Hironao Wakabayashi
| Title | Research Assistant Professor |
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| Institution | School of Medicine and Dentistry |
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| Department | Biochemistry and Biophysics |
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| Address | University of Rochester Medical Center
School of Medicine and Dentistry
601 Elmwood Ave, Box 712
Rochester NY 14642
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Structural and functional analyses of factor VIII
Factor VIII functions as a cofactor for the serine protease factor IXa in the surface-dependent conversion of zymogen factor X to the serine protease, factor Xa. Deficiency of factor VIII activity results in a marked reduction of factor IXa activity and in the subsequent rates of factor Xa generated during the propagation phase of coagulation.
My research focuses on the role of metal ions for structural integrity and function of factor VIII. Active factor VIII can be reconstituted by combining the isolated subunits in the presence of Ca2+ or Mn2+. Ca2+ had little effect on inter-subunit affinity yet converted the inactive dimer to an active form. Alternatively, Cu2+ enhanced the inter-subunit affinity ~100-fold but yielded a dimer lacking cofactor activity. Site-directed mutagenesis is employed to identify residues important in Ca2+ binding to factor VIII and resultant effects of this binding on activity. Several acidic amino acids within segment 110-126 of the A1 domain of factor VIII that likely participate in the coordination of Ca2+ necessary for generation of maximal cofactor specific activity.
Recent Publications
Wakabayashi, H., Zhou, Q., Varfaj, F., and Fay, P. J.
A3 Domain Residue Glu1829 Contributes to A2 Subunit Retention in Factor VIIIa J. Thromb. Haemost., in press.
Varfaj, F., Neuberg, J., Jenkins, P. V., Wakabayashi, H., and Fay, P. J.
Role of P1 Arg in Activated Protein C - Catalyzed Inactivation of Factor VIIIa. Biochemical J. 396, 355-362, 2006.
Wakabayashi, H., Zhou, Q., Nogami#, K., Ansong, C., Varfaj, F., Miles, S., and Fay, P. J.
pH-dependent association of factor VIII chains: Enhancement of affinity at physiological pH by Cu2+. Biochim. Biophys. Acta 1764,1094-1101, 2006.
Nogami, K., Zhou, Q., Myles, T., Leung, L. L. , Wakabayashi, H., and Fay, P. J.
Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain.
J. Biol. Chem. 280, 18476-18487, 2005.
Nogami, K., Zhou, Q., Wakabayashi, H., and Fay, P. J. Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site.
Blood 105, 4362-4368, 2005.
Wakabayashi, H., Su, Y. C. , Ahmad, S. S. , Walsh, P. N. , and Fay, P. J.
A Glu113Ala Mutation within a Factor VIII Ca(2+)-Binding Site Enhances Cofactor Interactions in Factor Xase.
Biochemistry 44, 10298-10304, 2005.
Nogami, K., Wakabayashi, H., Ansong, C., and Fay, P. J. Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit.
Biochim. Biophys. Acta 1701, 25-35, 2004.
Nogami, K., Freas, J., Manithody, C., Wakabayashi, H., Rezaie, A. R. , and Fay, P. J.
Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.
J. Biol. Chem. 279, 33104-33113, 2004.
Nogami K., Lapan K.A., Zhou Q., Wakabayashi H., and Fay P.J.
Identification of a Factor Xa-interactive Site within Residues 337-372 of the Factor VIII Heavy Chain.
J. Biol. Chem. 279, 15763-15771, 2004.
Wakabayashi H., Freas J., Zhou Q., and Fay P.J.
Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity.
J. Biol. Chem. 279, 12677-12684, 2004.
Wakabayashi H., Zhen Z., Schmidt K.M., and Fay P.J.
Mn2+ binding to factor VIII Subunits and its effect on cofactor activity.
Biochemistry 42, 145-153, 2003.
Wakabayashi H., Schmidt K.M., and Fay P.J.
Ca2+ binding to both the heavy and light chains of factor VIII is required for cofactor activity.
Biochemistry 41, 8485-8492, 2002.
Wakabayashi H., Koszelak M., Mastri, M., and Fay P.J Metal-ion independent factor VIII subunit association and the role of calcium and copper for its affinity and activity.
Biochemistry 40, 10293-10300, 2001.
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Takeyama M, Wakabayashi H, Fay PJ. Contribution of factor VIII light-chain residues 2007-2016 to an activated protein C-interactive site. Thromb Haemost. 2013 Jan 31; 109(2):187-98.
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Deangelis JP, Wakabayashi H, Fay PJ. Sequences Flanking Arg336 in Factor VIIIa Modulate Factor Xa-catalyzed Cleavage Rates at this Site and Cofactor Function. J Biol Chem. 2012 May 4; 287(19):15409-17.
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Wakabayashi H, Griffiths AE, Fay PJ. Enhancing factor VIII and VIIIa stability by combining mutations at the A2 domain interface and A1-C2 domain interface. J Thromb Haemost. 2012 Mar; 10(3):492-5.
