Cytokine receptor-mediated regulation of MAP kinases in human platelets

血小板生成素（TPO）受体在巨核细胞谱系中表达，从晚期祖细胞到血小板。我们研究了TPO对人血小板细胞外信号调节激酶（ERK）激活途径的影响。TPO本身不激活ERK1、ERK2和蛋白激酶C(PKC)，而TPO直接增强了凝血酶和佛波酯等其他激动剂诱导的ERKs的PKC依赖性激活，而不影响这些激动剂对PKC的激活。TPO没有激活丝裂原激活的蛋白激酶/ERK激酶MEK1和MEK2，但激活了Raf-1并直接增强了pkc介导的MEK激活，表明TPO主要通过调节MEK或MEK的上游步骤（包括Raf-1）来增强ERK途径。MEK抑制剂PD098059不仅不能影响凝血素诱导或磷酯诱导的聚集，也不能影响TPO增强的聚集，从而否认ERKs和MEKs在这些事件中的主要参与。更多的ERKs和MEKs主要位于洗涤剂可溶性/非细胞骨架部分。血小板聚集和肌动蛋白聚合后，ERKs而非MEKs被重新定位到细胞骨架上。这些数据表明，TPO与血小板ERK激活途径中的其他激动剂协同作用，这种协同作用可能影响ERK可能调节的细胞骨架功能。TPO本身不诱导聚集，但在体内增强其他激动剂诱导的阿司匹林治疗或未治疗的血小板聚集。由于我们发现ERKs和MEKs并不主要参与上述血小板聚集，因此我们研究了TPO对p38丝裂原活化蛋白激酶活化的影响，以研究TPO如何影响血小板功能。凝血酶而非phorbol 12,13 -dibutyrate（PDBu）激活p38，与阿司匹林预处理无关。TPO本身不激活p38，而TPO预处理增强了凝血酶诱导的p38激活，无论血小板是否被阿司匹林化。TPO还增强了凝血酶受体激动剂肽、血栓素A2类似物、胶原蛋白、交联糖蛋白VI、ADP和肾上腺素诱导的p38活化。TPO本身不促进Hsp27和胞质磷脂酶A2的磷酸化，但增强了凝血酶诱导的它们的磷酸化。特异性p38抑制剂SB203580强烈抑制这种磷酸化，证实其是通过p38途径介导的。无论血小板是否被阿斯匹林，SB203580都能抑制ADP或凝血素诱导的聚集以及TPO对其的增强作用，但不是完全抑制。相反，尽管TPO也能增强阿斯匹林血小板中pdbu诱导的聚集，但无论TPO是否预处理，SB203580都不能抑制ADP或凝血素诱导的聚集。p38途径可能是TPO在阿司匹林敏感和不敏感两种方式下增强激动剂诱导的聚集的机制之一。少

The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C(PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. … More ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.TPO does not induce aggregation by itself but potentiates other-agonist-induced aggregation in aspirin-treated or -untreated platelets in vivo. Since we found that both ERKs and MEKs were not primarily involved in platelet aggregation as described above, we investigated effects of TPO on activation of p38 mitogen-activated protein kinase to study how TPO affects platelet functions. Thrombin but not phorbol 12, 13-dibutyrate(PDBu) activated p38 irrespective of aspirin pretreatment TPO did not activate p38 by itself, whereas TPO pretreatment potentiated thrombin-induced activation of p38, whether platelet were aspirinized or not. TPO also potentiated p38 activation induced by a thrombin receptor agonist peptide, a thromboxane A2 analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A2 by itself but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation, confirming that it is mediated via the p38 pathway. SB203580 inhibited, but not completely, ADP- or thrombin-induced aggregation and its enhancement by TPO, whether platelets were aspirinized or not In contrast, although TPO also potentiated PDBu-induced aggregation in aspirinized platelets, SB203580 did not inhibit it irrespective of TPO pretreatment. The p38 pathway could be one of the mechanisms by which TPO potentiates agonist-induced aggregation in both aspirin-sensitive and -insensitive manners. Less