Agonist stimulation, talin-1, and kindlin-3 are crucial for αIIbβ3 activation in a human megakaryoblastic cell line, CMK

Platelet integrin αIIbβ3 activation is regulated by inside-out signaling via agonist stimulation. However, when αIIbβ3 was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced αIIbβ3 activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed αIIbβ3 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 αIIbβ3 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 αIIbβ3 activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed αIIbβ3 in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate αIIbβ3 in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce αIIbβ3 activation, but it significantly augmented agonist-induced αIIbβ3 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 β3 cytoplasmic tail was essential for talin-mediated αIIbβ3 activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced αIIbβ3 activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent αIIbβ3 activation.