Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin.
Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin.
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DOI：
10.1182/blood.v86.11.4054.bloodjournal86114054
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发表时间：
1995-12
期刊：
影响因子：
20.3
通讯作者：
Jichun Chen;Lidija Herceg-Harjacek;Jerome E. Groopman;Jadwiga Grabarek
中科院分区：
文献类型：
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作者：
Jichun Chen;Lidija Herceg-Harjacek;Jerome E. Groopman;Jadwiga Grabarek
血小板生成素（TPO）是最近发现的一种调节巨核细胞生成的生长因子。其受体c-Mpl在巨核祖细胞、成熟巨核细胞和人血小板中表达。我们已经观察到TPO处理人血小板导致几种细胞蛋白的酪氨酸磷酸化，包括c-Mpl受体和磷脂酰肌醇3-激酶（PI 3-K）的85-kD亚基。TPO以时间依赖性方式刺激这种酪氨酸磷酸化，在5分钟内达到最大值。PI 3-K的酪氨酸磷酸化依赖于TPO的浓度，并在50 - 100 ng/mL之间达到最大值。这种磷酸化不依赖于细胞外纤维蛋白原和α IIb β 3整合素的连接。与此相反，TPO，在外源性纤维蛋白原的存在下，诱导浓度依赖性血小板聚集，这是由可溶性c-Mpl受体阻断。TPO浓度的增加调制的程度的初级波的聚集和滞后相，但不是斜率或最大值的次级波的聚集。这种二次聚集是通过添加腺苷三磷酸双磷酸酶来控制的，这表明腺苷二磷酸（ADP）依赖性机制。血小板与TPO治疗导致125 I-纤维蛋白原与完整血小板的结合增强，50%效应（EC 50）发生在5和10 ng/mL之间。TPO诱导的纤维蛋白原与血小板的结合程度与用10 mumol/L ADP刺激所观察到的相当。在固定化胶原-血小板粘附试验中，观察到TPO刺激的血小板粘附显著增加。这种作用依赖于TPO的浓度。在50 ng/mL的TPO，血小板附着胶原蛋白增加了三倍相比，缓冲液对照。此外，纤维蛋白原的存在并没有显着改变血小板-胶原相互作用的TPO增强。这种相互作用是由Arg-Gly-Asp（RGD）粘附识别序列介导的，因为它被100 mumol/L的RGDS肽完全消除。TPO依赖性血小板附着在胶原蛋白包被表面的一部分对前列腺素E1治疗不敏感。此外，α IIb整合素抗体部分抑制血小板附着胶原蛋白，这表明整合素α IIb β 3参与了这种关联。这些数据表明，TPO可能不仅作为巨核细胞生长和分化的细胞因子，但也可能参与直接血小板活化和调节血小板-细胞外基质的相互作用。
Thrombopoietin (TPO) is a recently identified growth factor that regulates megakaryocytopoiesis. Its receptor, c-Mpl, is expressed in megakaryocyte progenitors, mature megakaryocytes, and human blood platelets. We have observed that TPO treatment of human platelets resulted in tyrosine phosphorylation of several cellular proteins, including the c-Mpl receptor and the 85-kD subunit of phosphatidylinositol 3-kinase (PI3-K). TPO stimulated this tyrosine phosphorylation in a time-dependent manner, reaching a maximum in 5 minutes. The tyrosine phosphorylation of PI 3-K was dependent on the concentration of TPO and reached a maximum at concentrations between 50 and 100 ng/mL. This phosphorylation was independent of extracellular fibrinogen and ligation of the alpha IIb beta 3 integrin. In contrast, TPO, in the presence of exogenous fibrinogen, induced concentration-dependent platelet aggregation, which was blocked by the soluble c-Mpl receptor. Increasing TPO concentrations modulated the degree of the primary wave of aggregation and the lag phase, but not the slope or maximum of the secondary wave of aggregation. This secondary aggregation was controlled by the addition of apyrase, suggesting an adenosine diphosphate (ADP)-dependent mechanism. Treatment of platelets with TPO resulted in augmented binding of 125I-fibrinogen to intact platelets, with a 50% effect (EC50) occurring between 5 and 10 ng/mL. TPO-induced binding of fibrinogen to platelets was comparable in degree with that observed by stimulation with 10 mumol/L ADP. In an immobilized collagen-platelet adhesion assay, a significant increase in the attachment of TPO-stimulated platelets was observed. This effect was dependent on the concentration of TPO. At 50 ng/mL of TPO, platelet attachment to collagen increased threefold compared with the buffer control. Furthermore, the presence of fibrinogen did not significantly alter TPO augmentation of the platelet-collagen interaction. This interaction was mediated by the Arg-Gly-Asp (RGD) adhesion recognition sequence, as it was completely abolished by 100 mumol/L of the RGDS peptide. A fraction of the TPO-dependent platelet attachment to a collagen-coated surface was insensitive to treatment with prostaglandin E1. Furthermore, antibody to alpha IIb integrin partially inhibited platelet attachment to collagen, suggesting that the integrin alpha IIb beta 3 participates in this association. These data indicate that TPO might function not only as a cytokine in megakaryocyte growth and differentiation, but may also participate in direct platelet activation and modulate platelet-extracellular matrix interactions.