Sphingosine-1-phosphate (S1P) is known to affect platelet responsiveness but the receptor mediating these effects and the mechanisms involved are poorly understood. This study was undertaken to examine S1P receptor expression in human platelets as well as potential changes associated with type 2 diabetes. S1P(2) receptor expression (Western blotting) was detected in washed human platelets from healthy volunteers. Stimulation of these platelets with exogenous S1P led to a concentration-dependent increase in intracellular Ca(2+) as well as to platelet aggregation. The S1P-induced increase in Ca(2+) was sensitive to the S1P(2) receptor antagonist JTE-013 but not the S1P(1/3) antagonist VPC23019. Both antagonists reduced the aggregation stimulated by S1P in a non-additive manner. S1P also elicited the translocation of RhoA to the membrane and RhoA activity was inhibited (by 50%) by the S1P receptor antagonists. Platelets from patients with type 2 diabetes demonstrated an attenuated aggregability to S1P as well as decreased levels of the full-length S1P(2) protein. The S1P(2) antibody used identified a 45 kDa receptor cleavage product in patients with diabetes that could also be generated from healthy human platelet lysates by the addition of the Ca(2+)-activated protease, mu-calpain. These results indicate that the S1P(2) receptor is involved in S1P-induced platelet aggregation and Rho kinase activation. Moreover, in platelets from patients with type 2 diabetes, responses to S1P are attenuated via a phenomenon attributed to the calpain-dependent cleavage of the S1P(2) receptor.