Inside-out αIIbβ3 signals require the N-terminal β3 NPxY motif. (A) ADP-stimulated fibrinogen binding by wild-type and mutant β3 integrins in platelets. β3-deficient platelets expressing wild-type (WT) and mutant β3 integrins were exposed to 50 μM ADP (red lines) or buffer (blue lines) and the binding of soluble fibrinogen used to measure inside-out signal transduction. Note the complete loss of inside-out integrin activation in β3Y747A platelets. The experiment shown is representative of more than 15 experiments in 7 individual animals performed for each mutant. (B) Relative inside-out signal transduction by β3 mutant receptors. To obtain a relative measure of inside-out signaling, the mean fluorescent intensity of bound fibrinogen was normalized to the mean fluorescent intensity of surface β3 expression. P values for values obtained in platelets reconstituted with wild-type versus individual mutant β3 integrins were calculated. Asterisk indicates P < .05; n = 7 animals studied for each group; error bars indicate mean ± standard deviation.