Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. We propose to define critical aspects of factor VIII structure and regulation that includes fine point details of inter-subunit interactions, the role of calcium in stabilizing factor VIII structure and mechanisms for the activation of factor VIII by thrombin.
Aim I focuses on interactions of A2 subunit with the A1/A3C1C2 dimer in the factor VIIIa heterotrimer. Specific studies will elucidate mechanisms for the retention of A2 subunit in factor VIIIa, expanding on our prior work that mapped a number of hydrogen bonds to the interface of A2 with A1 and A3 domains and identified high stability factor VIIIa variants resulting from removal of destabilizing charged residues from hydrophobic pockets. The identification of several residues that appear functionally important in bonding interactions in factor VIIIa but not factor VIII suggests sites for structural changes resulting from procofactor activation. These sites will be characterized regarding inter-subunit affinity parameters and identification of potential bonding partners.
Aim II studies focus on the functional role of a Ca2+ site we previously identified in the factor VIII A1 domain. Mutagenesis experiments are proposed to enhance stability at this site by altering noncovalent interactions in an effort to recapitulate our recent work stabilizing this region by covalent bonding. This study includes generation of gain-of-function variants possessing increased specific activity coupled with increased stability. A second line of investigation will explore Ca2+-dependent changes in conformation at the A1-C2 junction regarding spatial changes in A1 domain relative to the membrane surface and Ca2+-dependent protease protection.
Aim III studies will define a model for the exosite-dependent, thrombin-catalyzed activation of factor VIII following binding interactions using factor VIII chains and VIIIa subunits, as well as variant proteins constructed with point mutations at potentially critical exosite-interactive regions. Reciprocal binding and activity studies will use thrombin variants possessing point mutations in anion binding exosites I or II. A focal point of these studies is the rate-limiting cleavage at Arg372 which exposes cryptic factor IXa-sites in the cofactor. Additional studies will address the influence of P3-P3 These aims will be facilitated by use of novel reagents including recombinant protease forms, substrates comprised of highly purified factor VIII chains (VIIIa subunits) and variants possessing point mutations at residues of interest. We will accomplish these studies using high-resolution techniques including fluorescence energy transfer, surface plasmon resonance and MALDI-TOF mass spectrometry. Results from these studies will define mechanisms providing significant insights into factor VIII structure and its regulation, as well as provide important 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 factor VIII including inter-subunit interactions, the role of calcium in stabilizing structure and mechanisms for the activation of factor VIII by thrombin. These studies will define important mechanisms for factor VIII structure and regulation, aiding in our understanding of hemophilia, and providing significant insights into the design of new therapeutics for the treatment of this disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038199-26
Application #
8463581
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1988-04-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
26
Fiscal Year
2013
Total Cost
$364,033
Indirect Cost
$128,413
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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
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
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
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
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
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) Modification of interdomain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity. Biochemistry 52:3921-9
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
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

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