Liu Guoxing, Liu Guilai, Chen Hong, Borst Oliver, Gawaz Meinrad, Vortkamp Andrea, Schreiber Rainer, Kunzelmann Karl, Lang Florian
Cell Physiol Biochem. 2015;37(5):1934-44. doi: 10.1159/000438554. Epub 2015 Nov 20.
BACKGROUND/AIMS: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume.
Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter.
Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets.
Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.
背景/目的：广泛表达的钙离子激活氯离子通道Ano6参与刺激细胞膜磷脂翻转。Ano6功能缺陷是斯科特综合征的病因，这是一种遗传性出血性疾病，其特征为质膜磷脂翻转受损。至少在理论上，斯科特综合征的出血性疾病可能是由血小板功能受损导致的。血小板激活剂包括凝血酶和胶原相关肽（CRP），它们可引发胞质钙离子活性（[Ca2+]i）升高、活性氧（ROS）生成、脱颗粒、整合素激活，以及细胞收缩和细胞膜磷脂翻转。因此，本研究探讨了Ano6是否会改变激活诱导的胞质钙离子活性（[Ca2+]i）变化、脱颗粒（P-选择素暴露）、整合素激活、血小板表面磷脂酰丝氨酸暴露以及血小板体积。
将缺乏Ano6的小鼠（ano6-/-）的血小板与相应野生型小鼠（ano6+/+）的血小板进行比较。通过Fluo-3荧光估计[Ca2+]i，通过DCFDA荧光估计ROS，通过P-选择素丰度估计脱颗粒，通过αIIbβ3整合素丰度估计整合素激活，通过膜联蛋白-V结合估计磷脂酰丝氨酸丰度，通过前向散射估计细胞体积。
ano6-/-小鼠血液中的血小板数量略高于ano6+/+小鼠。在未激活的情况下，ano6-/-和ano6+/+血小板的[Ca2+]i和体积相似，并且两种基因型的ROS丰度、P-选择素丰度、αIIbβ3整合素激活和磷脂酰丝氨酸暴露均可忽略不计。凝血酶（0.01 U/ml）和CRP（2或5 μg/ml）可增加[Ca2+]i、ROS丰度、血小板脱颗粒、αIIbβ3整合素激活，并引发膜联蛋白-V结合以及细胞收缩，所有这些效应在ano6-/-血小板中比在ano6+/+血小板中更不明显。
Ano6基因敲除减弱了凝血酶和CRP诱导的血小板激活和凋亡。
