Abstract
Blood coagulation is regulated through protein–protein and protein–lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)—a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.
Highlights
Abbreviations PS Phosphatidylserine PE Phosphatidylethanolamine phosphatidic acid (PA) Phosphatidic acid PC Phosphatidylcholine PT Prothrombin factor X (fX) Factor X factor IX (fIX) Factor IX fVIIa Activated factor VII activated protein C (aPC) Activated protein C protein S (PrS) Protein S protein Z (PrZ) Protein Z
To date, the only GLA domain-containing protein whose binding to membrane surfaces has been directly shown to be enhanced by PE is fX, raising the question of how generally PE–PS synergy promotes the binding of GLA domain-containing proteins
In order to assess the influence of PE lipids on binding, the equilibrium dissociation constants, Kd values, of fVIIa, fIX, fX, PT, aPC, PrS, and PrZ, were determined for eight different lipid compositions using the same Nanodisc microarray to prevent experiment to experiment variation
Summary
Abbreviations PS Phosphatidylserine PE Phosphatidylethanolamine PA Phosphatidic acid PC Phosphatidylcholine PT Prothrombin fX Factor X fIX Factor IX fVIIa Activated factor VII aPC Activated protein C PrS Protein S PrZ Protein Z. Maximal rates of activation of fX by fVIIa bound to tissue factor[6,7,8], of activation of prothrombin by fXa bound to factor Va9, and of inactivation of factor Va by a PC10, have been reported to require much less PS when membranes contain phosphatidylethanolamine (PE) This phenomenon has been termed PE–PS synergy. Since most of the previous work on PE–PS synergy focused on enzyme activity assays using membrane-bound enzyme, cofactors and substrates, it is not clear which of the proteins within these multi-protein complexes exhibited enhanced membrane binding in the presence of PE This is especially important since some of the proteins in these complexes (e.g., factors Va and VIIIa), bind to PS in membrane surfaces via discoidin-type C2 domains, not GLA d omains[4]. To date, the only GLA domain-containing protein whose binding to membrane surfaces has been directly shown to be enhanced by PE is fX, raising the question of how generally PE–PS synergy promotes the binding of GLA domain-containing proteins
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