Functional and Structural Characterization of Factor Xa Dimer in Solution

Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca2+-dependent dimerization in solution. We report the effects of Ca2+, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 106- to 107-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in kcat. Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in α-helix upon dimerization. Mass spectrometry identifies a lysine (K270) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K351 (part of a reported FVa binding region), K242 (adjacent to the catalytic triad), and K420 (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex.