The small guanine-nucleotide-binding protein Rap1 plays a key role in platelet aggregation and hemostasis and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. Yeast-two-hybrid screening revealed 14-3-3 and synaptotagmin-like protein 1 (Slp1) as binding partners of Rap1GAP2. Using GST pull down assays in platelets and immunoprecipitation studies in transfected HeLa cells we could show that ADP and thrombin induce 14-3-3 binding to Rap1GAP2 whereas prostacyclin and nitric oxide induced platelet inhibition involves the detachment of 14-3-3 from Rap1GAP2. The interaction of Rap1GAP2 and 14-3-3 is regulated by two phosphorylation events at serines 7 and 9 of Rap1GAP2. In transfected HeLa cells 14-3-3 binding abolished Rap1GAP2 mediated inhibition of cell adhesion suggesting that the GTPase-activating function of Rap1GAP2 towards Rap1 might be regulated by 14-3-3. In contrast, binding of Slp1 appears to link Rap1GAP2 to dense granule release. Slp1 contains a Rab27-binding domain and by co-immunoprecipitation we demonstrate that Rap1GAP2, Slp1 and Rab27 form a trimeric complex in platelets. By mutagenesis studies we mapped the Rap1GAP2/Slp1 interaction sites to a new TKXT motif within Rap1GAP2 and to the C2A domain of Slp1. Purified recombinant Slp1 dose-dependently decreased dense granule secretion in streptolysin-O permeabilized platelets treated with calcium or GTPgammaS. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and could reverse the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets. Deleting the Slp1-binding TKXT motif in Rap1GAP2 abolished the effect of Rap1GAP2 on secretion. We conclude that Slp1 inhibits dense granule secretion in platelets whereas Rap1GAP2 has a modulatory function in secretion. Our data suggest possible connections between the regulation of granule secretion and aggregation in platelets.