Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C-2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C-2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C-2-Ceramide (15 mu M) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C-2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C-2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin, C-2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C-2-ceramide at higher ceramide:lipid ratios (0.5), whereas C-2-dihydroceramide did not induce lysis, suggesting that C-2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles, C-2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C-2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C-2-ceramide at a ratio of 0.2 (similar to 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C-2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C-2-Dihydroceramide did not alter membrane fluidity, The ability of C-2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C-2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C-2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C-2-ceramide possessed, suggesting that C-2-dihydroceramide is not an appropriate control for the nonspecific effects of C-2-ceramide.