Abstract 453: The S1p2 Receptor Is Expressed In Human Platelets,is Linked To The Rhoa-rho Kinase Pathway And Is Degraded By μ-calpain In Type 2 Diabetes
Sphingosine-1-phosphate (S1P) is a bioactive lipid involved in multiple biological processes; it is stored in platelets and released upon activation. Although S1P is known to exert extracellular effects on platelets such as shape change and aggregation, the receptor that mediates these effects and the molecular mechanisms involved are poorly understood. The aim of the present study was to characterize the S1P receptor expressed in platelets as well as the molecular mechanism underlying S1P-mediated platelet activation and eventual changes associated with the development of type 2 diabetes. We found that washed human platelets from healthy volunteers expressed mainly the S1P2 receptor and to a lesser extent the S1P1 receptor. Stimulation of these platelets with exogenous S1P led to a concentration-dependent increase in intracellular calcium as well as to platelet aggregation. The latter response was accompanied by the translocation of RhoA to the membrane and was inhibited (by 50%, P<0.01) by the Rho kinase inhibitor Y27632 (1 μmol/L). Neither the PKC inhibitor, Ro-318220, the MAPK inhibitor, PD98059, nor the tyrosine kinase inhibitor, tyrphostin A-23, affected the S1P-mediated platelet aggregation. Serum obtained from patients with type 2 diabetes contained 1.5-fold higher levels (P<0.01) of S1P that from healthy individuals. However, platelets from patients with type 2 diabetes demonstrated an attenuated aggregability to S1P as well as decreased levels of the S1P2 protein. The latter was found to be a target of the calcium-dependent protease μ-calpain which we found to be highly activated in platelets from patients with type 2 diabetes. Finally, stimulation of platelets from healthy donors with high concentration of S1P led to the activation of μ-calpain. Taken together, our results show that S1P can elicit platelet aggregation by activating the S1P2 receptor and the RhoA-Rho kinase pathway. In platelets from patients with type 2 diabetes, in which the circulating concentration of S1P is elevated, responses to S1P are damped, via a phenomenon that correlates with the activation of μ-calpain and the subsequent degradation of the S1P2 receptor. The latter may represent an auto-protective mechanism against exaggerated platelet activation in diabetes.