Agonist stimulation, talin-1, and kindlin-3 are crucial for alpha(IIb)beta(3) activation in a human megakaryoblastic cell line, CMK

作者：Nakazawa Tsuyoshi; Tadokoro Seiji; Kamae Tsuyoshi; Kiyomizu Kazunobu; Kashiwagi Hirokazu; Honda Shigenori; Kanakura Yuzuru; Tomiyama Yoshiaki^*

来源：Experimental Hematology, 2013, 41(1): 79-90.

DOI：10.1016/j.exphem.2012.09.011

Platelet integrin alpha(IIb)beta(3) activation is regulated by inside-out signaling via agonist stimulation. However, when alpha(IIb)beta(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced alpha(IIb)beta(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed alpha(IIb)beta(3) in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient alpha(IIb)beta(3) activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide-induced alpha(IIb)beta(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed alpha(IIb)beta(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate alpha(IIb)beta(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce alpha(IIb)beta(3) activation, but it significantly augmented agonist-induced alpha(IIb)beta(3) activation. Several mutants of FL-talin and THD suggested that the head-rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the beta(3) cytoplasmic tail was essential for talin-mediated alpha(II)b beta(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1 induced alpha(IIb)beta(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent alpha(IIb)beta(3) activation.

• 出版日期2013-1