Kuroda K, Ozaki Y, Qi R, Asazuma N, Yatomi Y, Satoh K, Nomura S, Suzuki M, Kume S
Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Japan.
J Immunol. 1995 Nov 1;155(9):4427-36.
Anti-human platelet CD9 mAb, NNKY1-19, induced platelet activation in a Fc gamma RII-dependent manner in terms of aggregation and secretion of intracellular granule contents. These responses were considerably suppressed by aspirin. [Ca2+]i elevation in the absence of extracellular Ca2+ ([Ca2+]e), which represents the amount of Ca2+ released from intracellular Ca2+ ([Ca2+]i) stores, was also greatly reduced, whereas Ca2+ influx was sustained at similar levels. We thus investigated the mechanism that leads to the opening of Ca2+ channels in platelets incubated with aspirin. IP3 production and Ca2+ efflux were below detectable levels. 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading of platelets to chelate [Ca2+]i did not reduce Ca2+ influx, as assessed by 45Ca2+ measurement. These findings suggested that NNKY1-19 induces Ca2+ channels to open without [Ca2+]i mobilization or by depleting the [Ca2+]i stores. The magnitude of Ca2+ influx was evaluated by adding [Ca2+]e to a platelet suspension activated by various agonists in the absence of [Ca2+]e. The dose dependence of the Ca2+ influx on [Ca2+]e concentrations differed according to the mode of activation. The ED50 value of Ca2+ after thrombin or thapsigargin stimulation was 0.6 mM, whereas that of NNKY1-19 activation was about 3 mM. The addition of anti-Fc gamma RII mAb, IV.3, even 10 min after the initiation of platelet activation induced by NNKY1-19, inhibited the Ca2+ influx. These findings suggest that the Fc gamma RII-dependent activation of platelets induced by NNKY1-19 directly opens Ca2+ channels, which are distinct from those opened by thrombin or thapsigargin.
抗人血小板CD9单克隆抗体NNKY1-19在血小板聚集和细胞内颗粒内容物分泌方面，以FcγRII依赖的方式诱导血小板活化。这些反应被阿司匹林显著抑制。在无细胞外Ca2+（[Ca2+]e）情况下细胞内Ca2+（[Ca2+]i）升高，这代表从细胞内Ca2+（[Ca2+]i）储存释放的Ca2+量，也大大降低，而Ca2+内流维持在相似水平。因此，我们研究了在与阿司匹林孵育的血小板中导致Ca2+通道开放的机制。IP3产生和Ca2+外流低于可检测水平。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸加载血小板以螯合[Ca2+]i，通过45Ca2+测量评估，并未降低Ca2+内流。这些发现表明，NNKY1-19诱导Ca2+通道开放而不伴有[Ca2+]i动员或通过耗尽[Ca2+]i储存。通过在无[Ca2+]e情况下向由各种激动剂激活的血小板悬液中添加[Ca2+]e来评估Ca2+内流的幅度。Ca2+内流对[Ca2+]e浓度的剂量依赖性根据激活模式而不同。凝血酶或毒胡萝卜素刺激后Ca2+的半数有效剂量（ED50）值为0.6 mM，而NNKY1-19激活后的ED50值约为3 mM。即使在NNKY1-19诱导血小板活化开始10分钟后添加抗FcγRII单克隆抗体IV.3，也抑制了Ca2+内流。这些发现表明，NNKY1-19诱导的FcγRII依赖的血小板活化直接打开Ca2+通道，这些通道与凝血酶或毒胡萝卜素打开的通道不同。
