Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes

Abstract

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.

A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex

“…23 The fact that even higher concentrations of FXa or C1 mutant rFVa 2 still do not produce full prothrombinase activity 23 reflects the much weaker binding of these proteins to membranes with low PS content. 40,41 Taken together, the current results along with our previous observations 23 indicate that the C6PS-binding pocket defined by the (Y1956,L1957)A mutation regulates the affinity of rFVa 2 for FXa but not the effect of FVa on FXa activity.It is worth noting that the turnover number of this membraneassembled C1 mutant rFVa 2 complex is within error limits the same as that seen with wild-type rFVa 2 complex. 23,25 Thus, although the (Y1956,L1957)A mutation clearly affects the assembly of the prothrombinase complex, 21 it does not affect significantly the activity of the prothrombinase complex once it is assembled.…”

“…It is well known that extrinsic membrane proteins, including FXa and FVa, can bind to membranes through multiple interaction sites. 40,41 In the case of rFVa 2 , the interaction that overcomes most of the unfavorable free energy of moving the protein from a 3-dimensional to 2-dimensional environment comes from the C2 domain. 21 However, once rFVa 2 is on the surface, the less avid C1 site can be occupied.…”

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)]

“…Furthermore, when the observed linear increase of nonspecific binding of BFA to non-imprinted p(EGDMA-co-MAA) nanospheres is taken into account (Fig. 1), we concluded, that the nonspecific binding would be better modelled by introduction of a linear term depending only on the ligand concentration in the investigated concentration regime [37,38]. Thus, instead of assuming precisely two different types of specific binding sites, as in the BLM, the extended Langmuir model (ELM) differentiated between a specific interaction based on the molecular recognition of the adsorbate with specific binding sites in the adsorbent generated by molecular imprinting and all other interactions between adsorbate and adsorbent subsumed as nonspecific binding, were modelled by two mathematically independent terms.…”

“…(3), for the mass of nonspecifically bound molecules c N was assumed to be in linear correlation to the free adsorbate after equilibration in the soluble phase [37,38]. By other means, as long as free surface is available during the adsorption process, the nonspecific binding will unabatedly take place parallel to the specific adsorption until the equilibrium is reached:…”

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…Near optimal binding of Xa and Va to model membranes occurs at ϳ10 -12% PS, 4 which supports optimal prothrombinase activity (7,8). Membrane PE has been shown to enhance prothrombin activation particularly in the presence of low concentrations of PS (1-5%) (9).…”