Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E(1) (PGE(1)) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE(1)-mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE(1)-stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromo-cAMP, indicating that it may regulate cAMP-mediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. Moreover, inhibition of Src kinases blocked TSP-1-mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway.