Brass L F, Shattil S J
J Clin Invest. 1984 Mar;73(3):626-32. doi: 10.1172/JCI111252.
Extracellular Ca2+ is required for platelet aggregation and secretion in response to ADP or epinephrine. Recently, we reported that the platelet surface contains two classes of high affinity binding sites for extracellular Ca2+. To identify these sites and clarify their role in platelet function, we have now (a) studied platelets congenitally deficient in surface membrane glycoproteins and (b) examined the effect of removing surface-bound Ca2+ on platelet responses to ADP and epinephrine. Unstimulated normal platelets contained 86,000 Ca2+-binding sites/platelet with a dissociation constant (Kd) of 9 nM and 389,000 sites with a Kd of 400 nM. In contrast, thrombasthenic platelets, which lack glycoproteins IIb and IIIa, exhibited a 92% reduction in the number of higher affinity Ca2+-binding sites and a 63% reduction in the number of lower affinity sites. Bernard-Soulier platelets, which lack glycoprotein Ib, were not deficient in Ca2+-binding sites. After stimulation with ADP, both normal and thrombasthenic platelets developed approximately 138,000 new Ca2+-binding sites/platelet (Kd = 400 nM), while the larger Bernard-Soulier platelets developed 216,000 new sites. These data suggest that IIb and IIIa represent the major Ca2+-binding glycoproteins on unstimulated platelets, while neither these glycoproteins nor Ib represent the new Ca2+-binding sites on stimulated platelets. Removal of Ca2+ from the platelet surface inhibited platelet function. Despite the presence of 1 mM Mg2+, ADP- and epinephrine-induced aggregation and [14C]serotonin release were markedly decreased at free Ca2+ concentrations less than 7 nM, a value similar to the Kd of the higher affinity Ca2+-binding sites. Moreover, gadolinium, a lanthanide that competed for these Ca2+-binding sites, also inhibited aggregation and serotonin release. These studies demonstrate, therefore, that the binding of extracellular Ca2+ to glycoproteins IIb/IIIa on unstimulated platelets or to additional membrane proteins on stimulated platelets is necessary for maximal platelet responses to ADP and epinephrine. Thus, the requirement for extracellular Ca2+ during platelet activation by these agonists may actually represent a requirement for surface-bound Ca2+.
细胞外钙离子是血小板对二磷酸腺苷（ADP）或肾上腺素产生聚集和分泌反应所必需的。最近，我们报道血小板表面含有两类对细胞外钙离子具有高亲和力的结合位点。为了鉴定这些位点并阐明它们在血小板功能中的作用，我们现在：（a）研究先天性缺乏表面膜糖蛋白的血小板，以及（b）检测去除表面结合的钙离子对血小板对ADP和肾上腺素反应的影响。未受刺激的正常血小板每个血小板含有86,000个钙离子结合位点，解离常数（Kd）为9 nM，还有389,000个位点，Kd为400 nM。相比之下，缺乏糖蛋白IIb和IIIa的血小板无力症血小板，其高亲和力钙离子结合位点数量减少了92%，低亲和力位点数量减少了63%。缺乏糖蛋白Ib的伯纳德 - 索利尔血小板在钙离子结合位点方面并无缺陷。在用ADP刺激后，正常血小板和血小板无力症血小板每个血小板均产生约138,000个新的钙离子结合位点（Kd = 400 nM），而较大的伯纳德 - 索利尔血小板则产生216,000个新位点。这些数据表明，IIb和IIIa代表未受刺激血小板上主要的钙离子结合糖蛋白，而无论是这些糖蛋白还是Ib都不代表受刺激血小板上的新钙离子结合位点。从血小板表面去除钙离子会抑制血小板功能。尽管存在1 mM的镁离子，但在游离钙离子浓度低于7 nM时，ADP和肾上腺素诱导的聚集以及[14C]5 - 羟色胺释放均显著降低，该值与高亲和力钙离子结合位点的Kd相似。此外，钆，一种竞争这些钙离子结合位点的镧系元素，也抑制聚集和5 - 羟色胺释放。因此，这些研究表明，细胞外钙离子与未受刺激血小板上的糖蛋白IIb/IIIa或与受刺激血小板上的其他膜蛋白结合，对于血小板对ADP和肾上腺素的最大反应是必要的。因此，这些激动剂在血小板激活过程中对细胞外钙离子的需求实际上可能代表对表面结合钙离子的需求。
