Membrane Phosphatidylserine and Plasma Calcium Levels Switch Factor Xa From An Inactive Dimer to An Active Monomer.
Authors: Koklic Tilen , Majumder Rinku , Lentz Barry 
Published: October 2019

Abstract
Abstract 3180
Poster Board III-103
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. 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 (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 Xa towards human prethrombin2 as a substrate both at fixed membrane concentration and increasing factor Xa concentration, and at fixed factor Xa concentration and increasing membrane concentration. Neither of these experiments showed the expected behavior of an increase in activity as factor Xa bound to membranes. Factor Xa 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 Xa activity did not increase proportionally with factor Xa 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 Xa. The binding of factor Xa to PS-containing membranes also appeared to be tighter at low than at high membrane concentration. Because factor Xa 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 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 = 9000 M-1s-1, kcat/KMsolution = 94 M-1s-1, and Kd,surfacedimer = ( 40±25) · 10-15 mol/(dm)2. This surface dimerization constant corresponds to a solution-phase Kddimer = 1 nM at 10 mM lipid concentration, nearly what we observed for formation of bovine factor Xa 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 Xa was activated by membrane binding below 1.5 mM Ca2+, but inactivated above this Ca2+ concentration. The transition of factor Xa 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 Xa 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.