MOLECULAR BASIS OF BLOOD COAGULATION REGULATION

Summary

Principal Investigator: Steven T Olson
Abstract: DESCRIPTION (provided by applicant): Serpin family protein protease inhibitors function as key regulators of blood coagulation proteases. Two serpins, anti-thrombin and protein Z-dependent protease inhibitor (ZPI), are known to inhibit procoagulant proteases in a manner that is regulated by cofactors and dependent on the functional state of the proteases, but the molecular details of this complex regulatory mechanism are poorly understood. The physiologic importance of these serpins in regulating coagulation proteases is borne out by the observations that knocking out the mouse genes results in embryonic lethality due to a consumptive coagulopathy in the case of anti-thrombin and increased thrombosis when mice are given a thrombotic challenge or are bred on a factor V Leiden background in the case of ZPI. The long-term goal of our studies is to understand how coagulation proteases are regulated by these two critical serpin inhibitors. Our proposed studies seek to build on our prior studies to advance the detailed molecular understanding of this cofactor-dependent regulation. With respect to anti-thrombin, our recent studies have suggested an important revision of the allosteric mechanism of activation of this serpin by heparin in showing that activation is mediated principally by the relief of repulsive interactions with factor Xa and factor IXa and secondarily by the relief of structural constraints of the reactive site that promote reactive site and exosite interactions with the protease. Our proposed studies seek to characterize the molecular determinants of the repulsive interactions, establish how certain mutations can decouple the structural changes accompanying activation to produce an intermediate activated state and to show how an induced-fit mechanism of heparin binding is coupled to allosteric activation. With respect to ZPI, our recent X- ray structure of a complex of ZPI with its cofactor protein, protein Z, has provided new insights into the mechanism by which the cofactor promotes the specific inhibition of membrane-associated factor Xa, despite the unfavorable P1 Tyr of ZPI. Our studies seek to map putative exosites on ZPI for factor Xa and factor XIa, validate the ZPI sites for protein Z binding that have been revealed by the X-ray structure, and assess the role of the unique N-terminal tail of ZPI in the inhibition of membrane-associated factor Xa. For both serpins, gain of function studies in which the serpin scaffold of 11-proteinase inhibitor is used to graft the molecular determinants of exosite interactions in anti-thrombin and of both protein Z and exosite interactions in ZPI will provide a stringent test of the minimal determinants of protease and cofactor recognition by these serpins.) PUBLIC HEALTH RELEVANCE: Cardiovascular diseases resulting in heart attack, stroke and other forms of abnormal clotting are the number one cause of death in humans. These diseases reflect an imbalance in the natural procoagulant and anticoagulant mechanisms that are responsible for hemostasis. Proteins of the serpin superfamily act as critical regulators of this dynamic balance but their molecular mechanisms are poorly understood. Our studies seek to advance understanding of these mechanisms and thereby to improve on currently available anticoagulant therapy. Novel anticoagulant drugs which target serpins and upregulate their normal downregulated function could greatly improve upon currently available anticoagulant drugs which lack specificity and are beset by risks of bleeding .
Funding Period: 1994-01-01 - 2015-02-28
more information: NIH RePORT

Top Publications

  1. pmc Exosite determinants of serpin specificity
    Peter G W Gettins
    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612, USA
    J Biol Chem 284:20441-5. 2009
  2. pmc Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin
    Ashoka A Maddur
    From the Center for Molecular Biology of Oral Diseases and Department of Periodontics and
    J Biol Chem 288:32020-35. 2013
  3. pmc Identification of serpin determinants of specificity and selectivity for furin inhibition through studies of α1PDX (α1-protease inhibitor Portland)-serpin B8 and furin active-site loop chimeras
    Gonzalo Izaguirre
    Center for Molecular Biology of Oral Diseases and the Department of Periodontics, University of Illinois, Chicago, Illinois 60612, USA
    J Biol Chem 288:21802-14. 2013
  4. pmc Structural basis for catalytic activation of protein Z-dependent protease inhibitor (ZPI) by protein Z
    Xin Huang
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, IL 60612, USA
    Blood 120:1726-33. 2012
  5. pmc Cathepsin B controls the persistence of memory CD8+ T lymphocytes
    Susan M Byrne
    Section of Immunobiology, Division of Immunology and Inflammation, Department of Medicine, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom
    J Immunol 189:1133-43. 2012
  6. pmc Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa
    Steven T Olson
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 60612, USA
    Blood 119:2187-95. 2012
  7. pmc Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells
    Feng Pai Chou
    Greenebaum Cancer Center, University of Maryland, Baltimore, MD 21201, USA
    Am J Physiol Cell Physiol 301:C1093-103. 2011
  8. ncbi Regulation of proteases by protein inhibitors of the serpin superfamily
    Steven T Olson
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois, USA
    Prog Mol Biol Transl Sci 99:185-240. 2011
  9. pmc Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor
    Xin Huang
    Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, Illinois 60612, USA
    J Biol Chem 286:8740-51. 2011
  10. pmc Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes
    Sophia Schedin-Weiss
    Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden
    Arch Biochem Biophys 504:169-76. 2010

