Abstract
We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca(2+), suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg(93), Lys(96), Arg(125), Arg(165), Lys(169), Lys(236), and Arg(240) were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k(2) with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg(240) > Lys(236) > Lys(169) > Arg(165) > Lys(96) > Arg(93) >/= Arg(125). Interestingly, further study suggested that certain basic residues of this site, particularly Arg(165) and Lys(169), play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.
Highlights
Factor Xa is a vitamin K-dependent coagulation serine proteinase which, upon binding to its cofactor factor Va on membrane surfaces in the presence of Ca2ϩ ions, rapidly activates prothrombin to thrombin in the clotting cascade [2, 3]
In antithrombin inactivation of GDFXa, we further showed that the cofactor effect of unfractionated heparin is mediated by a combination of an ϳ300-fold enhancement by the pentasaccharide-induced conformational change in the reactive site loop of antithrombin and an ϳ60 – 90-fold enhancement by a template mechanism [18]
Structural data revealed that seven basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in factor Xa (Fig. 6)
Summary
Mutagenesis and Expression of Recombinant Proteins—Construction and expression of factor X in Gla domainless form (GDFX) in the RSV-PL4 expression/purification vector system was described previously [7, 23]. The initial rate of activation was measured after 5 min of incubation at room temperature in the same buffer system described above Under these experimental conditions, it is expected that the effective concentration of the enzyme-cofactor complex would be equal with all GDFXa derivatives. The heparin concentration dependence of the inactivation reactions were determined by incubating 0.2 nM of each factor Xa derivative with 2.5 nM antithrombin and varying concentrations of heparin (0 – 64 M) in TBS buffer (I ϳ0.12) containing 1 mg/ml BSA and 0.1% PEG 8000 in 50-l reactions. This equation neglects the uncatalyzed rate constant because its contribution was found to be negligible within
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