Abstract
Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.
Highlights
This study shows that annexin V binds with high affinity to human platelets
The properties of annexin V binding to platelet membranes are largely identical to the properties of annexin V binding to PS-containing phospholipid surfaces [23, 24]: binding is reversible, calcium-dependent, and of high affinity
The maximum number of annexin V binding sites on platelets is consistent with a calculated PS content of 2 x lo7 molecules/ platelet [33]: A23187 induces approximately 2 X lo5 annexin V binding sites/platelet (Table I), which would require 8 x lo6 PS molecules at a stoichiometry of approximately 40 PS molecules/annexin binding site in phospholipid vesicles containing 20 mol % of PS [23]
Summary
E1, Russell’s viper venom, and BSA (Fraction V), Sigma; A23187, Calbiochem-Hoechst, San Diego, CA; Iodo-Gen, Pierce Chemical Co.; Na”“1 and ‘251-Bolton-Hunter reagent, ICN Radiochemicals; phospholipids, Avanti Polar Lipids. V was labeled by the Iodo-Gen method [27] to a specific activity of 3000 cpm/ng. The reaction tube was coated with 10 pg of Iodo-Gen; iodination was carried out for 10 min at room temperature with 100 wg of annexin V, 1 mCi of Na”“I in 43 mM Tris, pH 8.0, in a total volume of 115 ~1. Anticoagulant and phospholipid binding activities (determined as described [22]) were unchanged by iodination. Factor Xa (10 Fg) was labeled with the Bolton-Hunter reagent (0.25 mCi) as described [10] to a specific activity of 2000 cpm/ng. Platelet Binding Assay-Human platelets were isolated and washed twice according to Baenziger and Majerus [28] in the presence of 1
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