The small-GTPase Rho and its effector kinase ROCK control platelet production in vitro and in vivo through regulation of myosin-IIA functions. (A) MLC phosphorylation appears to be regulated by ROCK. Kinase inhibitors were added to primary MKs enriched over a BSA step-gradient on day 3 of fetal liver cell culture. PPF, assessed 24 hours later by the method described in Figure 2, was enhanced by the ROCK inhibitor Y-27632 but not the MLCK inhibitor ML-7. The effect of Y-27632 was completely reversed upon expression of D₁₈D₁₉-MLC. (B) Expression of activated Rho (RhoAV14) in MKs led to greatly reduced PPF, which was reversed by Y-27632 or expression of dominant-negative A₁₈A₁₉-MLC, but not by ML-7. (C) Mouse transplantation with blood progenitors transduced by retroviral constructs expressing wild-type MLC, D₁₈D₁₉-MLC, or RhoAV14. Comparison of peripheral blood leukocyte engraftment and bone marrow MK engraftment in animal groups transplanted with cells transduced by the indicated constructs. (D) Assessment of platelet production efficiency. Peripheral blood platelet recovery was measured 5 weeks after transplantation, followed by euthanasia to determine bone marrow MK and myeloid engraftment using flow cytometry. Only animals showing less than 15% MK engraftment were included in the analysis. Thrombopoietic efficiency is expressed as the ratio of (GFP⁺ platelets/total platelets) to (GFP⁺ MKs/total MKs). Statistical significance of the differences between groups was calculated by the 2-tailed Student t test. (E) Sdf-1 attenuates endogenous RhoA activity in mature MKs. Bone marrow–derived MKs, isolated over a BSA step-gradient 72 hours after culture initiation, were treated with 10 or 100 ng/mL recombinant Sdf-1. Activated and total Rho levels were evaluated after 3 hours. The right panel shows a time-course study of fetal liver–derived primary MKs treated with 100 ng/mL Sdf-1, with determination of Rho activity 30 minutes and 3 hours later.