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Thromb Haemost 2000; 83(02): 253-261
DOI: 10.1055/s-0037-1613796
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Schattauer GmbH

Oxidation of Human α-Thrombin by the Myeloperoxidase-H2O2-chloride System: Structural and Functional Effects

Raimondo De Cristofaro
1   From the Hemostasis Research Center, Department of Internal Medicine, Catholic University School of Medicine, Rome, Italy
,
Raffaele Landolfi
1   From the Hemostasis Research Center, Department of Internal Medicine, Catholic University School of Medicine, Rome, Italy
› Author Affiliations
Further Information

Publication History

Received 17 June 1999

Accepted after resubmission 22 October 1999

Publication Date:
11 December 2017 (online)

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Summary

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This study investigated the biochemical and functional effects of α-thrombin oxidation by MPOS. This system, in the presence of 100 µM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human α-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.

Abbreviations: DNPH: 2,4-dinitrophenylhydrazine; FpA: fibrinopeptide A; FpB: fibrinopeptide B; FRS: Fibrinogen recognition Site; GpIb: Glycoprotein Ib; HBS: Heparin Binding Site; MPO: myeloperoxidase; MPOS: Myeloperoxidase System; Pip: Pipecolynic acid; PMN: polymorphonuclear cells; pNA: para-nitroaniline; PPACK: Phenylalanine-Proline-Arginine-Chloromethyl-Ketone; TM: Thrombomodulin; TNB: 5-thio-2-nitrobenzoic acid; TPCK: N-tosyl-L-Phenylalanine-Chloromethyl-Ketone. Thrombin residues are numbered according to the chymotrypsin numbering system (31).

