Gaffet P, Bettache N, Bienvenüe A
URA 1856 CNRS, CP 107, Université Montpellier II, France.
Biochemistry. 1995 May 23;34(20):6762-9. doi: 10.1021/bi00020a022.
The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.
研究了钙离子载体激活人血小板过程中磷脂的重新分布动力学，以确定所观察到的磷脂外流的特异性[巴塞等人（1993年），《生物化学》32卷，2337页]。（1）进行了两项双标记实验，使用等量的自旋标记和荧光标记的磷脂酰丝氨酸与磷脂酰胆碱组合。在A23187诱导的激活过程中，50%的内部磷脂酰丝氨酸类似物迅速（t1/2 < 1分钟）重新暴露于血小板表面，而未观察到外部磷脂酰胆碱类似物的反向流入。（2）用氯丙嗪处理可使20%的外部自旋标记的鞘磷脂或自旋标记的磷脂酰胆碱内化，既不诱导血小板激活，也不干扰氨基磷脂转位酶活性或A23187诱导的激活（致密颗粒分泌和囊泡脱落）。在A23187诱导的激活过程中，先前内化的胆碱头部磷脂均未向外暴露，而无论血小板是否用氯丙嗪预处理，自旋标记的磷脂酰丝氨酸向外移动情况相似。我们的结果表明，在强烈的血小板激活过程中：（1）由于添加探针导致的内膜层磷脂过量并非其外流的原因；（2）快速的氨基磷脂外流绝对是一种单向外流，不会被胆碱头部磷脂的快速反向流入所抵消（即不会发生翻转）；（3）单向外流对氨基磷脂具有特异性，因为先前内化的鞘磷脂和磷脂酰胆碱不会向外移动。这表明氨基磷脂的特异性单向外流可能由“反向氨基磷脂转位酶”活性催化。