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Takeyama M, Wakabayashi H, Fay PJ. Factor VIII Light Chain Contains a Binding Site for Factor X That Contributes to the Catalytic Efficiency of Factor Xase. Biochemistry. 2012 Jan 24; 51(3):820-8.
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Wakabayashi H, Griffiths AE, Fay PJ. Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability. J Biol Chem. 2011 Jul 22; 286(29):25748-55.
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DeAngelis JP, Varfaj F, Wakabayashi H, Fay PJ. The role of P4-P3' residues flanking Arg336 in facilitating activated protein C-catalyzed cleavage and inactivation of factor VIIIa. Thromb Res. 2011 Nov; 128(5):470-6.
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Novakovic VA, Cullinan DB, Wakabayashi H, Fay PJ, Baleja JD, Gilbert GE. Membrane-binding properties of the Factor VIII C2 domain. Biochem J. 2011 Apr 1; 435(1):187-96.
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Wakabayashi H, Griffiths AE, Fay PJ. Factor VIII lacking the C2 domain retains cofactor activity in vitro. J Biol Chem. 2010 Aug 13; 285(33):25176-84.
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Wakabayashi H, Griffiths AE, Fay PJ. Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations. J Thromb Haemost. 2009 Mar; 7(3):438-44.
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Wakabayashi H, Varfaj F, Deangelis J, Fay PJ. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface. Blood. 2008 Oct 1; 112(7):2761-9.
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Wakabayashi H, Fay PJ. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa. J Biol Chem. 2008 Apr 25; 283(17):11645-51.
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Varfaj F, Wakabayashi H, Fay PJ. Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C. J Biol Chem. 2007 Jul 13; 282(28):20264-72.
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Wakabayashi H, Zhou Q, Varfaj F, Fay PJ. A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa. J Thromb Haemost. 2007 May; 5(5):996-1001.
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Varfaj F, Neuberg J, Jenkins PV, Wakabayashi H, Fay PJ. Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa. Biochem J. 2006 Jun 1; 396(2):355-62.
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Wakabayashi H, Zhou Q, Nogami K, Ansong C, Varfaj F, Miles S, Fay PJ. pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+. Biochim Biophys Acta. 2006 Jun; 1764(6):1094-101.
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Wakabayashi H, Su YC, Ahmad SS, Walsh PN, Fay PJ. A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase. Biochemistry. 2005 Aug 2; 44(30):10298-304.
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Nogami K, Zhou Q, Myles T, Leung LL, Wakabayashi H, Fay PJ. Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain. J Biol Chem. 2005 May 6; 280(18):18476-87.
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Nogami K, Zhou Q, Wakabayashi H, Fay PJ. Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site. Blood. 2005 Jun 1; 105(11):4362-8.
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Nogami K, Wakabayashi H, Ansong C, Fay PJ. Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit. Biochim Biophys Acta. 2004 Sep 1; 1701(1-2):25-35.
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Nogami K, Freas J, Manithody C, Wakabayashi H, Rezaie AR, Fay PJ. Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain. J Biol Chem. 2004 Aug 6; 279(32):33104-13.
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Nogami K, Lapan KA, Zhou Q, Wakabayashi H, Fay PJ. Identification of a factor Xa-interactive site within residues 337-372 of the factor VIII heavy chain. J Biol Chem. 2004 Apr 16; 279(16):15763-71.
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Wakabayashi H, Freas J, Zhou Q, Fay PJ. Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity. J Biol Chem. 2004 Mar 26; 279(13):12677-84.
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Nogami K, Wakabayashi H, Fay PJ. Mechanisms of factor Xa-catalyzed cleavage of the factor VIIIa A1 subunit resulting in cofactor inactivation. J Biol Chem. 2003 May 9; 278(19):16502-9.
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Wakabayashi H, Zhen Z, Schmidt KM, Fay PJ. Mn2+ binding to factor VIII subunits and its effect on cofactor activity. Biochemistry. 2003 Jan 14; 42(1):145-53.
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Nogami K, Wakabayashi H, Schmidt K, Fay PJ. Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa. J Biol Chem. 2003 Jan 17; 278(3):1634-41.
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Wakabayashi H, Schmidt KM, Fay PJ. Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity. Biochemistry. 2002 Jul 2; 41(26):8485-92.
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Wakabayashi H, Koszelak ME, Mastri M, Fay PJ. Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity. Biochemistry. 2001 Aug 28; 40(34):10293-300.
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Mino T, Nakajima M, Wakabayashi H, Yamato S, Shimada K. Determination of valproic acid in serum by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Anal Sci. 2001 Aug; 17(8):999-1001.
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Fay PJ, Mastri M, Koszelak ME, Wakabayashi H. Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA. J Biol Chem. 2001 Apr 13; 276(15):12434-9.
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Seto M, Wakabayashi H, Yamazaki T, Sonoda J, Shinto Y, Uchida A. Gene therapy of chondrosarcoma using retrovirus vectors encoding the herpes simplex virus thymidine kinase gene. Int J Oncol. 1999 Jun; 14(6):1137-41.
