Gabbeta J, Yang X, Kowalska M A, Sun L, Dhanasekaran N, Rao A K
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8750-5. doi: 10.1073/pnas.94.16.8750.
G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.
G蛋白在血小板激活后的信号转导中起主要作用。我们之前报道过一名患者，其激动剂诱导的聚集、分泌、花生四烯酸释放和Ca2+动员均受损。目前的研究表明，该患者血小板磷脂酶A2（胞质型和膜型）活性正常。尽管血小板上的糖蛋白（GP）IIb-IIIa含量正常，但通过使用抗体PAC-1的流式细胞术测量的受体介导的GPIIb-IIIa复合物激活减少。患者血小板中鸟苷5'-[γ-硫代]三磷酸（GTP[γS]）诱导的Ca2+释放减少，提示激动剂受体下游存在缺陷。血小板膜中的GTP酶活性（α亚基的功能）在静息状态下正常，但与正常受试者相比，在用凝血酶、血小板活化因子或血栓素A2类似物U46619刺激时降低。在基础状态和凝血酶刺激状态下，35S标记的GTP[γS]与血小板膜的结合均减少。依洛前列素（一种稳定的前列腺素I2类似物）诱导的患者血小板膜中cAMP升高（由Gαs介导）及其被凝血酶抑制（由Gαi介导）均正常。对患者血小板膜中Gα亚基的免疫印迹分析显示Gαq减少（<50%），但Gαi、Gαz、Gα12和Gα13未减少。这些研究为人类血小板G蛋白α亚基功能中一种迄今未描述的缺陷导致血小板反应受损提供了证据，并且它们为Gαq在凝血酶诱导的反应中的主要作用提供了进一步的证据。
