Lages B, Weiss H J
Department of Medicine, St Luke's-Roosevelt Hospital Center, New York, NY 10019.
Blood. 1994 May 1;83(9):2549-59.
Ionophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenic subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-induced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb-IIIa was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+]i increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-IIIa-dependent process that most likely involves a property of the ligand-occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation-independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.
发现在血小板无力症患者的枸橼酸化富血小板血浆（PRP）以及用糖蛋白（GP）IIb-IIIa复合物特异性单克隆抗体处理的正常PRP中，离子载体A23187诱导的14C-5-羟色胺分泌和血栓素B2（TxB2）形成均不存在。当存在5 mmol/L乙二胺四乙酸（EDTA）时，这两种反应均恢复到正常水平，表明它们的缺失并非由聚集本身的缺失所致。在与各种添加物一起孵育的凝胶过滤血小板（GFP）中，GPIIb-IIIa的阻断或缺失导致在含有1 mmol/L钙离子（无论有无纤维蛋白原）和1 mmol/L镁离子加纤维蛋白原的培养基中，A23187诱导的分泌和TxB2形成减少，但当单独存在无钙镁缓冲液、1 mmol/L EDTA或单独的纤维蛋白原时则不然。相反，在存在1 mmol/L钙离子、1 mmol/L EDTA或单独缓冲液的情况下，用凝血酶或佛波酯肉豆蔻酸酯刺激的GFP中未观察到这种对GPIIb-IIIa的依赖性。在用抗体处理的正常GFP和血小板无力症GFP中，离子载体诱导的反应受到抑制，但与离子载体介导的细胞内钙离子浓度（[Ca2+]i）增加的任何显著改变均无关，这在用A23187刺激的水母发光蛋白负载的GFP和用离子霉素刺激的fura-2负载的GFP中均有测量。在存在1 mmol/L钙离子的情况下，用单克隆抗体或配体结合位点肽RGDS孵育正常GFP，导致A23187诱导的聚集几乎完全受到抑制，以单个血小板的损失来衡量，但与抗体不同的是，RGDS并不抑制分泌或TxB形成。我们得出结论，在存在但非不存在细胞外二价阳离子的情况下，离子载体诱导的血小板活化涉及一个依赖GPIIb-IIIa的过程，该过程很可能涉及复合物配体占据形式的一种特性，与其支持聚集的能力不同。这可能代表了GPIIb-IIIa复合物受体占据状态在信号转导中不依赖聚集的活性的另一个例子。
