Myosin-IIA activity controls conversion to proplatelets and cleavage of normal platelets. Proplatelet-enriched fractions from peripheral blood collected in AJ buffer were incubated with DMSO, 10 μM blebbistatin, or 5 μM nocodazole for 6 hours at 37°C. (A-B) Imaging after drug treatment shows at high magnification of the myosin IIA distribution as well as its morphology. Line scans and surface plots show that myosin localization to the periphery with DMSO and nocodazole treatment is disrupted by blebbistatin. Staining for MIIB was done in parallel using the same secondary antibody, which indicated, on average, MIIA:MIIB = 250 (N = 25 across 5 fields of view for each isoform). (C) Quantification of immunofluorescence of 6-hour–treated proplatelet-enriched fractions shows a shift toward preplatelets at the expense of proplatelets after blebbistatin or nocodazole incubation. One-hour treatments done in parallel did not show a significant difference from 6-hour treatments. Gates are based on previously published values.2 (D) Flow cytometry scatter plots with platelet populations determined based on scatter and gated on CD41+ Hoechst– GPA–. The bar graph summarizes flow cytometry.
