MEMBRANE-DESTABILIZING PROPERTIES OF C-2-CERAMIDE MAY BE RESPONSIBLE FOR ITS ABILITY TO INHIBIT PLATELET-AGGREGATION

We have studied the effects of short-chain ceramides on platelet struc ture and function. N-Acetylsphingosine (C-2-ceramide), a cell-permeabl e short-chain analogue, and N-acetyldihydrosphingosine (C-2-dihydrocer amide), which lacks the 4-5 double bond, have been investigated. C-2-C eramide (15 mu M) inhibited ADP-induced aggregation by 50% at a platel et concentration of 1.25 x 10(8)/mL, while it took twice that concentr ation to inhibit aggregation by 50% when the platelet concentration wa s doubled. This indicates that the effect of C-2-ceramide on ADP-induc ed platelet aggregation depends on the ratio of ceramide to total plat elet lipid, with a ratio of 0.2 giving significant inhibition. C-2-Cer amide at a ceramide: lipid ratio of 0.2 caused platelets to form fenes trations and pseudopodia which were longer and thinner than those caus ed by agonists such as ADP or thrombin, C-2-Dihydroceramide had no eff ect on ADP-induced aggregation or platelet morphology at any ceramide: lipid ratio. Platelet lysis was induced by C-2-ceramide at higher cera mide:lipid ratios (0.5), whereas C-2-dihydroceramide did not induce ly sis, suggesting that C-2-ceramide is able to destabilize membranes. Th is was tested directly by assessing whether the ceramides induced leak age of 6-carboxyfluorescein from lipid vesicles, C-2-Ceramide caused n early 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 membran es was also examined by measuring changes in fluorescence anisotropy o f the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C-2-Ceramide induced a larger decrease in anisot ropy 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 pl atelet pseudopodia, platelet lysis, lipid vesicle leakage, and increas es in the fluidity of lipid vesicles all suggest that C-2-ceramide inh ibits platelet aggregation because it destabilizes the platelet membra ne. C-2-Dihydroceramide did not inhibit platelet aggregation and lacke d the nonspecific effects on membranes that C-2-ceramide possessed, su ggesting that C-2-dihydroceramide is not an appropriate control for th e nonspecific effects of C-2-ceramide.