Rho/ROCK pathway negatively regulates proplatelet formation mainly through α2 integrin. (A) MKs were plated onto PLL (Ai-ii) or GFOGER-coated coverslips in serum-free medium and incubated for 18 hours without (Aiii-iv) or with Y-27632 (Av-vi). Adherent cells were fixed, permeabilized, and stained with polyclonal anti-VWF antibody (Ai,iii,v) and phalloidin (Aii,iv,vi). Phase contrast images merged with VWF staining show the distribution of VWF (Ai,iii,v) in MKs forming proplatelets. Phalloidin staining shows irregular F-actin distribution in the same cells (Aii,iv,vi). The arrowheads indicate branching emanating from VWF positive proplatelets (Av-vi). The graphs represent the mean ± SD of 300 cells showing stress fibers (B) or VWF⁺ proplatelets (C) in the indicated substrates without and with TAT-C3 or Y-27632 treatment in 3 independent experiments. Scale bar equals 10 μm.