ADAP regulates VASP localization during outside-in αIIbβ3 signaling under shear flow. (A) Perfusion-fixed ADAP^+/+ (left) and ADAP^−/− (right) platelets captured onto fibrinogen were stained for VASP (green) and GP IX (red) to delineate membranes. Three-dimensional reconstructions were made from acquired stacks of 0.1-μm image slices by the use of Volocity software. Note VASP clusters near the membrane (arrowhead). (B) Quantification of VASP fluorescence at the membrane, determined from 3-dimensional images prepared as in panel A. Platelets were costained with antibodies against VASP and GPIX, and the average overlapping volume fluorescence was calculated per platelet. Results shown are the average of 3 experiments ± SEM. *P < .05. (C-D) Spreading on fibrinogen under shear flow was examined in platelets from VASP-deficient mice and their littermate controls. The average close surface contact area per platelet over the course of 1.5 minutes, quantified from RICM images acquired in real time (see “Image analysis” and the legend to Figure 2 for details), is shown in panel C. *Statistically significant increase in the area of VASP^−/− platelets relative to controls at this time point. (D) The colocalization of actin (red) and ADAP (blue) is shown. Note that actin-rich structures to which ADAP also localizes are present in both VASP^+/+ (left) and VASP^−/− (right) platelets. Results are representative of at least 3 separate experiments.