Slp1 and Rap1GAP2 effects on platelet secretion are not mediated by Rap1. (A) Ca²⁺-induced dense granule secretion after incubation of permeabilized platelets with Rap1. Permeabilized platelets were incubated with 1 μM of either BSA as control or purified recombinant native Rap1b or Rap1b loaded with GTP. Then, platelets were stimulated with Ca²⁺ for 1 minute. Baseline and Ca²⁺-induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 5 independent experiments performed in triplicate. (B) In vitro GAP assay. Epitope-tagged Slp1, Rap1GAP2, and Rap1GAP2 in complex with Slp1 were expressed in HeLa cells and purified using tag-specific affinity agarose. In parallel, His₆-tagged Rap1b was purified from E coli and loaded with [³²P]-GTP. Precipitated Slp1 and Rap1GAP2 proteins were added to the GTP-loaded Rap1b, and reactions were incubated at 25°C. Aliquots were removed at the indicated time points, and amounts of released [³²P] were determined by liquid scintillation counting and plotted as a percentage of input Rap1b-bound [³²P]-GTP counts. Data are the mean ± SE of 3 independent experiments performed in triplicate.