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 beta (2)-glycoprotein I (beta (2)GPI) 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 trot 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.