Thrombin-generation potential of RBCs. WB was drawn via phlebotomy into 0.1 mg/mL of CTI and washed as described in “Methods.” Washed RBCs were counted and stored at 37°C. (A) IIase (20nM fVa, 200pM fXa final concentrations) was assembled in samples containing 250nM PCPS (■), 40% washed RBCs (●), RBCs pretreated with 40nM bovine lactadherin (*), RBC wash supernatant (▾), or no exogenous surface (▴), and the reactions were initiated with a solution containing prothrombin and AT (1.4 and 3.4μM final concentrations, respectively). Aliquots were removed at selected time points, quenched with EDTA and Phe-Pro-Arg chloromethylketone, and the soluble fraction was analyzed via αTAT ELISA and Western blotting. Data are shown as the means ± SEM (n = 5). Western blotting was performed on nonreduced samples probing with the burro anti-AT polyclonal Ab. Lane 1 shows the mTAT/mTAT des F1/αTAT standard; lanes 2-, 0- to 20-minute time-course samples. (B) Representative Western blot from PCPS prothrombinase time course. (C) Representative Western blot from RBC prothrombinase time course. (D) Representative Western blot from a PMA-treated RBC prothrombinase time course.