RESUMO
Plasma lipid composition, platelet aggregation, cholesterol (Ch)/glycoprotein IIb-IIIa (GP) and phospholipid (Ph)/GP molar ratios, fatty acid composition and structural order (1, 6-diphenyl-1, 3, 5-hexatriene (DPH) fluorescence anisotropy at 35 degrees C (r(DPH,35)) of human platelet plasma membranes (HPPM) were measured in four DPH,35 groups of hyperlipidemic patients (II: plasma Ch < 250 mg/dl and TG (triglycerides) <220 mg/dl, n = 21; III: Ch > 250 mg/dl and TG < 220 mg/dl, n = 23; IV: Ch < 250 mg/dl and TG > 220 mg/dl, n = 18; and V: Ch > 250 mg/dl and TG > 220 mg/dl, n = 12) and compared with those of the control group (I). Our results were: (i) in groups III, IV and V the HPPM (Ch + Ph)/GP molar ratio increased 7.0+/-7.7% (mean SD); (ii) the Ph/GP molar ratio increased significantly in groups III, IV and V, but most in IV and V, while the Ch/GP molar ratio increased only in groups III and V; (iii) the mean relative increase of Ch with respect to Ph in the HPPM of groups III, IV and V was 140% 21% and 54%, respectively; (iv) the Ch/GP molar ratio was correlated with LDL-Ch (0.41+/-0.16, P < 0.002, n = 55, for all the subjects and 0.60+/-0.11, P < 2.10(-4), n = 33, for subjects with TG < 220 mg/dl), however, it was totally uncorrelated with HDL-Ch; (v) the HPPM Ch/Ph molar ratio was positively correlated with plasma Ch (r = 0.51+/-0.08, P < 1.10(-6), n = 83) and with (LDL + HDL) Ch (r = 0.64+/-0.07, P < 1.10(-6), n = 73), the former correlation increased significantly ( r = 0.67+/-0.07, P < 1.10(-6), n = 53) when done only for subjects with TG < 220 mg/dl; (vi) the Ch/Ph molar ratio was only increased in group III (0.70+/-0.03, P < 3.10(-5), n = 23) and decreased in group IV (0.62+/-0.02, P < 0.001, n = 18); (vii) the fatty acid/GP molar ratio was significantly increased in groups IV and V, however, a significant absolute and relative increase of C16:0 and C18:1 was observed only in severe hypertriglyceridemia (> 500 mg/dl), together with a relative decrease of C18:0 and C20:4 ( n - 6); (viii) the HPPM structural order, as probed by r(DPH,35), was negatively correlated with DPH,35 plasma TG (r =- 0.61+/-0.10, P < 4.10(-5), n = 39), the Ph/GP molar ratio (r =-0.58+/-0.10, P < 2.10(-4), n = 39) and the the (C18:1 + C18:2))/GP molar ratio (r =- 0.80+/-0.05, P < 1.10(-6), n = 39), however, it was independent of plasma and HPPM Ch; (ix) the higher HPPM Ch/Ph molar ratio in group III was associated (r = 0.58+/-0.12, P < 0.005, n = 22) with a moderately higher platelet reactivity to collagen. We conclude that Ch and Ph were distinctly incorporated to HPPM in the different groups of hyperlipidemia and, therefore, that the absolute increase of Ch and Ph was more informative to understand the structural and functional modifications of the HPPM in hyperlipidemias, than the Ch/Ph molar ratio. On the other hand, the r was sensitive to the DPH,35 increase in the content of HPPM Ph and C18:1 + C18:2 and it was insensitive to the increase in the Ch content.
RESUMO
The total lipids of the human platelet plasma membrane (HPPM) from 50 ml of blood of healthy subjects were extracted, quantified and related to the mass content of the major intrinsic membrane protein, the glycoprotein IIb/IIIa (GPIIb/IIIa). The HPPM total lipid/GPIIb/IIIa weight ratio determined was 5.40 ± 0.20, independently of the membrane washing procedure used, with the cholesterol/GPIIb/IIIa and phospholipid/GPIIb/IIIa molar ratios of 800 ± 50 and 1200 ± 40, respectively. If the distribution of lipids around each intrinsic protein were proportional to its mass, the lipids around a molecule of GPIIb/IIIa will occupy about 120 nm(2) of the membrane plane, which is about one and a half times the cross-sectional area of the extracellular head of GPIIb/IIIa, as estimated by electron microscopy. The lipid extracts were further subjected to thin-layer chromatography to separate and quantify the different phospholipid fractions, the free fatty acids and the neutral lipid fraction and the distribution of fatty acids in each fraction was determined by gas chromatography after methanolysis. The phospholipid molar distribution was SPM(22.3 ± 0.9%), PC(36.2 ± 1.0%), PE(24.9 ± 0.9%), PS(12.1 ± 0.6%) and PI(4.5 ± 0.4%) and the free fatty acid fraction represented 2.9 ± 0.4% of the total fatty acids in HPPM. The fatty acid chain length ranged from 14 to 24 carbons, comprising unsaturated fatty acids (47.3% molar per cent of the total) of which 40.7 ± 2.0% were monosaturated and 40.7 ± 0.9% tetraunsaturated. Palmitic, stearic, oleic and arachidonic acids represent 66% of the total fatty acids of HPPM, being: 68.9 ± 5.3% of palmitic acid and 63.3 ± 6.9% of oleic acid in PC; 50.9 ± 3.8% of arachidonic acid in PE; and 30.5 ± 2.4% of stearic acid in PS. We discuss the methodological modifications and the new data in relation with the major differences in HPPM lipid composition found in the literature. The data obtained provides a comprehensive and accurate description of the lipid composition of HPPM on which to rely as a reference for basic and medical studies.
