Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein, factor VIII. There is no cure for hemophilia A and treatment consists of replacement using (purified) factor VIII preparations. During the last funding period we have made significant strides in characterizing sequences and ligands, and determining mechanisms important for factor VIIL/factor Villa subunit structure and function. We will use this information as a platform to identify fine point structural details of intra-protein interactions that will define mechanisms for the regulation of this critical protein.
The first aim will study inter-factor Villa subunit interactions related to the retention and stimulation of A2 subunit. This subunit is critical as demonstrated by the limited cofactor activity observed for isolated A2. Further, the relatively weak affinity interaction of A2 subunit in factor VIIIa governs the subunit structure and function of the active protein. Goals are to identify and determine the roles of residues in A1 and A2 that serve in the inter-subunit interaction and are responsible for the stimulation of A2-dependent cofactor activity.
Specific aims i nclude: (i) elucidation of salt bridging between basic residues in A2 and the acidic C-terminus of Al and (ii) examination of interactive sites in Al that are independent of this C-terminal tail. Methods will employ peptide analysis and molecular biology/cell culture techniques to generate novel factor VIII/subunit molecules. Alterations in activity will be correlated with changes in structure using a variety of physical methods amenable to low levels of protein.
The second aim studies metal ion-dependent and -independent interactions in factor VIII involved in association of heavy and light chains. The goal of this aim is to define and model the association of these two chains in the active conformation. Our studies will: (i) examine the role of Cu in the high affinity inter-chain interaction following characterization of factor VIH molecules possessing altered consensus type 1 and type 2 Cu binding Sites, (ii) determine the role of Ca2+ in yielding the active cofactor conformation following quantitation of Ca sites and physical analyses of the conversion of inactive to active cofactor conformation, and (iii) assess metal ion-independent interactions including the role of exposed hydrophobic sites in heavy and light chains, and contribution of the A2 domain to the inter-factor VIII interaction. Definition of these issues will yield valuable insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Link, Rebecca P
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University of Rochester
Internal Medicine/Medicine
Schools of Dentistry
United States
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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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