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Satoh N, Hibasami H, Mori K, Kaneko H, Wakabayashi H, Hirata K, Sonoda J, Nakashima K, Uchida A. Growth inhibition of human osteosarcoma HuO9 cells by methylglyoxal bis(cyclopentylamidinohydrazone) in vitro and in vivo. Oncol Rep. 1999 May-Jun; 6(3):627-30.
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Yachida S, Kokudo Y, Wakabayashi H, Maeba T, Kaneda K, Maeta H. Morphological and functional alterations to sinusoidal endothelial cells in the early phase of endotoxin-induced liver failure after partial hepatectomy in rats. Virchows Arch. 1998 Aug; 433(2):173-81.
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Nicolson GL, Nakajima M, Wakabayashi H, Boyd DD, Diaz D, Irimura T. Cancer cell heparanase activity associated with invasion and metastasis. Adv Enzyme Regul. 1998; 38:19-32.
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Sawada H, Wakabayashi H, Nawa A, Mora E, Cavanaugh PG, Nicolson GL. Differential motility stimulation but not growth stimulation or adhesion of metastatic human colorectal carcinoma cells by target organ-derived liver sinusoidal endothelial cells. Clin Exp Metastasis. 1996 May; 14(3):308-13.
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Nakajima M, Yamato S, Wakabayashi H, Shimada K. High-performance liquid chromatographic determination of cholesterol and cholestanol in human serum by precolumn derivatization with 2-[2-(isocyanate)ethyl]-3-methyl-1,4-naphthoquinone combined with platinum catalyst reduction and electrochemical detection. Biol Pharm Bull. 1995 Dec; 18(12):1762-4.
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Wakabayashi H, Cavanaugh PG, Nicolson GL. Purification and identification of mouse lung microvessel endothelial cell-derived chemoattractant for lung-metastasizing murine RAW117 large-cell lymphoma cells: identification as mouse monocyte chemotactic protein 1. Cancer Res. 1995 Oct 1; 55(19):4458-64.
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Wakabayashi H, Cavanaugh PG, Nicolson GL. Responses to paracrine chemotactic and autocrine chemokinetic factors and lung metastatic capability of mouse RAW117 large-cell lymphoma cells. Br J Cancer. 1994 Dec; 70(6):1089-94.
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Shimada K, Mino T, Nakajima M, Wakabayashi H, Yamato S. Application of the desulfurization of phenothiazines for a sensitive detection method by high-performance liquid chromatography. J Chromatogr B Biomed Appl. 1994 Nov 4; 661(1):85-91.
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Wakabayashi H, Yamato S, Nakajima M, Shimada K. Application of an electrochemical detector with a graphite electrode to liquid chromatographic determination of penicillamine and captopril in biological samples. J Pharm Biomed Anal. 1994 Sep; 12(9):1147-52.
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Wakabayashi H, Yamato S, Nakajima M, Shimada K. Simultaneous determination of oxidized and reduced coenzyme Q and alpha-tocopherol in biological samples by high performance liquid chromatography with platinum catalyst reduction and electrochemical detection. Biol Pharm Bull. 1994 Aug; 17(8):997-1002.
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Yamato S, Wakabayashi H, Nakajima M, Shimada K. Amperometric determination of oxalate in plasma and urine by liquid chromatography with immobilized oxalate oxidase. J Chromatogr B Biomed Appl. 1994 Jun 3; 656(1):29-35.
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Kawaguchi T, Ono T, Wakabayashi H, Igarashi S. Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential. Clin Exp Metastasis. 1994 May; 12(3):203-12.
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Watanabe K, Wakabayashi H, Veerkamp JH, Ono T, Suzuki T. Immunohistochemical distribution of heart-type fatty acid-binding protein immunoreactivity in normal human tissues and in acute myocardial infarct. J Pathol. 1993 May; 170(1):59-65.
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Kawaguchi T, Igarashi S, Wakabayashi H, Yokoya S, Fukui K. Substrate adhesiveness and experimental metastatic potential of rat ascites hepatoma AH7974-derived variant sublines. Clin Exp Metastasis. 1992 Jul; 10(4):225-38.
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Yamato S, Nakajima M, Wakabayashi H, Shimada K. Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor. J Chromatogr. 1992 Jan 31; 590(2):241-5.
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Wakabayashi H, Nakajima M, Yamato S, Shimada K. Determination of idebenone in rat serum and brain by high-performance liquid chromatography using platinum catalyst reduction and electrochemical detection. J Chromatogr. 1992 Jan 3; 573(1):154-7.
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Wakabayashi H, Kawaguchi T. Fibronectin degradation by human gastric carcinoma cell lines and its associated proteases in relation to stromal invasion in nude mice. Invasion Metastasis. 1992; 12(5-6):284-300.
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Watanabe K, Saito A, Wakabayashi H, Kawaguchi T, Suzuki T. Two autopsy cases of primary leiomyosarcoma of the liver. Superiority of muscle-specific actin immunoreactivity in diagnosis. Acta Pathol Jpn. 1991 Jun; 41(6):461-5.
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