Myosin-IIA inhibition of MKs enhances generation of fragments that are similar in size to normal human pre/proplatelets. (A) Platelet generation scheme: MKs in bone marrow extend membrane processes into the flowing blood where shear stress fragments large pre/proplatelets. Conversion to dumbbell-shaped proplatelets allows fission to small, normal platelets. (B) MKs enriched from fresh human bone marrow were stained with Hoechst 33342 and anti-CD41 and treated ± blebbistatin before micropipette aspiration. Fragmentation occurs within seconds and persists for minutes (scale bar = 10 μm). (C) Blebbistatin enhances fragment frequency and results in a higher percentage of total MK volume release vs DMSO control (N = 4 cells, n > 10 fragments/cell for control, n > 30 fragments/cell for blebb, ± standard error of the mean (SEM), *P < .01). The single-parameter fit goes through both data points. (D) Released fragment size is unaffected by blebbistatin, and fragments are similar in size to normal human pre/proplatelets (n = 35 fragments for control, n = 135 fragments for blebb, ±SEM, P = .5). The pink shaded region denotes the calculated range of projected area from normal human platelet diameters. The gray shaded region denotes estimations from reported preplatelet diameters.²  (E) MEG-01 cells nucleofected with GFP-MIIA and treated ± blebbistatin and fluorescent images of GFP-MIIA MEG-01 cells undergoing fragmentation in micropipette aspiration (scale bar = 10 μm). Intensity of cell body has been adjusted to allow visualization of aspirated fragment. Intensity is plotted below each micrograph. (F) Blebbistatin treatment of non-nucleofected MEG-01 cells increases the number of fragments per cell as well as permission of fragmentation at smaller-radius pipettes. Linear fit of DMSO: y = 0.57x − 0.8, R² = 0.99, n = 2, ±SEM. 20 μM blebbistatin: n > 15 ±SEM. Statistical significance determined between DMSO and 20 μM blebbistatin for a given R_p, *P < .05. (G) The fragment area is not affected by blebbistatin, but treatment facilitates fragmentation of smaller fragments from smaller pipettes. Linear fit of 20 μM blebbistatin: y = 138x−187 (R² = 0.99, n < 40, ±SEM). DMSO: n > 6 ±SEM. The pink shaded region denotes calculated range of area from reported normal human platelet diameters. The gray shaded region denotes estimations from reported preplatelet diameters.²