reversible modulation of human factor xa activity with phosphonate esters: media effects Original Research Article

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X=H, PMN; CH3 and CH3O) inactivate human factor Xa with rate constants 8–86 M−1 s−1 at pH 6.75 in 0.025 M Hepes buffer, 0.15 M NaCl and 2 mM CaCl2 at 7.0±0.1 °C. The stereoselectivity of the inactivation of factor Xa is 2–10 and favors the levorotatory enantiomers. The pH-dependence of inactivation of factor Xa by (−)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 6.2±0.1. Factor Xa reactivates from its phosphonyl adducts through a self-catalyzed intramolecular reaction, which is much influenced by the presence of phospholipids. The rate of reactivation in the absence of phospholipids is not pH dependent at pH <9, but it increases very much at pH >9. In the presence of phospholipids, the pH dependence of the rate constant for reactivation is sigmoidal in the pH 6.5–10.3 range and levels off at pH >9 indicating that the enzyme catalyzes its reactivation. The kinetic pKa for the recovery of factor Xa from its adducts with the PMNs is in the range of 6.7–8.1 and is consistent with the participation of the catalytic His57 in the reactivation process.