Key words

Thrombin - myeloperoxidase - protein oxidation
 

  • References

  • 1 Hazell LJ, Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high uptake form for macrophages. Biochem J 1993; 290: 165-72.
    CrossrefPubMedGoogle Scholar
  • 2 Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994; 94: 437-44.
    CrossrefPubMedGoogle Scholar
  • 3 Hazen S, Heinecke JW. 3-chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J Clin Invest 1997; 99: 2075-81.
    CrossrefPubMedGoogle Scholar
  • 4 Harrison JE, Schultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem 1976; 251: 1371-4.
    PubMedGoogle Scholar
  • 5 Foote CS, Goine TE, Lehrer RI. Assessment of chlorination by human neutrophils. Nature 1981; 301: 715-6.
    PubMedGoogle Scholar
  • 6 Albrich JM, Mc Carthy CA, Hurst JK. Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase. Proc Natl Acad Sci USA 1981; 78: 210-4.
    CrossrefPubMedGoogle Scholar
  • 7 Ames RA, Shingenaga MK, Park EM. In: Davies KGA. (ed): Oxidation Damage and Repair: Chemical, Biological and Medical Aspects. Pergamon, Elmsford, NY; 1991
    Google Scholar
  • 8 Weiss SJ, Klein R, Slivka A, Wei M. Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation J Clin Invest 1982; 70: 598-607.
    PubMedGoogle Scholar
  • 9 Stadtman ER. Protein oxidation and aging. Science 1992; 257: 1220-4.
    CrossrefPubMedGoogle Scholar
  • 10 Heinecke JW, Li W, Daehnke HL, Goldstein JA. Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages. J Biol Chem 1993; 268: 4069-77.
    PubMedGoogle Scholar
  • 11 Savenkova MI, Mueller DM, Heinecke JW. Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for initiation of lipid peroxidation in low density lipoprotein. J Biol Chem 1994; 269: 20394-400.
    PubMedGoogle Scholar
  • 12 Anderson MM, Hazen SL, Hsu FF, Heinecke JW. Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride systemto convert hydroxy-amino acids into glycolaldehyde, 2-hydroxypropanal, and acrolein. J Clin Invest 1997; 99: 424-32.
    CrossrefPubMedGoogle Scholar
  • 13 Drozdz R, Naskalski JW, Sznajd J. Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide. Biochim Biophys Acta 1988; 957: 47-52.
    CrossrefPubMedGoogle Scholar
  • 14 Thomas EL, Grisham MB, Jefferson MM. Preparation and characterization of chloramines. Methods Enzymol 1986; 32: 569-85.
    PubMedGoogle Scholar
  • 15 Schwager I, Jungi TW. Effect of human recombinant cytokines on the induction of macrophage procoagulant activity. Blood 1994; 83: 152-60.
    PubMedGoogle Scholar
  • 16 Plescia J, Altieri DC. Activation of Mac-1 (CS11b/CD18)-bound Factor X by released cathepsin G defines an alternative pathway of leukocyte initiation of coagulation. Biochem J 1996; 319: 873-9.
    CrossrefPubMedGoogle Scholar
  • 17 Oates AM, Salem HH. The binding and regulation of Protein S by neutrophils. Blood Coagul Fibrinol 1991; 02: 601-7.
    CrossrefPubMedGoogle Scholar
  • 18 Stief TW, Aab A, Heimburger N. Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III and C1-inhibitor. Thromb Res 1988; 49: 581-9.
    CrossrefPubMedGoogle Scholar
  • 19 Glaser BB, Morser J, Clarke JH, Blasko E, McLean K, Kuhn I, Chang RJ, Lin JH, Vilander L, Andrews WH, Light DR. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. J Clin Invest 1992; 90: 2565-73.
    CrossrefPubMedGoogle Scholar
  • 20 Shacter E, Williams JA, Levine RL. Oxidative modification of fibrinogen inhibits thrombin-catalyzed clot formation. Free Rad Med Biol 1995; 18: 815-21.
    CrossrefPubMedGoogle Scholar
  • 21 Uhteg LC, Lundblad RL. The modification of tryptophan in bovine thrombin. Biochim Biophys Acta 1977; 491: 551-7.
    CrossrefPubMedGoogle Scholar
  • 22 Thompson EA, Salem H. Modification of human thrombin: effect on thrombomodulin binding. Thromb Haemost 1988; 59: 415-20.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 De Cristofaro R, Rocca B, Bizzi B, Landolfi R. The linkage between the binding of the C-terminal domain of hirudin and amidase activity in human a-thrombin. Biochem J 1993; 289: 475-80.
    CrossrefPubMedGoogle Scholar
  • 24 Marquez LA, Dunford HB. Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II Implications for lipoprotein peroxidation studies. J Biol Chem 1995; 270: 30434-40.
    CrossrefPubMedGoogle Scholar
  • 25 Scott AI. Oxidative coupling of phenolic compounds Q Rev Chem Soc (Lond). 1965; 19: 1-35.
    PubMedGoogle Scholar
  • 26 Prince RC. Tyrosine radicals. Trend Biochem Sci 1988; 13: 286-8.
    CrossrefPubMedGoogle Scholar
  • 27 Heinecke JW, Li W, Daenke III HL, Goldstein JA. Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages. J Biol Chem 1993; 268: 4069-77.
    PubMedGoogle Scholar
  • 28 Morris JC. The acid ionization constant of HOCl from 5 to 35° C. J Phys Chem 1966; 70: 3798-805.
    CrossrefPubMedGoogle Scholar
  • 29 Oliver CN, Ahn B, Moerman EJ, Goldstein S, Stadtman ER. Age-related changes in oxidized proteins. J Biol Chem 1987; 262: 5488-91.
    PubMedGoogle Scholar
  • 30 Griffith MJ. Covalent modification of human α-thrombin with pyridoxal5’-phosphate Effect of phosphopyridoxylation on the interaction of thrombin with heparin. J Biol Chem 1978; 254: 3401-6.
    PubMedGoogle Scholar
