Schootemeijer A, Gorter G, Tertoolen L G, De Laat S W, Akkerman J W
Department of Haematology, University Hospital Utrecht, The Netherlands.
Biochim Biophys Acta. 1995 May 24;1236(1):128-34. doi: 10.1016/0005-2736(95)00104-b.
The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-01, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol/phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC14 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 x 10(-9) cm2/s at a low C/P decreasing to 2.55 x 10(-9) cm2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22 degrees C. There were only little differences in phosphatidylinositol metabolism, Ca2+ influx or mobilization and prostaglandin I2-induced formation of cyclic AMP. At 37 degrees C, cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20-0.31 the influence on signal processing is minor.
细胞膜的流动性被认为会影响血小板的反应性。我们通过亲脂性探针DiIC14的光漂白后荧光恢复（FRAP）技术在单个细胞中测量了膜流动性。由于血小板对于该技术来说太小，我们使用了人巨核母细胞系MEG-01，它与血小板具有许多共同特性。将MEG-01细胞用辛伐他汀或甲羟戊酸培养44小时以改变胆固醇含量，从而能够分析胆固醇/磷脂比（C/P）在0.20至0.31之间时的信号处理情况。DiIC14的扩散与C/P比呈负相关，低C/P时横向扩散系数（D）为3.28×10⁻⁹ cm²/s，高C/P比时降至2.55×10⁻⁹ cm²/s。可移动部分为65%，在不同C/P比下保持恒定。在22℃用10 U/ml凝血酶刺激后测量脂质扩散与信号处理之间的关系。在磷脂酰肌醇代谢、Ca²⁺内流或动员以及前列腺素I2诱导的环磷酸腺苷形成方面只有很小的差异。在37℃时，高C/P比的细胞显示出磷脂酰肌醇代谢增加，但这些差异对Ca²⁺反应没有重大影响。这些数据表明，在巨核母细胞中，脂质的横向扩散与C/P比呈负相关，但在0.20 - 0.31范围内对信号处理的影响较小。