Detail Information

Publications15

  1. pmc Exosite determinants of serpin specificity
    Peter G W Gettins
    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612, USA
    J Biol Chem 284:20441-5. 2009
    ..A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered...
  2. pmc Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin
    Ashoka A Maddur
    From the Center for Molecular Biology of Oral Diseases and Department of Periodontics and
    J Biol Chem 288:32020-35. 2013
    ....
  3. pmc Identification of serpin determinants of specificity and selectivity for furin inhibition through studies of α1PDX (α1-protease inhibitor Portland)-serpin B8 and furin active-site loop chimeras
    Gonzalo Izaguirre
    Center for Molecular Biology of Oral Diseases and the Department of Periodontics, University of Illinois, Chicago, Illinois 60612, USA
    J Biol Chem 288:21802-14. 2013
    ....
  4. pmc Structural basis for catalytic activation of protein Z-dependent protease inhibitor (ZPI) by protein Z
    Xin Huang
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, IL 60612, USA
    Blood 120:1726-33. 2012
    ..These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction...
  5. pmc Cathepsin B controls the persistence of memory CD8+ T lymphocytes
    Susan M Byrne
    Section of Immunobiology, Division of Immunology and Inflammation, Department of Medicine, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom
    J Immunol 189:1133-43. 2012
    ..Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells...
  6. pmc Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa
    Steven T Olson
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 60612, USA
    Blood 119:2187-95. 2012
    ..These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone...
  7. pmc Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells
    Feng Pai Chou
    Greenebaum Cancer Center, University of Maryland, Baltimore, MD 21201, USA
    Am J Physiol Cell Physiol 301:C1093-103. 2011
    ..Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells...
  8. ncbi Regulation of proteases by protein inhibitors of the serpin superfamily
    Steven T Olson
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois, USA
    Prog Mol Biol Transl Sci 99:185-240. 2011
    ..An understanding of the structure and function of serpins has suggested that they may provide novel scaffolds for engineering protease inhibitors of desired specificity for therapeutic use...
  9. pmc Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor
    Xin Huang
    Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, Illinois 60612, USA
    J Biol Chem 286:8740-51. 2011
    ....
  10. pmc Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes
    Sophia Schedin-Weiss
    Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden
    Arch Biochem Biophys 504:169-76. 2010
    ....
  11. pmc Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors
    Steven T Olson
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, IL 60612, USA
    Biochimie 92:1587-96. 2010
    ....
  12. pmc Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa
    Xin Huang
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60607, USA
    J Biol Chem 285:20399-409. 2010
    ....
  13. pmc Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement
    Peter G W Gettins
    Department of Biochemistry and Molecular Genetics, and Center for Structural Biology, University of Illinois at Chicago, IL 60612 4316, USA
    FEBS Lett 583:3397-400. 2009
    ....
  14. pmc The signature 3-O-sulfo group of the anticoagulant heparin sequence is critical for heparin binding to antithrombin but is not required for allosteric activation
    Benjamin Richard
    Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612, USA
    J Biol Chem 284:27054-64. 2009
    ....
  15. pmc The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D
    Alexey Dementiev
    From the Department of Biochemistry and Molecular Genetics and
    J Biol Chem 288:33611-9. 2013
    ....

Research Grants30

  1. Recombinant and Chemo-/Bio-Orthogonal Synthesis of Liposomal Thrombomodulin
    Xue Long Sun; Fiscal Year: 2013
    ....
  2. Interactions Between Inflammation, Oxidant Stress and Cardiovascular Disease
    David G Harrison; Fiscal Year: 2013
    ..Overall, these studies will promote our understanding of the interplay between inflammation, oxidant stress and cardiovascular disease. ..
  3. Blood Coagulation Protein - Metal Ion - Lipid Interactions
    Francis J Castellino; Fiscal Year: 2013
    ..This proposal is concerned with the complex mechanisms of action of the conantokins in vitro and in vivo and relating these properties to their neuroprotective effects in a model of occlusive stroke. ..
  4. Dopamine and Angiotensin Receptor Interactions in Genetic Hypertension
    Robin A Felder; Fiscal Year: 2013
    ..abstract_text> ..