Effect of mild acid wash on annexin V binding and thrombin generation on polarized Caco-2 cells. (A) Annexin V binding: The cells were treated with mild acid as described in the legend to Fig5. Cells were then incubated with 10 nmol/L of ¹²⁵I-annexin V on the apical and basolateral side at 4°C. Total cell-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 a 50-fold excess of cold annexin V (500 nmol/L) before adding ¹²⁵I-annexin V. Specific binding (total-nonspecific) on the apical (□) and basolateral (▪) surfaces are presented (n = 4). (B) Thrombin generation: The polarized epithelial Caco-2 cells in transwells were subjected to a mild acid wash (0.1 mol/L glycine, pH 3.0) or to buffer B (control) on the apical or basolateral for 3 minutes. After washing, the cells were incubated with factor Va (20 nmol/L) and factor 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 samples were removed from the apical (open symbols) or the basolateral (closed symbols) side and assayed for the amount of thrombin generated (n = 4).