Padoin E, Alexandre A, Cavallini L, Polverino de Laureto P, Rao G H, Doni M G
Institute of Human Physiology, Faculty of Medicine and Surgery, University of Padova, Italy.
Arch Biochem Biophys. 1996 Sep 15;333(2):407-13. doi: 10.1006/abbi.1996.0408.
We studied the effect of glycoprotein GPIIb/IIIa (integrin alpha IIb beta 3) receptor occupancy by adenosine 5',1-thiotriphosphate (ATP alpha S), a competitive inhibitor of the ADP receptor, by fibrinogen, and by peptides containing the RGD (Arg-Gly-Asp) sequence as RGDW (Arg-Gly-Asp-Trp), RGDS (Arg-Gly-Asp-Ser), or the negative control RGGW (Arg-Gly-Gly-Trp) on human platelet physiological functions: aggregation, ATP secretion, and [Ca2+]in. As the presence of a nucleotide binding site on GPIIb alpha has been demonstrated in platelets [N. J. Greco, N. Yamamoto, B. W. Jackson, N. N. Tandon, M. Moos, and G. A. Jamieson (1991) J. Biol. Chem. 266, 13627-13633], we studied the effect of ATP alpha S, which specifically binds to this site, on platelet activation. We observed that ATP alpha S inhibited aggregation by thrombin, ADP, PMA, and ionophore A23187. Moreover, ATP alpha S dose dependently inhibited ATP secretion by ionophore A23187 and Ca2+ transients by thrombin and vasopressin in both the presence and absence of external Ca2+. Fibrinogen, although induced by a potentiation of platelet aggregation, inhibited ATP secretion and [Ca2+]in elevation induced by low thrombin concentrations or by vasopressin, interfering with both Ca2+ entry and Ca2+ release by the intracellular stores. RGD peptides, which specifically bind to GPIIb/IIIa, inhibited aggregation, secretion, and Ca2+ transients by thrombin, whereas the negative control RGGW did not exert any effect. We conclude that the occupancy of the GPIIb/IIIa receptor binding sites modulates platelet function by giving an inhibitory outside-in signal in platelets, particularly effective in platelets stimulated with low agonist doses. We suggest that ATP alpha S, fibrinogen, or RGD compounds, by interacting with GPIIb/IIIa receptor, prime some intracellular negative feedback mechanisms, which prevent further activation of circulating platelets by low-intensity stimuli and intravascular aggregation.
我们研究了5'-三磷酸腺苷硫代物（ATPαS，一种ADP受体的竞争性抑制剂）、纤维蛋白原以及含有RGD（精氨酸-甘氨酸-天冬氨酸）序列的肽（如RGDW，即精氨酸-甘氨酸-天冬氨酸-色氨酸；RGDS，即精氨酸-甘氨酸-天冬氨酸-丝氨酸）或阴性对照RGGW（精氨酸-甘氨酸-甘氨酸-色氨酸）占据糖蛋白GPIIb/IIIa（整合素αIIbβ3）受体对人血小板生理功能的影响：聚集、ATP分泌以及细胞内钙离子浓度（[Ca2+]in）。由于已在血小板中证实GPIIbα上存在核苷酸结合位点[N. J. 格雷科、N. 山本、B. W. 杰克逊、N. N. 坦登、M. 穆斯和G. A. 贾米森（1991年）《生物化学杂志》266, 13627 - 13633]，我们研究了特异性结合该位点的ATPαS对血小板活化的影响。我们观察到ATPαS抑制凝血酶、ADP、佛波酯（PMA）和离子载体A23187诱导的聚集。此外，无论有无细胞外钙离子，ATPαS均剂量依赖性地抑制离子载体A23187诱导的ATP分泌以及凝血酶和血管加压素诱导的钙离子瞬变。纤维蛋白原虽然可增强血小板聚集，但抑制低浓度凝血酶或血管加压素诱导的ATP分泌以及细胞内钙离子浓度升高，干扰钙离子内流和细胞内储存库的钙离子释放。特异性结合GPIIb/IIIa的RGD肽抑制凝血酶诱导的聚集、分泌和钙离子瞬变，而阴性对照RGGW则无任何作用。我们得出结论，GPIIb/IIIa受体结合位点的占据通过在血小板中产生抑制性的由外向内信号来调节血小板功能，这在低剂量激动剂刺激的血小板中尤为有效。我们认为，ATPαS、纤维蛋白原或RGD化合物通过与GPIIb/IIIa受体相互作用，启动一些细胞内负反馈机制，从而防止低强度刺激和血管内聚集对循环血小板的进一步激活。
