Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor

 

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Summary

Protein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05–3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.

• References

• 1 Han X, Fiehler R, Broze Jr GJ. Isolation of a protein Z-dependent plasma protease inhibitor. Proc Natl Acad Sci USA 1998; 95: 9250-9255.
  CrossrefPubMedGoogle Scholar
• 2 Han X, Fiehler R, Broze Jr GJ. Characterization of the protein Z-dependent protease inhibitor. Blood 2000; 96: 3049-3055.
  PubMedGoogle Scholar
• 3 Wei Z, Yan Y, Carrell RW. et al. Crystal structure of protein Z-dependent inhibitor complex shows how protein Z functions as a cofactor in the membrane inhibition of factor X. Blood 2009; 114: 3662-3667.
  CrossrefPubMedGoogle Scholar
• 4 Huang X, Dementiev A, Olson ST. et al. Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa. J Biol Chem 2010; 285: 20399-20409.
  CrossrefPubMedGoogle Scholar
• 5 Huang X, Swanson R, Broze Jr GJ. et al. Kinetic characterization of the protein Z-dependent protease inhibitor reaction with blood coagulation factor Xa. J Biol Chem 2008; 283: 29770-29783.
  CrossrefPubMedGoogle Scholar
• 6 Heeb MJ, Cabral KM, Ruan L. Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor. J Biol Chem 2005; 280: 33819-33825.
  CrossrefPubMedGoogle Scholar
• 7 Zhang J, Tu Y, Lu L. et al. Protein Z-dependent protease inhibitor deficiency produces a more severe murine phenotype than protein Z deficiency. Blood 2008; 111: 4973-4978.
  CrossrefPubMedGoogle Scholar
• 8 Sofi F, Cesari F, Tu Y. et al. Protein Z-dependent protease inhibitor and protein Z in peripheral arterial disease patients. J Thromb Haemost 2009; 07: 731-735.
  CrossrefPubMedGoogle Scholar
• 9 Heeb MJ, Paganini-Hill A, Griffin JH. et al. Low protein Z levels and risk of ischemic stroke: differences by diabetic status and gender. Blood Cells Mol Dis 2002; 29: 139-144.
  CrossrefPubMedGoogle Scholar
• 10 Gris J-C, Quere I, Dechaud H. et al. High frequency of protein Z deficiency in patients with unexplained early fetal loss.. Blood 2002; 99: 2606-2608.
  CrossrefPubMedGoogle Scholar
• 11 Kobelt K, Biasiutti FD, Mattle HP. et al. Protein Z in ischaemic stroke. Br J Haema-tol 2001; 114: 169-173.
  CrossrefPubMedGoogle Scholar
• 12 McQuillan AM, Eikelboom JW, Hankey GJ. et al. Protein Z in ischemic stroke and its etiologic subtypes. Stroke 2003; 34: 2415-2419.
  CrossrefPubMedGoogle Scholar
• 13 Refaai MA, Ahn C, Lu L. et al. Protein Z and ZPI levels and cardiovascular events. J Thromb Haemost 2006; 04: 1628-1629.
  CrossrefPubMedGoogle Scholar
• 14 Van de Water N, Tan T, Ashton F. et al. Mutations within the protein Z-dependent protease inhibitor gene are associated with venous thromboembolic disease: a new form of thrombophilia.[see comment]. Br J Haematol 2004; 127: 190-194.
  CrossrefPubMedGoogle Scholar
• 15 Heeb MJ, Mesters RM, Tans G. et al. Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-2877.
  PubMedGoogle Scholar
• 16 Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and un-catalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-559.
  PubMedGoogle Scholar
• 17 Rezaie AR, Manithody C, Yang L. Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and ex-osite interactions are required for inhibition. J Biol Chem 2005; 280: 32722-32728.
  CrossrefPubMedGoogle Scholar
• 18 Dafforn TR, Pike RN, Bottomley SP. Physical characterization of serpin conformations. Methods 2004; 32: 150-158.
  CrossrefPubMedGoogle Scholar
• 19 Rezaie AR. Determinants of specificity of factor xa interaction with its physiological inhibitors. Mini Rev Med Chem 2006; 06: 859-865.
  CrossrefPubMedGoogle Scholar
• 20 Gettins PG. Serpin structure, mechanism, and function. Chem Rev 2002; 102: 4751-4804.
  CrossrefPubMedGoogle Scholar
• 21 Rezaie AR, Sun M-f, Gailani D. Contributions of basic amino acids in the autoly-sis loop of factor XIa to serpin specificity. Biochemistry 2006; 45: 9427-9433.
  CrossrefPubMedGoogle Scholar
• 22 Lu BG, Sofian T, Law RH. et al. Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition. J Biol Chem 2011; 286: 24544-24552.
  CrossrefPubMedGoogle Scholar
• 23 Rehault SM, Zechmeister-Machhart M, Fortenberry YM. et al. Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. Biochim Biophys Acta 2005; 1748: 57-65.
  CrossrefPubMedGoogle Scholar
• 24 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.
  CrossrefPubMedGoogle Scholar
• 25 Rezaie AR, Bae JS, Manithody C. et al. Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z. J Biol Chem 2008; 283: 19922-19926.
  CrossrefPubMedGoogle Scholar
• 26 Huang X, Rezaie AR, Broze Jr GJ. et al. Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI). J Biol Chem 2011; 286: 8740-8751.
  CrossrefPubMedGoogle Scholar
• 27 Soons H, Tans G, Hemker HC. The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type. Biochem J 1988; 256: 815-820.
