title: Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites.
abstract: Blood coagulation factor IXa has been presumed to be regulated by the serpin, antithrombin, and its polysaccharide activator, heparin, but it has not been clear whether factor IXa is inhibited by the serpin with a specificity comparable to that for thrombin and factor Xa or what determinants govern this specificity. Here we show that antithrombin is essentially unreactive with factor IXa in the absence of heparin (k(ass) approximately 10 M(-1) s(-1)) but undergoes a remarkable approximately 1 million-fold enhancement in reactivity with this proteinase to the physiologically relevant range (k(ass) approximately 10(7) M(-1) s(-1)) when activated by heparin in the presence of physiologic levels of calcium. This rate enhancement is shown to derive from three sources: (i) allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (300-500-fold), (ii) allosteric activation of factor IXa by calcium ions (4-8-fold), and (iii) heparin bridging of antithrombin and factor IXa augmented by calcium ions (130-1000-fold depending on heparin chain length). Mutagenesis of P6-P3' reactive loop residues of antithrombin further reveals that the reactivity of the unactivated inhibitor is principally determined by the P1 Arg residue, whereas exosites outside the loop which are present on the activated serpin and on heparin are responsible for heparin enhancement of this reactivity. These results together with our previous findings demonstrate that exosites are responsible for the unusual specificity of antithrombin and heparin for three clotting proteases with quite distinct substrate specificities.
authors.affiliation: Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Bedsted
authors.forename: Richard
authors.initials: R
authors.lastname: Swanson
authors.forename: Yung-Jen
authors.initials: YJ
authors.lastname: Chuang
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
authors.forename: Ingemar
authors.initials: I
authors.lastname: Björk
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
mesh_terms: D000991:Antithrombins / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000991:Antithrombins; D009844:Oligosaccharides; D006493:Heparin; D015949:Factor IXa; D002118:Calcium