Abstract
Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.
Highlights
Factor IXa (FIXa)1 is a vitamin K-dependent coagulation serine protease, which upon binding to factor VIIIa on membrane surfaces in the presence of Ca2ϩ forms the intrinsic Xase complex and rapidly activates factor X to factor Xa (FXa) during the blood coagulation process [1,2,3]
By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin
Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg233 > Arg165 > Lys230 > Lys126 > Arg170 are critical for binding to heparin
Summary
Mutagenesis and Expression of Recombinant Proteins—The wild type, K98A, K126A, R165A, R170A, K173A, K230A, R233A, and K239A mutants of FIX in Gla-domainless forms (GD-FIX) were constructed by PCR mutagenesis methods as described previously [23]. Binding to Heparin-Sepharose—Factor IX derivatives (ϳ250 g) in 0.02 M Tris-HCl (pH 7.5), 0.1 M NaCl (TBS buffer) containing 5 mM Ca2ϩ were converted to their activated forms with 25 g of RVV-X for 3 h at 37 °C as described previously [24]. The apparent Km and kcat values for substrate hydrolysis were calculated from the MichaelisMenten equation, and the catalytic efficiencies were expressed as the ratios of kcat/Km. Reaction with Antithrombin—The rate of inactivation of FIXa derivatives by AT in both the absence and presence of heparin and the pentasaccharide fragment of heparin was measured under pseudo firstorder rate conditions by a discontinuous assay as described previously [20]. All values are the average of at least 3 independent measurements Ϯ S.E
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