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首页> 外文期刊>Biochemistry >PAR-1-stimulated factor IXa binding to a small platelet subpopulation requires a pronounced and sustained increase of cytoplasmic calcium
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PAR-1-stimulated factor IXa binding to a small platelet subpopulation requires a pronounced and sustained increase of cytoplasmic calcium

机译：PAR-1刺激的因子IXa与少量血小板亚群的结合需要细胞质钙的持续显着增加

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摘要

We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM- loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5-20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 AM BAPTA/AM (K-d 160 nM) had minimal effects, 100 mu M 5,5'-dimethylBAPTA/AM (K-d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.
机译：我们先前曾报道过，由于其他实验室的证实，只有PAR-1刺激的血小板亚群与凝血因子IXa结合。由于钙的变化与促凝性氨基磷脂的暴露有关，因此我们现在通过测量PAR-1刺激后富拉红/ AM加载血小板的荧光变化来检查该亚群中的钙通量。虽然所有血小板的荧光变化都表明钙从内部储存中释放出来，并且外部钙大量涌入，但血小板亚群显示钙瞬变显着增加15 s，正因子IXa结合持续2分钟，钙瞬变持续45分钟。用Xestospongin C预处理血小板以抑制IP3介导的致密小管钙释放，以及不可渗透的钙通道阻滞剂硝苯地平，SKF96365或LaCl3的存在，可抑制PAR-1诱导的具有明显钙瞬变，因子IXa结合的亚群的发育，以及FXa生成的血小板支持，提示钙从内部储存中释放和细胞外钙流入的重要性。当在添加钙之前在EDTA中刺激血小板5-20分钟时，因子IXa结合位点在较小的亚群上发育，但速率不变，表明钙通道的持续开放和结合位点暴露所需的信号元件的持续可用性。虽然用100 AM BAPTA / AM（Kd 160 nM）预处理血小板的作用很小，但100μM 5,5'-二甲基BAPTA / AM（Kd 40 nM）完全抑制了血小板亚群的出现和功能，表明了细胞质钙的少量增加。我们得出的结论是，在血小板亚群中PAR-1-刺激因子IXa结合位点的发育取决于钙从内部存储中释放出来，从而导致持续且明显的钙瞬变。

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