ADAP−/− platelets exhibit unstable adhesion in the presence of shear stress. (A) In vitro adhesion of platelets labeled with 10 μg/mL mepacrine to visualize platelets, under shear flow. Quantification of unfixed ADAP+/+ or ADAP−/− platelets captured onto fibrinogen at γ = 500 s−1. Movie clips were prepared to cover the period 10 to 80 seconds after flow initiation, and images were processed at 6 fps. The first frame was subtracted to remove already attached platelets. All platelets perfused over fibrinogen under shear flow, which appeared on the surface within the first 30 seconds, were individually tracked, and platelets remaining surface-bound for a period of 2 to 30 seconds are depicted as a percent of total surface-contacting cells. Results presented are the average of 4 separate experiments ± SEM (*P < .05; **P < .01, paired Student t test). (B) Platelet adhesion to fibrinogen under static conditions. Washed platelets were plated for 60 minutes in wells coated with increasing concentrations of fibrinogen, and adhesion was determined by an alkaline phosphatase assay. Results show the percent of total platelets added that remained adherent, and are an average of at least 3 separate experiments ± SEM. (C) Mean surface areas of platelets spread onto fibrinogen under static conditions. Washed platelets were plated onto fibrinogen with or without 250μM PAR4-activating peptide or 0.5mM MnCl2, allowed to spread for 60 minutes at 37°C, then fixed, permeabilized and stained for F-actin with the use of rhodamine phalloidin. Results presented are the average of 3 separate experiments ± SEM.
