A, Hortelano G. et al. Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits. Br J Haematol 2004; 126: 565-573.
• 32 Hopkins PC, Crowther DC, Carrell RW. et al. Development of a novel recombinant serpin with potential antithrombotic properties. J Biol Chem 1995; 270: 11866-11871.
• 33 Rogers SJ, Pratt CW, Whinna HC. et al. Role of thrombin exosites in inhibition by heparin cofactor II. J Biol Chem 1992; 267: 3613-3617.
• 34 Hoffmann JN, Wiedermann CJ, Juers M. et al. Benefit/risk profile of high-dose antithrombin in patients with severe sepsis treated with and without concomitant heparin. Thromb Haemost 2006; 95: 850-856.
• 35 Huntington JA, Gettins PG. Conformational conversion of antithrombin to a fully activated substrate of factor Xa without need for heparin. Biochemistry 1998; 37: 3272-3277.
• 36 Liaw PC, Austin RC, Fredenburgh JC. et al. Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II. J Biol Chem 1999; 274: 27597-27604.
• 37 Janciauskiene S, Lindgren S. Human monocyte activation by cleaved form of alpha-1-antitrypsin involvement of the phagocytic pathway. Eur J Biochem 1999; 265: 875-882.
• 38 Bottomley SP, Lawrenson ID, Tew D. et al. The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin. Protein Sci 2001; 10: 2518-2524.
• 39 Pratt CW, Tobin RB, Church FC. Interaction of heparin cofactor II with neutrophil elastase and cathepsin G. J Biol Chem 1990; 265: 6092-6097.
• 40 Rubin H, Plotnick M, Wang ZM. et al. Conversion of alpha 1-antichymotrypsin into a human neutrophil elastase inhibitor: demonstration of variants with different association rate constants, stoichiometries of inhibition, and complex stabilities. Biochemistry 1994; 33: 7627-7633.
• 41 Sands H, Hook JB. Pharmacology and pharmacokinetics of LEX 032, a bioengineered serpin: the first of a potential new class of drugs. Drug Metab Rev 1997; 29: 309-328.
• 42 Viswanathan K, Liu L, Vaziri S. et al. Myxoma viral serpin, Serp-1, a unique interceptor of coagulation and innate immune pathways. Thromb Haemost 2006; 95: 499-510.
• 43 Bernard GR, Vincent JL, Laterre PF. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 44 Blinder MA, Marasa JC, Reynolds CH. et al. Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli. Biochemistry 1988; 27: 752-759.
• 45 Kurachi K, Chandra T, Degen SJ. et al. Cloning and sequence of cDNA coding for alpha 1-antitrypsin. Proc Natl Acad Sci USA 1981; 78: 6826-6830.
• 46 Bock SC, Wion KL, Vehar GA. et al. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res 1982; 10: 8113-8125.