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

Platelet integrin α(IIb)β(3) activation is regulated by inside-out signaling via agonist stimulation. However, when α(IIb)β(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α(IIb)β(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α(IIb)β(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 α(IIb)β(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 α(IIb)β(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α(IIb)β(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α(IIb)β(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α(IIb)β(3) activation, but it significantly augmented agonist-induced α(IIb)β(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 β(3) cytoplasmic tail was essential for talin-mediated α(IIb)β(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced α(IIb)β(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α(IIb)β(3) activation.