Bassé F, Gaffet P, Rendu F, Bienvenüe A
URA 530 CNRS, CP 107, Université Montpellier II, France.
Biochemistry. 1993 Mar 9;32(9):2337-44. doi: 10.1021/bi00060a027.
After incorporation of spin-labeled phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine analogues in the outer leaflet of the plasma membrane in resting platelets, more than 90% amino-head analogues accumulated within 30 min in the inner leaflet by aminophospholipid translocase activity, while choline analogues mostly remained on the outer leaflet. Platelets were then activated by thrombin or Ca2+ ionophore A23187. No outward movement of internally located spin-labeled aminophospholipids was observed during thrombin-induced activation, whereas the influx of externally located probes increased slightly. During A23187-mediated activation, similar slightly increased influx was observed, while 40-50% of the initially internally located aminophospholipids could then be extracted from the outer leaflet. This sudden exposure on the outer face was dependent on an increase in intracellular Ca2+ and achieved in less than 2 min at 37 degrees C. Inhibition of translocase activity by N-ethylmaleimide did not induce any aminophospholipid outflux. When probes were incorporated on the outer face of the plasma membrane in resting platelets, they were still fully accessible from the extracellular medium after A23187-induced activation. Moreover, they were distributed between the vesicles and remnant platelets in proportion to the external membrane phospholipidic content in each structure. This suggested that no scrambling of plasma membrane leaflets occurred during the vesicle blebbing. Moreover, the spin-labeled aminophospholipids exposure rate and amplitude were unchanged when vesicle formation was inhibited by the calpain inhibitor calpeptin. These results indicate that loss of asymmetry thus inducing generation of a catalytic surface is not the consequence of vesicle formation. Conversely, we propose that vesicle shedding is an effect of PL transverse redistribution and calpain-mediated proteolysis during activation.
在静息血小板质膜外小叶中掺入自旋标记的磷脂酰胆碱、磷脂酰丝氨酸和磷脂酰乙醇胺类似物后，超过90%的氨基头部类似物在30分钟内通过氨基磷脂转位酶活性积累到内小叶中，而胆碱类似物大多仍留在外小叶。然后用凝血酶或Ca2+离子载体A23187激活血小板。在凝血酶诱导的激活过程中，未观察到内部定位的自旋标记氨基磷脂向外移动，而外部定位的探针流入量略有增加。在A23187介导的激活过程中，观察到类似的略有增加的流入量，而最初位于内部的氨基磷脂的40-50%随后可从外小叶中提取出来。这种在外表面的突然暴露依赖于细胞内Ca2+的增加，并在37℃下不到2分钟内实现。N-乙基马来酰亚胺对转位酶活性的抑制未诱导任何氨基磷脂外流。当探针掺入静息血小板质膜的外表面时，在A23187诱导的激活后，它们仍可从细胞外培养基中完全获取。此外，它们在囊泡和残余血小板之间的分布与每个结构中的外膜磷脂含量成比例。这表明在囊泡出泡过程中质膜小叶没有发生翻转。此外，当用钙蛋白酶抑制剂钙肽素抑制囊泡形成时，自旋标记的氨基磷脂暴露率和幅度没有变化。这些结果表明，不对称性的丧失从而诱导催化表面的产生不是囊泡形成的结果。相反，我们提出囊泡脱落是激活过程中磷脂横向重新分布和钙蛋白酶介导的蛋白水解的结果。
