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 Ca 2+ , 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 Ca 2+ concentration and that this Ca 2+ switch occurs just below plasma Ca 2+ 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 -1 s -1 , k cat /K M monomer = 9000 M -1 s -1 , k cat /K M solution = 94 M -1 s -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 Ca 2+ dependent. Unlike in solution, factor X a was activated by membrane binding below 1.5 mM Ca 2+ , but inactivated above this Ca 2+ concentration. The transition of factor X a from monomer to inactive dimer state on PS-containing membranes is a sensitive function of Ca 2+ concentration. Just below the normal range of plasma Ca 2+ concentration, addition of PS-containing membranes promotes factor Xa activity, while just above this level PS-containing membranes inhibit factor Xa. This suggests that Ca 2+ -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. Disclosures No relevant conflicts of interest to declare.