  • 31 Church FC, Pratt CW, Noyas CM, Katanyanami T, Sherril GB, Tobin RB, Meade JB. Structural and functional properties of human a-thrombin, phosphopyridoxylated a-thrombin, and ‘γT-thrombin: identification of lysyl residues in α-thrombin that are critical for heparin and fibrin(ogen) interactions. J Biol Chem 1989; 264: 18419-25.
    PubMedGoogle Scholar
  • 32 De Candia E, De Cristofaro R, De Marco L, Mazzucato M, Picozzi M, Landolfi R. Thrombin interaction with platelet GpIb: role of the heparin binding domain. Thromb Haemost 1997; 77: 735-40.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 De Cristofaro R, De Candia E, Croce G, Morosetti R, Landolfi R. Binding of human κ-thrombin to platelet GpIb: energeticsand functional effects. Biochem J 1998; 332: 643-50.
    CrossrefPubMedGoogle Scholar
  • 34 De Cristofaro R, Landolfi R. Thermodynamics of amide substrates and reversible inhibitors binding to the active site cleft of human a-thrombin. J Mol Biol 1994; 239: 569-77.
    CrossrefPubMedGoogle Scholar
  • 35 De Cristofaro R, De Candia E, Landolfi R. Effect of high and low molecular weight heparins on thrombin-thrombomodulin interaction and Protein C activation. Circulation 1998; 98: 1297-301.
    CrossrefPubMedGoogle Scholar
  • 36 De Cristofaro R, Picozzi M, Morosetti R, Landolfi R. Effect of sodium on the energetics of thrombin-thrombomodulin interaction and its relevance for Protein C hydrolysis. J Mol Biol 1996; 258: 190-200.
    CrossrefPubMedGoogle Scholar
  • 37 Picozzi M, Landolfi R, De Cristofaro R. Effect of protons on the thrombinfibrinogen interaction. Eur J Biochem 219: 1013-21.
    PubMedGoogle Scholar
  • 38 Olson ST, Björk I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-59.
    PubMedGoogle Scholar
  • 39 Olson ST, Shore JD. Binding of high affinity heparin to antithrombin III. J Biol Chem 1981; 256: 11065-72.
    PubMedGoogle Scholar
  • 40 De Cristofaro R, Landolfi R, De Candia E, Castagnola M, Di Cera E, Wyman J. Allosteric equilibria in the binding of fibrinogen to platelets. Proc Natl Acad Sci USA 1988; 85: 8473-6.
    CrossrefPubMedGoogle Scholar
  • 41 Hawkins CL, Davies MJ. Hypochlorite-induced damage to proteins: formation of nitrogen-centred radicals from lysine residues and their role in protein fragmentation. Biochem J 1998; 332: 617-25.
    CrossrefPubMedGoogle Scholar
  • 42 Bode W, Turk D, Karshikov A. The refined 1.9-Å X-ray crystal structure of D-Ph-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active site geometry, and structure-function relationships. Protein Sci 1992; 01: 426-71.
    PubMedGoogle Scholar
  • 43 Hazell LJ, Arnold L, Flowers D, Waeg G, Malle E, Stocker R. Presence of hypochlorite-modified proteins in human atherosclerotic lesions. J Clin Invest 1996; 97: 1535-44.
    CrossrefPubMedGoogle Scholar
  • 44 Le Bonniec BF, Guinto ER, Esmon CT. Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and Protein C Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin. J Biol Chem 1992; 267: 19341-8.
    PubMedGoogle Scholar
  • 45 Le Bonniec BF, Guinto ER, MacGillivray RTA, Stone SR, Esmon CT. 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.
    PubMedGoogle Scholar
  • 46 Stubbs MT, Oschkinat H, Mayr I, Huber R, Angliker H, Stone SR, Bode W. The interaction of thrombin with fibrinogen A structural basis for its specificity. Eur J Biochem 1992; 206: 187-95.
    CrossrefPubMedGoogle Scholar
  • 47 Stubbs MT, Bode W. The clot thickens: clues provided by thrombin structure. Trends Biochem Sci 1995; 20: 23-7.
    CrossrefPubMedGoogle Scholar
  • 48 De Cristofaro R, De Candia R. Thrombin interaction with platelet GpIb: structural mapping and effects on platelet activation (Review). Int J Mol Med 1999; 03: 363-71.
    PubMedGoogle Scholar
  • 49 Arni RK, Padmanabhan K, Padmanabhan KP, Wu TP, Tulinsky A. Structures of the noncovalent complexes of human and bovine prothrombin factor 2 with human PPACK-thrombin Biochemistry. 1993; 32: 4727-37.
    PubMedGoogle Scholar
  • 50 De Cristofaro R, De Candia E, Croce G, Morosetti R, Landolfi R. Binding of human κ-thrombin to platelet GpIb: energetics and functional effects. Biochem J 1998; 332: 643-50.
    CrossrefPubMedGoogle Scholar
  • 51 van de Locht A, Bode W, Huber R, Le Bonniec BF, Stone SR, Esmon CT, Stubbs MT. The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin. EMBO J 1997; 16: 2977-84.
    CrossrefPubMedGoogle Scholar
  • 52 De Cristofaro R, Landolfi R. Allosteric modulation of BPTI interaction with human α-and ζ-thrombin. Eur J Biochem 1999; 260: 97-102.
    CrossrefPubMedGoogle Scholar
  • 53 Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-87.
    CrossrefPubMedGoogle Scholar
  • 54 De Candia E, De Cristofaro R, Landolfi R. Thrombin induced platelet activation is inhibited by high and low molecular weight heparin. Circulation 1999; 99: 3308-14.
    CrossrefPubMedGoogle Scholar
  • 55 Olson ST, Halvorson HR, Björk I. Quantitative characterization of the thrombin-heparin interaction. J Biol Chem 1991; 266: 6342-52.
    PubMedGoogle Scholar
  • 56 Marquez LA, Dunford HB. Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II Implications for lipoprotein peroxidation studies. J Biol Chem 1995; 270: 30434-40.
    CrossrefPubMedGoogle Scholar
  • 57 Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994; 94: 437-44.
    CrossrefPubMedGoogle Scholar