The influence of lipid composition and divalent cations on annexin V binding to phospholipid mixtures.

Annexin V is a 36-kDa protein which, it has been suggested, is a factor in protecting the vascular endothelium from attack by antibodies to other phospholipid-binding proteins. Competition between annexin V and beta2-glycoprotein I (beta2GPI) for phospholipid surfaces is complicated by empirical observations regarding alterations in binding to anionic phospholipid, primarily phosphatidylserine. In order to elucidate the effect of phospholipid composition and divalent cations (Ca(+2) and Mg(+2)) on annexin V binding to phospholipid, we used biotinylated annexin V and peroxidase-conjugated avidin D to probe the binding of annexin V to phospholipid-coated wells of polystyrene microtiter plates. Binding of annexin V to anionic phospholipid is Ca(+2)-dependent and, in its absence, annexin V was found to bind most avidly to 100% phosphatidylcholine in a saturable manner, followed by decreasing percentages of phosphatidylcholine. Ca(+2) was found to inhibit phosphatidylcholine binding and promote the binding of phospholipid mixtures containing phosphatidylserine. Phosphatidylserine (100%) did not bind annexin V as strongly as mixtures of 50% and 75% phosphatidylserine. The effect with Ca(+2) suggests saturation of Ca(+2)-binding sites on annexin V, reached under our experimental conditions at approximately 1 mM. Under the same conditions, Mg(+2) slightly enhanced the binding of all of the phospholipid compositions studied. Ca(+2)-dependent binding of annexin V was competitively inhibited by Mg(+2); 5 mM Mg(+2) reduced binding significantly (p < 0.0001 by ANOVA, p < 0.05 for post hoc test of 5 mM vs 0 mM). These data suggest that the translocation of membrane phospholipid under the dynamics of ion transport in vascular endothelium may alter annexin V binding.