title: Heparin modulates the 99-loop of factor IXa: effects on reactivity with isolated Kunitz-type inhibitor domains.
abstract: Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.
authors.affiliation: Department of Biochemistry, Biomedical Research Program, The University of Texas Health Science Center, Tyler, Texas 75708, USA.
authors.forename: Pierre F
authors.initials: PF
authors.lastname: Neuenschwander
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Williamson
authors.forename: Armen
authors.initials: A
authors.lastname: Nalian
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Baker-Deadmond
mesh_terms: D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D007611:Aprotinin / Q000737:chemistry*; D002460:Cell Line; D017984:Enoxaparin / Q000737:chemistry; D015949:Factor IXa / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D017984:Enoxaparin; D006493:Heparin; D007611:Aprotinin; D015949:Factor IXa; D000409:Alanine