Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. We propose to elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa-dependent catalytic rate enhancement of factor IXa and the down- regulation of its activity leading to dampening of the intrinsic factor Xase.
Aim I studies mechanisms by which factor VIIIa modulates catalytic efficiency within factor Xase. We continue to study the role of the factor VIIIa A2 subunit as this subunit directly influences events at the active site of factor IXa. Use of novel recombinant A2 reagents will facilitate identification of critical factor IXa-interactive residues. A new focus of these studies now examines the role of the factor VIIIa A3C1C2 subunit in factor Xase formation.
This aim i s significant since this subunit provides the majority of the binding energy for the interaction of factor VIIIa with factor IXa. Our development of a baculovirus expression system yielding fully functional A3C1C2 subunit will be instrumental in addressing this aim. Factor VIIIa enhances substrate factor X binding in Xase and we will investigate the stabilization of a factor X-interactive site in the factor VIIIa A1 subunit by the A3C1C2 subunit. The role of positive charge potential in the A2 subunit in contributing to substrate turnover and product release will be evaluated using recombinant reagents.
Aim II focuses on proteolytic inactivation of factor VIIIa and is based upon our recent studies showing exosite-dependence for cofactor inactivation by activated protein C (APC) and factor Xa, as well as an important contribution of sequences flanking the P1 Arg residues in APC-catalyzed inactivation of factor VIIIa. The molecular interactions leading to reactions of these enzymes responsible for factor VIIIa inactivation and consequent dampening of factor Xase remain poorly understood. We will probe these interactions using native and mutant factor VIIIa variants possessing alterations in putative exosite-interactive regions as well as with cleavage-resistant forms. A focal point is proteolysis at Arg336 in the A1 subunit, the predominant site cleaved by both APC and factor Xa, and elucidation of mechanisms for this catalytic event.
These aims will be facilitated by use of novel reagents including recombinant protease forms, substrate comprised of highly purified factor VIII(a) chains/subunits, as well as variants possessing point mutations at residues of interest. We will accomplish these studies using high resolution techniques including fluorescence energy transfer and anisotropy, surface plasmon resonance and MALDI-TOF mass spectrometry. We anticipate our results will define mechanisms providing significant insights into cofactor-mediated catalysis and its regulation, as well as provide useful information for the design of superior therapeutics for the treatment of hemophilia A.

Public Health Relevance

Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. In this application we will elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa cofactor function and the regulation of its activity in factor Xase.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Link, Rebecca P
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University of Rochester
Schools of Dentistry
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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
Griffiths, Amy E; Rydkin, Ivan; Fay, Philip J (2013) Factor VIIIa A2 subunit shows a high affinity interaction with factor IXa: contribution of A2 subunit residues 707-714 to the interaction with factor IXa. J Biol Chem 288:15057-64
Wakabayashi, Hironao; Fay, Philip J (2013) Modification of interdomain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity. Biochemistry 52:3921-9
Takeyama, Masahiro; Wintermute, Jennifer M; Manithody, Chandrashekhara et al. (2013) Variable contributions of basic residues forming an APC exosite in the binding and inactivation of factor VIIIa. Biochemistry 52:2228-35
Newell-Caito, Jennifer L; Griffiths, Amy E; Fay, Philip J (2012) P3-P3' residues flanking scissile bonds in factor VIII modulate rates of substrate cleavage and procofactor activation by thrombin. Biochemistry 51:3451-9
Wakabayashi, H; Griffiths, A E; Fay, P J (2012) Enhancing factor VIII and VIIIa stability by combining mutations at the A2 domain interface and A1-C2 domain interface. J Thromb Haemost 10:492-5
DeAngelis, Jennifer P; Wakabayashi, Hironao; Fay, Philip J (2012) Sequences flanking Arg336 in factor VIIIa modulate factor Xa-catalyzed cleavage rates at this site and cofactor function. J Biol Chem 287:15409-17
Takeyama, Masahiro; Wakabayashi, Hironao; Fay, Philip J (2012) Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase. Biochemistry 51:820-8
Wakabayashi, Hironao; Griffiths, Amy E; Fay, Philip J (2011) Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability. J Biol Chem 286:25748-55
Griffiths, A E; Wang, W; Hagen, F K et al. (2011) Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction. J Thromb Haemost 9:1534-40

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