Membrane-Destabilizing Properties of C2-Ceramide May Be Responsible for Its Ability To Inhibit Platelet Aggregation

• Published in: Biochemistry (Easton) 1998-02, Vol.37 (7), p.2059-2069
• Main Authors: Simon, Carl G, Gear, Adrian R. L
• Format: Article
• Language: English
• Subjects: Adenine - metabolism; Blood Platelets - drug effects; Blood Platelets - metabolism; Blood Platelets - ultrastructure; Cell Membrane Permeability - drug effects; Humans; Membrane Fluidity - drug effects; Microscopy, Electron, Scanning; Platelet Aggregation - drug effects; Platelet Aggregation Inhibitors - chemistry; Platelet Aggregation Inhibitors - pharmacology; Pseudopodia - drug effects; Sphingosine - analogs & derivatives; Sphingosine - chemistry; Sphingosine - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9710636

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4−5 double bond, have been investigated. C2-Ceramide (15 μM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 × 108/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-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. C2-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. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (∼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. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-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 C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.