Cytokine receptor-mediated regulation of MAP kinases in human platelets

血小板生成素(TPO)受体在巨核细胞系中表达，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直接增强其他激动剂(包括凝血酶和佛波酯)对ERKs依赖的PKC激活，而不影响这些激动剂对PKC的激活。TPO不激活丝裂原激活的蛋白激酶/ERK激酶MEK1和MEK2，但激活Raf-1，并直接增强PKC介导的MEK激活，提示TPO主要通过调节MEKs或包括Raf-1在内的MEKs上游步骤来增强ERK通路。MEK抑制剂PD098059不仅不影响凝血酶或佛波酯诱导的聚集，而且不影响TPO诱导的聚集，否认ERKs和meks在这些事件中的主要参与。…更多的ERK和MEK主要分布在洗涤剂可溶/非细胞骨架部分。在血小板聚集和肌动蛋白聚合后，ERK而不是MEK被重新定位到细胞骨架上。这些数据表明，TPO与其他激动剂在血小板ERK激活途径中具有协同作用，这种协同作用可能影响细胞骨架的功能，可能受ERK的调节。在体内，TPO本身不诱导聚集，但增强了其他激动剂诱导的聚集。由于我们发现ERKs和meks并不是如上所述主要参与血小板聚集，因此我们研究了TPO对p38丝裂原活化蛋白激酶激活的影响，以研究TPO如何影响血小板功能。凝血酶而不是佛波酯(PDBu)激活p38，而不是阿司匹林处理，TPO本身不能激活p38，而TPO预处理增强了凝血酶诱导的p38激活，无论血小板是否阿司匹林。TPO还增强了凝血酶受体激动肽、血栓素A2类似物、胶原、交联糖蛋白VI、ADP和肾上腺素诱导的p38激活。TPO本身不促进HSP27和胞浆磷脂酶A2的磷酸化，但增强凝血酶诱导的HSP27和胞浆磷脂酶A2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。SB203580抑制但不完全抑制ADP或凝血酶诱导的聚集以及TPO对其的促进作用，而无论血小板是否被阿司匹林化，TPO也能增强PDBu诱导的阿司匹林诱导的血小板聚集，但SB203580不能抑制PDBu诱导的聚集，而不受TPO的影响。P38通路可能是TPO以阿司匹林敏感和不敏感的方式增强激动剂诱导的聚集的机制之一。较少

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