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The Influence of Lipid Composition and Divalent Cations on Annexin V Binding to Phospholipid Mixtures

Correspondence to Vincent A. DeBari, PhD, St. Joseph’s Hospital Medical Center, 703 Main Street, Paterson, NJ 07503, USA; tel 973 754 3561; fax 973 754 3555; e-mail debariv{at}sjhmc.org

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 ß₂-glycoprotein I (ß₂GPI) 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.

Keywords: Annexin V, phospholipids, ß₂-glycoprotein I, antiphospholipid syndrome

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