Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets.
Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets.
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血小板表面 Ca2 高亲和力结合位点的鉴定和功能。
DOI：
10.1172/jci111252
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Shattil,SJ
中科院分区：
文献类型：
--
作者：
Brass,LF;Shattil,SJ
细胞外钙离子是ADP或肾上腺素引起的血小板聚集和分泌所必需的。最近，我们报道了血小板表面含有两类与细胞外钙离子高亲和力的结合部位。为了确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)研究了先天缺乏表面膜糖蛋白的血小板，以及(B)检测了去除表面结合的钙离子对血小板对ADP和肾上腺素的反应的影响。未经刺激的正常血小板含有86,000个钙结合位点，解离常数(Kd)为9 nM；389,000个结合位点，解离常数(Kd)为400 nM。相比之下，缺乏糖蛋白IIb和IIIa的血小板，其高亲和力钙结合部位的数量减少了92%，低亲和力部位的数量减少了63%。缺乏糖蛋白Ib的Bernard-Soulier血小板并不缺乏钙结合部位。经ADP刺激后，正常和血栓减少的血小板均形成了约138,000个新的钙结合位点/血小板(Kd=400 nM)，而较大的Bernard-Soulier血小板形成了216,000个新的结合位点。这些结果表明，IIb和IIIa是未受刺激的血小板上主要的钙结合糖蛋白，而这些糖蛋白和Ib都不代表受刺激的血小板上新的钙结合部位。去除血小板表面的钙离子会抑制血小板的功能。尽管有1 mM的镁离子存在，ADP和肾上腺素诱导的聚集和[14C]5-羟色胺的释放在游离钙浓度小于7 nM时显著减少，这一值与高亲和力钙结合部位的Kd值相似。此外，竞争这些钙结合部位的稀土元素Gd也抑制了聚集和5-羟色胺的释放。因此，这些研究表明，细胞外钙离子与未经刺激的血小板上的糖蛋白IIb/IIIa或刺激后的血小板上的额外膜蛋白结合是最大限度地促进血小板对ADP和肾上腺素的反应所必需的。因此，这些激动剂在激活血小板过程中对细胞外钙的需求实际上可能代表了对表面结合钙的需求。
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+.