Download PDF
Thromb Haemost 2007; 98(05): 1014-1023
DOI: 10.1160/TH07-03-0197
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

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

Jason S. Sutherland
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
,
Varsha Bhakta
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
,
William P. Sheffield
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
› Author AffiliationsFinancial support: This work was made possible by Grant-In-Aid T5597 from the Heart and Stroke Foundation of Ontario to WPS. JSS was the recipient of a Graduate Fellowship Award from Canadian Blood Services.
Further Information

Publication History

Received 15 March 2007

Accepted after resubmission 23 August 2007

Publication Date:
30 November 2017 (online)

Also available at
    Permissions and Reprints

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.
    CrossrefPubMedSearch in Google Scholar
  • 2 Bates SM, Weitz JI. The status of new anticoagulants. Br J Haematol 2006; 134: 3-19.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 6 Gettins PG. Serpin structure, mechanism, and function. Chem Rev 2002; 102: 4751-4804.
    CrossrefPubMedSearch in Google Scholar
  • 7 Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 157-162.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 10 Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood 2005; 106: 2605-2612.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 12 Luisetti M, Travis J. Bioengineering: alpha 1-proteinase inhibitor site-specific mutagenesis. The prospect for improving the inhibitor. Chest 1996; 110: 278S-283S.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 17 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 30 Francischetti IM, Valenzuela JG, Ribeiro JM. Anophelin: kinetics and mechanism of thrombin inhibition. Biochemistry 1999; 38: 16678-16685.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    PubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    Thieme ConnectPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar
  • 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.
    CrossrefPubMedSearch in Google Scholar