A Ca2+ Switch Triggers Formation of Inactive Factor Xa Dimers on Phosphatidylserine-Containing Membranes.

Factor Xa has a prominent role in amplifying both inflammation and coagulation cascades. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. PS, in the presence of Ca2+, triggers tight association of factor Xa with its cofactor, factor Va. An interesting complication is that PS also triggers tight association of factor Xa with factor Xa, at least in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251), to form an inactive factor Xa dimer (Sen S, and Lentz BR, unpublished). In this work, we ask whether Ca2+ and PS also trigger formation of an inactive factor Xa dimer on a membrane and explore the possible physiological significance of this. We have determined the proteolytic activity of human factor Xa towards human prethrombin2 as a substrate both at fixed membrane concentration (increasing factor Xa concentration) and at fixed factor Xa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as factor Xa bound to membranes. The total factor Xa activity actually decreased as low concentrations of PS-containing membranes were added, and increased at higher membrane concentrations. At fixed membrane concentrations, the total factor Xa activity did not increase proportionally with factor Xa concentration. Both observations suggested the existence of membrane-bound and inactive multimeric forms of factor Xa. Because we have observed factor Xa to form dimers in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251), we tried to fit globally four such data sets to a model that takes into account dimerization of factor Xa after binding to a membrane. This dimer model successfully described all our data, with the parameters of best fit being kcat/KMdimer = 0 M−1s−1, kcat/KMmonomer = 430 M−1s−1, kcat/KMsolution = 38 M−1s−1, and Kd,surfacedimer = 4·10−12 mol/(dm)2. This surface dimerization constant corresponds to a solution-phase Kddimer = 10 nM at 100 μM lipid concentration, nearly what we observed for formation of bovine factor Xa dimer in the presence of short-chain PS (20 nM; Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251). Also consistent with the dimer hypothesis, we observed that the binding of factor Xa to PS-containing membranes appears to be tighter at low than at high membrane concentration. As we observed for soluble-PS-induced dimer formation in solution, dimer formation on a membrane was Ca2+ dependent. Unlike in solution, factor Xa was activated by membrane binding below 1.5 mM Ca2+, but inactivated above this Ca2+ concentration. This suggests that factor Xa activity may be regulated by Ca2+ concentrations close to plasma Ca2+ levels. We conclude that:factor Xa dimerizes on PS-containing membranes;, factor Xa dimer is inactive; and, the transition from monomer to dimer state depends critically on Ca2+ concentration.