Previous studies have focused attention upon FXI and its interactions with other plasma proteins, i.e., thrombin, FXla, FXIla, prothrombin, high molecular weight kininogen (HK), and FIX, with the platelet plasma membrane and with various inhibitory molecules, e.g., alpha-l-protease inhibitor (alpha1PI), and protease nexin II (PN2), in the initiation and regulation of blood coagulation. The long term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXI/FXla with protein and cell surface ligands involved in its activation and with plasma protein and cell surface ligands involved in the expression and regulation of FXla enzymatic activity. Specifically we propose: 1) To examine the hypothesis that GPIb/IX/V comprises the platelet membrane receptor for FXI that colocalizes it with thrombin for efficient activation on the activated platelet surface, a) To investigate the hypothesis that the N-terminal globular domain of GPIbalpha is the major binding site for FXI that colocalizes it with thrombin for efficient activation on the activated platelet surface, b) To identify the FXI domain that mediates the interaction of FXI with GPIb/IX/V, to test the hypothesis that the FXI A3 domain serves this function and to determine the amino acid residues that mediate this interaction, c) To investigate the hypothesis that the FXI/GPIb complex colocalizes within lipid microdomains (membrane rafts) on the activated platelet membrane for efficient activation by thrombin. 2) To determine the structure of the A4 domain of FXI and the mechanism and structural determinants of homodimer formation by the A4 domain, a) To determine the A4 domain solution structure and the morphology of the interface mediating dimer formation, b) To carry out hydrogen/deuterium exchange experiments as an experimental approach to the analysis of interracial contacts between A4 domains, c) To carry out a mutational analysis of the mechanism and structural determinants of A4-mediated homodimer formation. 3) To determine the mechanism and physiological importance of homodimer formation in the assembly of the FXI-activation complex and the FIX-activation complex on the surface of activated platelets, a) To determine whether the dimeric structure of FXI is required for FXI activation to FXla by thrombin or by FXIIa in the presence or absence of activated platelets or glycocalicin, b) To determine whether the dimeric structure of FXla is required for FIX activation to FIXa in the presence of glycocalicin as it is in the presence of activated platelets, c) To differentiate between alternative mechanisms to account for the failure of monomeric FXla to activate FIX on the activated platelet surface.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL046213-14
Application #
6791383
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
1991-09-30
Project End
2007-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
14
Fiscal Year
2004
Total Cost
$411,019
Indirect Cost
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Navaneetham, Duraiswamy; Wu, Wenman; Li, Hongbo et al. (2013) P1 and P2' site mutations convert protease nexin-2 from a factor XIa inhibitor to a plasmin inhibitor. J Biochem 153:221-31
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Wu, Wenman; Li, Hongbo; Navaneetham, Duraiswamy et al. (2012) The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis. Blood 120:671-7
Su, Ya-Chi; Miller, Tara N; Navaneetham, Duraiswamy et al. (2011) The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2. J Biol Chem 286:31904-14
Navaneetham, Duraiswamy; Sinha, Dipali; Walsh, Peter N (2010) Mechanisms and specificity of factor XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin inhibitor. J Biochem 148:467-79
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Salameh, Moh'd A; Robinson, Jessica L; Navaneetham, Duraiswamy et al. (2010) The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin. J Biol Chem 285:1939-49
Kravtsov, Dmitri V; Matafonov, Anton; Tucker, Erik I et al. (2009) Factor XI contributes to thrombin generation in the absence of factor XII. Blood 114:452-8
Wu, Wenman; Sinha, Dipali; Shikov, Sergei et al. (2008) Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa. J Biol Chem 283:18655-64
Miller, Tara N; Sinha, Dipali; Baird, T Regan et al. (2007) A catalytic domain exosite (Cys527-Cys542) in factor XIa mediates binding to a site on activated platelets. Biochemistry 46:14450-60

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