  CrossrefPubMedGoogle Scholar
• 28 Wuillemin WA, Eldering E, Citarella F. et al. Modulation of contact system proteases by glycosaminoglycans. Selective enhancement of the inhibition of factor XIa.. J Biol Chem 1996; 271: 12913-12918.
  CrossrefPubMedGoogle Scholar
• 29 Steiner M, Steiner B, Rolfs A. et al. Antithrombin gene mutation 5356-5364*delCTT with type I deficiency and early-onset thrombophilia and a brief review of the antithrombin alpha-helix D molecular pathology. Ann Hema-tol 2005; 84: 56-58.
  CrossrefPubMedGoogle Scholar
• 30 Chowdhury V, Olds RJ, Lane DA. et al. Identification of nine novel mutations in type I antithrombin deficiency by heteroduplex screening. Br J Haematol 1993; 84: 656-661.
  CrossrefPubMedGoogle Scholar
• 31 Millar DS, Wacey AI, Ribando J. et al. Three novel missense mutations in the anti-thrombin III (AT3) gene causing recurrent venous thrombosis. Hum Genet 1994; 94: 509-512.
  PubMedGoogle Scholar
• 32 Frazier GC, Siewertsen MA, Hofker MH. et al. A null deficiency allele of alpha 1-antitrypsin, QOludwigshafen, with altered tertiary structure. J Clin Invest 1990; 86: 1878-1884.
  CrossrefPubMedGoogle Scholar
• 33 Robertson AS, Belorgey D, Gubb D. et al. Inhibitory activity of the Drosophila melanogaster serpin Necrotic is dependent on lysine residues in the D-helix. J Biol Chem 2006; 281: 26437-26443.
  CrossrefPubMedGoogle Scholar
• 34 Baglin TP, Carrell RW, Church FC. et al. Crystal structures of native and throm-bin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proc Natl Acad Sci USA 2002; 99: 11079-11084.
  CrossrefPubMedGoogle Scholar
• 35 Jin L, Abrahams JP, Skinner R. et al. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci USA 1997; 94: 14683-1468.
  CrossrefPubMedGoogle Scholar
• 36 Im H, Yu MH. Role of Lys335 in the metastability and function of inhibitory ser-pins. Protein Sci 2000; 09: 934-941.
  CrossrefPubMedGoogle Scholar
• 37 Bertenshaw R, Takeda K, Refetoff S. Sequencing of the variant thyroxine-binding globulin (TBG)-Quebec reveals two nucleotide substitutions. Am J Hum Genet 1991; 48: 741-744.
  PubMedGoogle Scholar
• 38 Davis RL, Shrimpton AE, Holohan PD. et al. Familial dementia caused by polymerisation of mutant neuroserpin. Nature 1999; 401: 376-379.
  PubMedGoogle Scholar
• 39 Holmes MD, Brantly ML, Fells GA. et al. Alpha 1-antitrypsin Wbethesda: molecular basis of an unusual alpha 1-antitrypsin deficiency variant. Biochem Biophys Res Commun 1990; 170: 1013-1020.
  CrossrefPubMedGoogle Scholar
• 40 Corral J, Gonzalez-Conejero R, Soria JM. et al. A nonsense polymorphism in the protein Z-dependent protease inhibitor increases the risk for venous thrombosis. Blood 2006; 108: 177-183.
  CrossrefPubMedGoogle Scholar
• 41 Folsom AR, Cushman M, Rasmussen-Torvik LJ. et al. Prospective study of polymorphisms of the protein Z-dependent protease inhibitor and risk of venous thromboembolism. Thromb Haemost 2007; 97: 493-494.
  Article in Thieme ConnectPubMedGoogle Scholar
• 42 Razzari C, Martinelli I, Bucciarelli P. et al. Polymorphisms of the protein Z-de-pendent protease inhibitor (ZPI) gene and the risk of venous thromboembolism. Thromb Haemost 2006; 95: 909-910.
  Article in Thieme ConnectPubMedGoogle Scholar
• 43 Dentali F, Gianni M, Lussana F. et al. Polymorphisms of the Z protein protease inhibitor and risk of venous thromboembolism: a meta-analysis. Br J Haematol 2008; 143: 284-287.
  CrossrefPubMedGoogle Scholar
• 44 Fabbro D, Barillari G, Damante G. Mutations R67X and W303X of the protein Z-dependent protease inhibitor gene and venous thromboembolic disease: a case-control study in Italian subjects. J Thromb Thrombolysis 2007; 23: 77-78.
  CrossrefPubMedGoogle Scholar
• 45 Sofi F, Cesari F, Abbate R. et al. A meta-analysis of potential risks of low levels of protein Z for diseases related to vascular thrombosis. Thromb Haemost 2010; 103: 749-756.
  Article in Thieme ConnectPubMedGoogle Scholar
• 46 Alsheikh FS, Finan RR, Almawi AW. et al. Association of the R67X and W303X nonsense polymorphisms in the protein Z-dependent protease inhibitor gene with idiopathic recurrent miscarriage. Mol Hum Reprod 2011; 18: 156-160.
  PubMedGoogle Scholar
• 47 Sierko E, Wojtukiewicz MZ, Ostrowska-Cichocka K. et al. Protein Z-dependent protease inhibitor (ZPI) is present in loco in human breast cancer tissue. Thromb Haemost 2010; 104: 183-185.
  Article in Thieme ConnectPubMedGoogle Scholar
• 48 Sierko E, Wojtukiewicz MZ, Zimnoch L. et al. Protein Z/protein Z-dependent protease inhibitor system in human non-small-cell lung cancer tissue. Thromb Res. 2011 epub ahead of print.
  PubMedGoogle Scholar
• 49 Broze Jr GJ, Tu Y. Protein Z and protein Z-dependent protease inhibitor and renal tubules. Thromb Haemost 2010; 103: 473-474.
  Article in Thieme ConnectPubMedGoogle Scholar