PAF formation specifically correlates with SCIP formation. (A) Washed platelets (5 × 107/mL) were resuspended in Tyrode buffer containing either 1 mM CaCl2/MgCl2 (Ca/Mg) or 1 mM EGTA and 2 mM MgCl2 (EGTA/Mg). Platelets were left unstimulated (Resting) or stimulated with 1 U/mL thrombin (Thr) or 1 μM ionophore A23187 (Iono) for 15 minutes at 37°C in the presence of 5 μg/mL FITC–annexin 5A. Platelets were then subjected to fluorescence-activated cell sorting (FACS) and the geometric mean of the fluorescence intensity per sample was calculated. Results are presented as means (± SEM; n = 4). NS indicates no significant difference; ***P < .001. (B) Washed platelets (2.5 × 108/mL) were resuspended in Tyrode buffer containing either 1 mM CaCl2/MgCl2 (Ca/Mg) or 1 mM EGTA and 2 mM MgCl2 (EGTA/Mg). Platelets were allowed to adhere to collagen-coated (2 mg/mL) 10-cm glass dishes to form monolayers for 30 minutes at 37°C. To obtain monolayers comprising 100% SCIPs, collagen-adherent platelets were exposed to 1 μM ionophore A23817 in the presence of calcium for 10 minutes (Iono − Ca/Mg). Nonadherent platelets were removed and the membrane lipids from the adherent platelets extracted and the level of PAF quantitatively assessed as described in “Materials and methods, Quantitation of platelet chemokines.” Results are expressed as means (± SEM; n = 3). *P < .05, **P < .01.
