Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

In order to isolate pre-beta 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre-beta 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double-dimension gel electrophoresis. Apo A-I in pre-beta 1 HDL represented 17.6% of total apo A-I. Stokes' radii corresponded to 3.42 nm in follicular fluid pre-beta 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre-beta 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre-beta 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidylcholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre-beta 1 HDL total weight. Thus, a lipid core is present in pre-beta 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism.