Deletion of α_IIbβ₃and Rho GTPase FLNa interaction domains, but not of GPIbα-interacting domain, deeply affects proplatelet formation. (A) Schematic representation of FLNa mutants introduced by zinc finger nuclease–mediated gene editing. (B) Representative immunoblot for FLNa expression in the iPSC-edited clones using an N-terminus (left) and C-terminus (right) antibody. (C) Representative pictures of proximity ligation assay for FLNa/β₃ and FLNa/RhoA interactions. Red staining represents the interaction between β₃ and FLNa and between RhoA and FLNa. The nucleus is stained with 49,6-diamidino-2-phenylindole (blue); scale bar = 30 μm. Two independent experiments for each interaction were performed, with 50 cells analyzed in each experiment. No specific signal was detected when cells were incubated without primary antibodies (data not shown). (D) Flow cytometry analysis of CD41a and CD42a expression in the edited clones at day 15 of differentiation. Results are presented as mean ± standard error of the mean; unpaired Student t test with Welch’s correction was used; each point represents 1 independent experiment. (E) Proplatelet formation potential evaluated at day 18 of MK differentiation for each edited clone (n = 3). Paired Student t test *P < .05, **P < .01.