Evaluation of anionic phospholipid availability by thrombin generation and annexin V binding on the surface of polarized Caco-2 epithelial cells. (A) Thrombin generation: Polarized cells in transwells were incubated with factor Va (20 nmol/L) and Xa (2 nmol/L) on both the apical and basolateral side for 15 minutes at 37°C before prothrombin (1,400 nmol/L) was added. At various time points, 20-μL aliquots were removed from the apical or the basolateral side and assayed for thrombin generated. Data are presented as initial rates of thrombin generation (n = 4). (B) Annexin V binding: Polarized Caco-2 cell layers were incubated with 10 nmol/L of125I-annexin V on the apical and basolateral side at 4°C. Cell-surface–associated radioactivity was removed from the apical (□) and basolateral (▪) surfaces by washing with an EDTA-containing buffer. Nonspecific binding was determined in parallel duplicate wells in which the cells were preincubated for 30 minutes at 4°C with 50-fold molar excess of cold annexin V (500 nmol/L) before adding 125I-annexin V (n = 3).
