Wang C N, Chen J C, Shiao M S, Wang C T
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, China.
Biochim Biophys Acta. 1989 Nov 17;986(1):151-60. doi: 10.1016/0005-2736(89)90285-x.
Incubation of gel-filtered human platelets in ganodermic acid S (lanosta-7,9(11),24-trien-3 beta,15 alpha-diacetoxy-26-oic acid) showed that within a min 80% of the agent was taken up by the cells. The process of uptake was a simple diffusion, and the partition coefficient was about 10(5). The agent caused platelet aggregation at a concentration above 20 microM. Above the threshold, the extent of cell aggregation was in a linear relationship to the agent concentration. Also, the % of cell aggregation was comparable to the elevation of: (1) cytosolic free Ca2+ concentration [( Ca2+]i); (2) protein phosphorylation; and (3) serotonin release. Also, it was correlated with the change in the interconversion of phosphoinositides. Moreover, platelets in various concentrations of ganodermic acid S appeared to show different time-course profiles in the changes of [32P]phosphoinositides and [32P]phosphatidic acid (PA). Upon addition of the agent, platelets showed an initial increase in all of the [32P]phosphoinositides, and then the level of each kind of phosphoinositide decreased sequentially in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol (PI). Below the aggregation threshold, platelets showed neither the resynthesis of [32P]PIP2 and [32P]PIP nor the accumulation of [32P]PA. However, at 25 and 50 microM, platelets showed not only the resynthesis of [32P]PIP2 and [32P]PIP but also the accumulation of [32P]PA. Interestingly, at 100 microM ganodermic acid S, platelets did not show the resynthesis of [32P]PIP2 and [32P]PIP. In this case, the level of [32P]PA accumulation and that of [32P]PI decrease were less than those found in platelets at 50 microM ganodermic acid S. The results suggested that ganodermic acid S caused the activation of PIP2 hydrolysis. Scanning electron microscopy (scanning EM) revealed that the morphology of platelets below the aggregation threshold appeared to be spiculate discoid shape. Above the threshold, the cells rounded up to spiculate irregular forms, which showed an elongation of filopodia after prolonged 30-s incubation. In addition, platelets at greater than or equal to 50 microM ganodermic acid S showed the occurrence of membrane vesiculation. Hence, the incorporation of ganodermic acid S into platelet membrane resulted in the change of membrane morphology.
将凝胶过滤的人血小板与灵芝酸S（羊毛甾-7,9(11),24-三烯-3β,15α-二乙酰氧基-26-酸）一起孵育，结果显示在1分钟内80%的该试剂被细胞摄取。摄取过程为简单扩散，分配系数约为10⁵。该试剂在浓度高于20μM时可引起血小板聚集。高于阈值时，细胞聚集程度与试剂浓度呈线性关系。此外，细胞聚集百分比与以下各项的升高相当：(1) 胞质游离Ca²⁺浓度[Ca²⁺]i；(2) 蛋白质磷酸化；(3) 5-羟色胺释放。它还与磷酸肌醇相互转化的变化相关。此外，处于不同浓度灵芝酸S中的血小板在[³²P]磷酸肌醇和[³²P]磷脂酸（PA）变化方面似乎呈现出不同的时间进程曲线。加入该试剂后，血小板中所有[³²P]磷酸肌醇均出现初始增加，然后磷脂酰肌醇4,5-二磷酸（PIP2）、磷脂酰肌醇4-磷酸（PIP）和磷脂酰肌醇（PI）中的每种磷酸肌醇水平依次下降。在聚集阈值以下，血小板既未出现[³²P]PIP2和[³²P]PIP的再合成，也未出现[³²P]PA的积累。然而，在25μM和50μM时，血小板不仅出现了[³²P]PIP2和[³²P]PIP的再合成，还出现了[³²P]PA的积累。有趣的是，在100μM灵芝酸S时，血小板未出现[³²P]PIP2和[³²P]PIP的再合成。在这种情况下，[³²P]PA积累水平和[³²P]PI下降水平低于50μM灵芝酸S处理的血小板。结果表明灵芝酸S导致了PIP2水解的激活。扫描电子显微镜（扫描电镜）显示，聚集阈值以下的血小板形态呈有刺的盘状。高于阈值时，细胞变圆形成有刺的不规则形态，在延长孵育30秒后显示丝状伪足伸长。此外，灵芝酸S浓度大于或等于50μM时的血小板出现了膜泡化现象。因此，灵芝酸S掺入血小板膜导致了膜形态的改变。
