This proposal will determine the structure and conformation of human factor VIII and elucidate the molecular mechanisms which result in the regulation of factor VIII activity. Factor VIII is believed to exist as a series of active heterodimers consisting of a variable length heavy chain noncovalently linked to an invariant light chain. Selected homogeneous heterodimers will be prepared and characterized in terms of native size and polypeptide composition. Detailed physico-chemical analyses will be performed on the homogeneous preparations to obtain information on conformation and intra-chain disulfide pairing. a second major focus will involve study of the structure of factor VIIIa following proteolytic activation by thrombin and will elucidate those events that result in the spontaneous inactivation of factor VIIIa. Additional studies will evaluate, both kinetically and structurally, the activation of factor VIII by factor IXa. Binding of factor VIII to factor IX (IXa) will be studied using kinetic methods, cross- linking reagents and monoclonal antibodies to determine dissociation constants, stoichiometry and location of binding sites. Sequence analysis of the transient and terminal fragments generated by proteolytic inactivation of factor VIII by activated protein C will elucidate product-precursor relationships. Comparison of rates of inactivation of factors VIII with factor VIIIa will be evaluated in terms of preferred substrates for activated protein C. These structural and mechanistic studies will offer insights into a coagulation protein that is central to the hemostatic process and will have implications for the understanding and management of hemophilia A, the most common of the congenital bleeding disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038199-02
Application #
3354294
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
School of Medicine & Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Monaghan, M; Wakabayashi, H; Griffiths, A E et al. (2016) Stabilizing interactions between D666-S1787 and T657-Y1792 at the A2-A3 interface support factor VIIIa stability in the blood clotting pathway. J Thromb Haemost 14:1021-30
Leong, Lilley; Sim, Derek; Patel, Chandra et al. (2015) Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models. Blood 125:392-8
Monaghan, M; Wakabayashi, H; Griffiths, A et al. (2014) Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit. Results from an increased apparent affinity for the A2 subunit. Thromb Haemost 112:495-502
Kosloski, Matthew P; Shetty, Krithika A; Wakabayashi, Hironao et al. (2014) Effects of replacement of factor VIII amino acids Asp519 and Glu665 with Val on plasma survival and efficacy in vivo. AAPS J 16:1038-45
Wakabayashi, Hironao; Monaghan, Morgan; Fay, Philip J (2014) Cofactor activity in factor VIIIa of the blood clotting pathway is stabilized by an interdomain bond between His281 and Ser524 formed in factor VIII. J Biol Chem 289:14020-9
Wakabayashi, H; Wintermute, J M; Fay, P J (2014) Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa. Thromb Haemost 112:43-52
Griffiths, Amy E; Wintermute, Jennifer; Newell-Caito, Jennifer L et al. (2013) Residues flanking scissile bonds in Factor VIII modulate rates of cleavage and proteolytic activation catalyzed by Factor Xa. Biochemistry 52:8060-8
Wakabayashi, Hironao; Fay, Philip J (2013) Replacing the factor VIII C1 domain with a second C2 domain reduces factor VIII stability and affinity for factor IXa. J Biol Chem 288:31289-97
Wakabayashi, Hironao; Fay, Philip J (2013) Molecular orientation of factor VIIIa on the phospholipid membrane surface determined by fluorescence resonance energy transfer. Biochem J 452:293-301
Takeyama, Masahiro; Wakabayashi, Hironao; Fay, Philip J (2013) Contribution of factor VIII light-chain residues 2007-2016 to an activated protein C-interactive site. Thromb Haemost 109:187-98

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