ADAP^−/− platelets have an outside-in spreading defect under shear flow. (A) Deconvolved microscopic images of adherent ADAP^+/+ (top) and ADAP^−/− (bottom) platelets fixed without cessation of flow, permeabilized, and stained with an antibody against the membrane receptor, GP IX (acquired at 100× magnification). (B) Areas of perfusion-fixed platelets were quantified by the use of Image Pro Plus software. Results are the average of 5 separate experiments ± SEM (*P < .05). (C-G). Images of unfixed spreading platelets under shear flow conditions were acquired in real time by RICM. (C) Close platelet/surface contacts (dark regions) in platelets captured onto fibrinogen after 1.5 minutes of flow (acquired at 60× magnification). (D) Close platelet/surface contact areas in an image acquired 20 seconds after initiation of flow: shown as the original unthresholded image (top) or after thresholding and binarization (bottom) for quantification. (E) Average close surface contact area per platelet as a function of time upon interaction with immobilized fibrinogen (F-G) or a dmA1A2 VWF fragment (quantified with Image Pro Plus). Results are representative of at least 3 separate experiments ± SEM. (F) RICM images of close platelet-surface contact areas in unfixed platelets captured onto fibrinogen and stimulated with a PAR4 agonist for 1 minute under shear flow. (G) Quantification of close surface contact areas in platelets captured onto fibrinogen and directly stimulated with the extrinsic integrin activator, MnCl₂ for 1 minute under shear flow. Results are the average of 3 separate experiments ± SEM (*P < .05; **P < .01).