Recent published work on the biochemistry, molecular biology and physiology of FXI has refocused attention on the biological importance of this unique and interesting protein. Previous studies from our laboratory (currently supported by NIH HL46213 """"""""Molecular Interactions of Factor XI""""""""), for which we now seek continued support, as well as work from other laboratories have focused attention upon FXI and its interactions with other plasma proteins [i.e., thrombin, FXIa, FXIIa, prothrombin, HK, and FIX], with the platelet plasma membrane and with various inhibitory molecules [e.g., alpha-1-protease inhibitor (alpha1PI), and protease nexin II (PN2)] in the initiation and regulation of blood coagulation. Recent observations resulting in the recognition of the essential role of FXI in hemostasis concern the relationship of its domain structure to its biological function, the molecular genetics of FXI, its molecular and cellular interactions, the elucidation of newly discovered pathways for activation of FXI, and the expression and regulation of its enzymatic activity. The long term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXI/FXIa 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 FXIa enzymatic activity. Specifically we propose: 1) To define the mechanism of homodimer formation mediated by the Apple 4 (A4) domain of FXI by carrying out a detailed physicochemical characterization of dimer formation using the rA4 domain. 2) To determine the morphology of the interface mediating dimer formation between the A4 domains of FXI. 3) To ascertain the structural determinants of dimer formation in full-length FXI using loss of function and gain of function chimeric rFXI/PK hybrids and FXI mutants using homology scanning and alanine scanning mutagenesis. 4) To determine the functional significance of dimer formation using conformationally constrained synthetic peptides and rFXI mutants designed to produce a monomeric FXI molecule. 5) To determine the most physiologically relevant surface(s), enzyme(s), cofactor(s), and FXI subdomains required for FXI activation. These proposed studies will enable us to utilize combined immunochemical, biochemical and molecular biological techniques to provide definitive information about the structure of FXI/FXIa domains essential for these important intermolecular interactions and to define the structure/function relationships involved in the initiation and regulation of intrinsic coagulation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Temple University
Internal Medicine/Medicine
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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
Marcinkiewicz, Mariola M; Sinha, Dipali; Walsh, Peter N (2012) Productive recognition of factor IX by factor XIa exosites requires disulfide linkage between heavy and light chains of factor XIa. J Biol Chem 287:6187-95
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
Salameh, Moh'd A; Soares, Alexei S; Navaneetham, Duraiswamy et al. (2010) Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin. J Biol Chem 285:36884-96
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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