Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

Abstract

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X a by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X a using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X a . Our results demonstrate that the structural responses of human and bovine factor X a to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X a are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the ␥-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca 2؉ (k d ϳ 1 mM), although this PS site does not influence the functional response of factor X a . Second, a Ca 2؉ -dependent binding site is in the epidermal growth factor domains (EGF NC ) that are linked by Ca 2؉ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X a . Third, a Ca 2؉ -requiring site seems to be in the EGF C -catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF NC , and catalytic domains in the presence of Ca 2؉ , meaning that PS regulation of factor X a involves linkage between widely separated parts of the protein.The substantial effects of soluble phosphatidylserine (C6PS 1 ) on the kinetics of prothrombin activation by factor X a(1) and on the structure of factor X a , as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles (2, 3). The implication of this PS exposure and of the effect of PS on factor X a and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation. To better define this regulatory process, it is important to know the locations of the PS binding sites on factor X a . The organization of factor X into structural domains is illustrated below in Fig. 1. Factor X consists of two peptides. The light chain consists of an N terminus ␥-carboxyglutamic acidrich region (Gla module) and two Cys-rich cassette modules. The heavy chain consists of the serine protease catalytic domain. The two cassette modules of the light chain show strong sequence and structural homology to epidermal growth factor (EGF) (4) and are thus referred to as EGF N and EGF C , where N and C indicate the domain nearer to the N and C termini, respectively. Crystal structures o...

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

“…This makes it very unlikely that very small and hydrodynamically undetectable C6PS aggregates could explain our observations. The second, and even stronger, argument against this possibility is provided by the observations that only two and four C6PS molecules were bound at saturation per FX a and FV a , respectively (10,32). This small amount of lipid could not form even a local surface for the binding of these proteins, and we must conclude that specific sites on both FX a and FV a bind C6PS in a way that regulates the structure, function, and assembly of the components of the prothrombinase complex.…”

“…At first glance, this is quite surprising, but it is actually not an unreasonable possibility, because we have shown that binding of C6PS to the second FX a site competes with synthetic substrate binding to the active site of FX a (32). Although this does not seem to interfere with the FX a proteolytic activity in the absence of FV a (8), 2 our results indicate that C6PS binding to the second site on FX a bound to FV a2 may interfere with formation of a productive enzymesubstrate complex.…”

“…5 and 7. This approach acknowledges that FX a binds two molecules of C6PS (32), the first with a k d…”

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…This could be explained by C6PE binding to two linked sites, as has been reported for C6PS (41). Our current hypothesis is that C6PS and C6PE share the regulatory site on Xa (non-additive effects on activity) but also bind to the anomalous site in the catalytic domain to which the regulatory site is linked (41). More extensive experiments are needed to prove this hypothesis.…”

“…In this picture, the effect of PE on membrane interfacial packing contributes only to bringing factor Va to the membrane, but not for the reduction in K m , the increase in k cat , and the decrease in K d app , which result from PE regulation of both factors Xa and Va. The lipid-binding regions through which C6PS influences factors Va and Xa have been identified (14,27,41). Are these the same sites occupied by PE?…”