Membrane Phosphatidylserine and Plasma Calcium Levels Switch Factor Xa From An Inactive Dimer to An Active Monomer.

Abstract 3180

Poster Board III-103

Factor X_a 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 X_a with its cofactor, factor V_a. PS also triggers tight association of factor X_a with factor X_a, at least in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238-1251) to form an inactive factor X_a dimer (Chattopadhyay et al. Biophys. J. 96(3) pp. 974 – 986, 2009). We report here that PS-triggered factor Xa dimer formation is a sharp sigmoidal function of Ca²⁺ concentration and that this Ca²⁺ switch occurs just below plasma Ca²⁺ concentrations.

We have determined the proteolytic activity of human factor X_a towards human prethrombin2 as a substrate both at fixed membrane concentration and increasing factor X_a concentration, and at fixed factor X_a concentration and increasing membrane concentration. Neither of these experiments showed the expected behavior of an increase in activity as factor X_a bound to membranes. Factor X_a activity actually decreased as low concentrations of PS-containing membranes were added, and increased only at higher membrane concentrations. At fixed membrane concentrations, the total factor X_a activity did not increase proportionally with factor X_a concentration. These observations showed that membranes actually inhibited factor Xa activity under conditions of high factor Xa or low membrane concentrations, suggesting the existence of inactive membrane-bound oligomers of factor X_a. The binding of factor X_a to PS-containing membranes also appeared to be tighter at low than at high membrane concentration. Because factor X_a forms dimers in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238-1251), we attempted to explain these observations in terms of a model that takes into account dimerization of factor X_a after binding to a membrane. This dimer model successfully described all our data, with the parameters of best fit being k_cat/K_M^dimer = 0 M^-1s^-1, k_cat/K_M^monomer = 9000 M^-1s^-1, k_cat/K_M^solution = 94 M^-1s^-1, and K_d,surface^dimer = ( 40±25) · 10^-15 mol/(dm)². This surface dimerization constant corresponds to a solution-phase K_d^dimer = 1 nM at 10 mM lipid concentration, nearly what we observed for formation of bovine factor X_a dimer in the presence of short-chain PS (20 nM; Majumder et al. Biophys. J. 2003, 84:1238-1251). As we observed for soluble-PS-induced dimer formation in solution, dimer formation on a membrane was Ca²⁺ dependent. Unlike in solution, factor X_a was activated by membrane binding below 1.5 mM Ca²⁺, but inactivated above this Ca²⁺ concentration. The transition of factor X_a from monomer to inactive dimer state on PS-containing membranes is a sensitive function of Ca²⁺ concentration. Just below the normal range of plasma Ca²⁺ concentration, addition of PS-containing membranes promotes factor Xa activity, while just above this level PS-containing membranes inhibit factor Xa. This suggests that Ca²⁺-dependent formation of inactive factor X_a dimers might have an important role in factor Xa activity during the initial phase of the blood coagulation process when generation of a small amount of thrombin in a short period of time activates platelets.

Supported by USPHS grant HL072827.