Agonist Stimulation, Talin-1, and Kindlin-3 Are Crucial for α¶bβ3 activation in a Human Megakaryoblastic Cell Line, CMK
Authors: Nakazawa Tsuyoshi , Tadokoro Seiji , Kiyomizu Kazunobu , Kashiwagi Hirokazu , Honda Shigenori , Tomiyama Yoshiaki , Kanakura Yuzuru 
Published: October 2019

Abstract
Abstract 1137
Integrin αIIbβ3 plays a central role in platelet aggregation, by changing its conformation from low-affinity to high-affinity state for ligand via inside-out signaling (integrin activation). However, detailed mechanism of αIIbβ3 activation remains to be determined. We have developed a powerful system to analyze the mechanism of physiological agonist-induced αIIbβ3 activation, by employing a human megakaryoblastic leukemia cell line, CMK. In contrast to exogenously expressed αIIbβ3 in CHO cells, endogenously expressed αIIbβ3 in GPIb-positive CMK cells can be activated by physiological agonists such as thrombin and protease-activated receptor-actibvating peptide (PAR-AP). An activated state of αIIbβ3 induced by agonists was only transient and the activated αIIbβ3 gradually returned inactive state. A robust αIIbβ3 activation was detected by an initial velocity analysis for FITC-PAC-1 binding. In this study, we performed direct genetic manipulation to examine an effect of talin-1 and kindlin-3 on αIIbβ3 activation in CMK. We measured the initial PAC-1 binding velocity by incubating FITC-PAC-1 and PAR1-TRAP with CMK cells only for 2 minutes. Over-expression of full length talin-1 (FL-TLN), talin head domain (THD) or kindlin-3 significantly augmented PAC-1 binding after 50 μM PAR1-TRAP stimulation. In sharp contrast to the CHO system, αIIbβ3 activation was not induced without any agonist stimulation in CMK system even if THD was over-expressed.
Overexpression of FL-TLN mutant in critical residues for its head-rod interaction (FL-TLN-K318A or FL-TLN-M319A) more strongly augmented αIIbβ3 activation, as compared with wild-type FL-TLN. In contrast, overexpression of THD with the same mutation did not augment it, as compared with wild-type THD. These findings confirmed an autoinhibition mechanism for talin-1. FL-TLN mutants or THD mutants in critical residues for the interaction with β3 cytoplasmic tail (W359A, L325R, S365D, S379R, Q381V or K324D) or in the critical residue for the interaction with membrane (K322D) failed to augnent αIIbβ3 activation. In order to gain stable talin-1 or kindlin-3 knocked-down cell line, we used lentivirus mediated RNAi technology. In brief, lentiviral particles containing short-hairpin RNAs targeting talin-1 mRNA were integrated in CMK, and DsRed2 which is expressed with shRNA was used as transfection marker. Stable clones were obtained by limitting dilution. In these clones the expression of endogenous talin-1 or kindlin-3 was reduced to approximately half of non-silencing cells. Knock-down each molecule resulted in a robust decrease in PAC-1 binding induced by PAR1-TRAP. The impaired αIIbβ3 activation in kindlin-3 knocked down cells was rescued by exogenously expressed kindlin-3. Interestingly, the impaired αIIbβ3 activation in kindlin-3 knocked down CMK cells was also rescued by overexpression of THD. However, the increase in αIIbβ3 activation by PAR1-TRAP after over-expression of THD in kindlin-3 knocked-down cells was still smaller, as compared with that in THD overexpressed wild-type cells. Those results suggest that talin-1 and kindlin-3 play a crucial role in αIIbβ3 activation co-operatively. Taken together, this experimental system with CMK using RNAi and overexpression technology provides a newly alternative way to investigate molecular mechanism for the αIIbβ3 activation induced by physiological agonists.

Disclosures:
No relevant conflicts of interest to declare.