Kashiwagi H, Shiraga M, Honda S, Kosugi S, Kamae T, Kato H, Kurata Y, Tomiyama Y
Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University and Department of Blood Transfusion, Osaka University Hospital, Suita, Osaka, Japan.
J Thromb Haemost. 2004 Jan;2(1):177-86. doi: 10.1111/j.1538-7836.2003.00529.x.
Affinity/avidity state of integrin alpha IIb beta 3 is regulated by intracellular inside-out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to alpha IIb beta 3 expressed in these cells by cellular agonists has not been demonstrated. We have re-examined agonist-induced alpha IIb beta 3 activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of alpha IIb beta 3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that alpha IIb beta 3 expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIbhigh cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA-reactive GPIbhigh cells. Not only PMA but also a calcium ionophore, A23187, induced alpha IIb beta 3 activation, and PMA and A23187 had an additive effect on alpha IIb beta 3 activation. Ligand binding to the activated alpha IIb beta 3 in the GPIbhigh CMK cells is totally abolished by an alpha IIb beta 3-specific antagonist, and inhibited by wortmannin, cytochalasin-D and prostaglandin E1, and the effects of these inhibitors on alpha IIb beta 3 activation in the GPIbhigh CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin-treated CMK cells express PKC-alpha, -beta, -delta and -theta, and suggested that PKC-alpha and/or -beta appear to be responsible for PMA-induced activation of alpha IIb beta 3 in CMK cells.
整合素αIIbβ3的亲和力/亲合力状态受细胞内由外向内信号传导调控。尽管已经建立了几种巨核细胞系，但尚未证实细胞激动剂诱导的可溶性配体与这些细胞中表达的αIIbβ3结合。我们利用巨核细胞分化后期标志物糖蛋白Ib（GPIb），重新研究了激动剂诱导的巨核细胞系上αIIbβ3的激活情况。通过PAC1和可溶性纤维蛋白原与细胞的结合来评估αIIbβ3的激活。我们发现，在高表达GPIb的CMK细胞中表达的αIIbβ3可被佛波酯十四酰佛波醇乙酸酯（PMA）激活。尽管GPIb高表达细胞群体占总细胞的比例小于0.5%，但与核苷类似物利巴韦林孵育可有效增加对PMA反应性的GPIb高表达细胞。不仅PMA，而且钙离子载体A23187也可诱导αIIbβ3激活，并且PMA和A23187对αIIbβ3激活具有相加作用。αIIbβ3特异性拮抗剂可完全消除GPIb高表达CMK细胞中活化的αIIbβ3与配体的结合，渥曼青霉素、细胞松弛素-D和前列腺素E1可抑制这种结合，并且这些抑制剂对GPIb高表达CMK细胞中αIIbβ3激活的影响与对血小板中的影响一致。我们还证实，经利巴韦林处理的CMK细胞表达蛋白激酶C-α、-β、-δ和-θ，并提示蛋白激酶C-α和/或-β似乎是CMK细胞中PMA诱导的αIIbβ3激活的原因。
