The SPL knockout produces a loss of function, whereas preventing SHP-1 activation produces a gain of function. (A) Ca²⁺ mobilization. (Left) Platelets from matched WT and SPL^−/− mice stimulated with the PAR4 agonist peptide, AYPGKF. (Right) Summary of data from 3 studies (mean ± SEM). (B) Rap1 activation. Rap1-GTP levels were measured in unstimulated platelets and in platelets stimulated with AYPGKF for 5 minutes (mean ± SEM; N = 3). (C) cAMP formation. (Left) Basal cAMP concentration in SPL^−/− platelets (N = 3) and matched controls (N = 8). (Right) cAMP levels in platelets preincubated with PGI₂ and stimulated with ADP at the final concentrations indicated (mean ± SEM; N = 3). (D) Aggregation traces comparing platelets from SPL^−/− and matched control mice (WT). These data are representative of 8-10 separate experiments. (E) Time to carotid artery occlusion after a 2-minute exposure to 10% FeCl₃ in 6 SPL^−/− mice and 9 matched WT controls. (F top) CHO cells transfected with Myc-SPL (177-817), RGS18, and either WT or Y536F SHP-1. After loading with Fura-2, the cells were stimulated with thrombin and changes in the cytosolic Ca²⁺ were recorded. (F middle) Summary of 3 experiments (mean ± SEM). (F bottom) Western blot analyses showing equal protein expression in cells receiving WT and Y536F SHP-1.