Yang X, Disa J, Rao A K
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Thromb Res. 1992 Mar 1;65(4-5):549-58. doi: 10.1016/0049-3848(92)90205-o.
Guanosine 5'-triphosphate (GTP) and its nonhydrolyzable analogs, such as guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), induce several responses in platelets including secretion, production of inositol 1,4,5-triphosphate (IP3) and mobilization of Ca2+ from intracellular sites. Because IP3 is well established as a second messenger in mobilizing Ca2+ from intracellular stores it has been generally assumed that Ca2+ release by GTP/GTP gamma S in platelets is mediated by IP3. However, studies in neuronal, hepatic and smooth muscle cells have suggested that IP3 and GTP/GTP gamma S activate Ca2+ release by distinct mechanisms and that IP3-independent mechanisms mediate GTP/GTP gamma S-induced Ca2+ release. In several tissues heparin inhibits binding of IP3 and blocks IP3-stimulated Ca2+ release in a competitive and specific manner. In the present studies, IP3 and GTP gamma S induced Ca2+ release and their relationship was examined in human platelets using heparin as a probe. In saponin permeabilized platelets, IP3 (0.05-5 microM) induced a prompt, dose-dependent release of Ca2+ (EC50 0.5 microM). GTP gamma S (1-50 microM) released Ca2+ in a dose-dependent manner with EC50 of 2 microM but with a time lag of 30-90 seconds. Exposure of platelets to 1 microM IP3 following a submaximal response with GTP gamma S (1 microM) resulted in a further increase in Ca2+ release but no further increase was noted on adding 1 microM IP3 following a maximal response with GTP gamma S (10 microM); similar findings were noted on reversing the order of addition of GTP gamma S and IP3 suggesting that these effectors release Ca2+ from the same source. IP3 (0.5 microM) induced Ca2+ release was blocked by low molecular weight (4000-6000) heparin (IC50 30 micrograms/ml). More importantly, heparin abolished GTP gamma S (2.5 microM) induced Ca2+ release (IC50 10 micrograms/ml). These results indicate that, in contrast to the findings in some other cells, in human platelets GTP gamma S-induced Ca2+ release is mediated largely by a mechanism involving IP3.
鸟苷 5'-三磷酸（GTP）及其不可水解的类似物，如鸟苷 5'-O-(3-硫代三磷酸)（GTPγS），可在血小板中引发多种反应，包括分泌、肌醇 1,4,5-三磷酸（IP3）的产生以及细胞内 Ca2+ 的动员。由于 IP3 作为从细胞内储存库动员 Ca2+ 的第二信使已得到充分证实，因此一般认为血小板中 GTP/GTPγS 诱导的 Ca2+ 释放是由 IP3 介导的。然而，对神经元、肝细胞和平滑肌细胞的研究表明，IP3 和 GTP/GTPγS 通过不同机制激活 Ca2+ 释放，且 IP3 非依赖机制介导 GTP/GTPγS 诱导的 Ca2+ 释放。在多个组织中，肝素以竞争和特异性方式抑制 IP3 的结合并阻断 IP3 刺激的 Ca2+ 释放。在本研究中，使用肝素作为探针，在人血小板中检测了 IP3 和 GTPγS 诱导的 Ca2+ 释放及其关系。在皂素通透的血小板中，IP3（0.05 - 5 μM）诱导 Ca2+ 迅速、剂量依赖性释放（EC50 为 0.5 μM）。GTPγS（1 - 50 μM）以剂量依赖性方式释放 Ca2+，EC50 为 2 μM，但有 30 - 90 秒的时间延迟。在对 GTPγS（1 μM）产生次最大反应后，将血小板暴露于 1 μM IP3 导致 Ca2+ 释放进一步增加，但在对 GTPγS（10 μM）产生最大反应后添加 1 μM IP3 则未观察到进一步增加；在颠倒 GTPγS 和 IP3 添加顺序时也有类似发现，表明这些效应物从同一来源释放 Ca2+。IP3（0.5 μM）诱导的 Ca2+ 释放被低分子量（4000 - 6000）肝素（IC50 为 30 μg/ml）阻断。更重要的是，肝素消除了 GTPγS（2.5 μM）诱导的 Ca2+ 释放（IC50 为 10 μg/ml）。这些结果表明，与其他一些细胞中的发现相反，在人血小板中，GTPγS 诱导的 Ca2+ 释放主要由涉及 IP3 的机制介导。
