Shear stress and pharmacologic inhibition of myosin-II synergistically enhance PLP generation in vitro. (A) Myosin inhibition scheme and schematic of cone and plate rheometer. Primary human CD34⁺ bone marrow–derived cells were cultured for 3 to 4 days in the presence of stem cell factor and thrombopoietin to drive differentiation toward MKs, followed by an additional 3 days of culture in the presence of 15 μM blebbistatin to promote polyploidy and MK maturation. In other experiments, MEG-01 cells were cultured for 3 days with 20 μM blebbistatin (IC₅₀ ≈ 5 μM). In either case, cell suspensions placed on the rheometer are subjected to controlled temperature (37°C for these studies), shear stress, stress duration, and gap size (60 μm for these studies). Samples collected from the rheometer are analyzed by flow cytometry to quantify PLP (R1) and nucleated cell (R2) fractions. (B) PLP quantitation after cone-and-plate rheometry of CD34⁺-derived CD41⁺ MKs shows an enhanced effect by both shear and MII inhibitions. Values are relative to unsheared, untreated cell culture (n = 3, ±SEM, * P < .05). (C) Quantitation of PLP generation after cone-and-plate imparted shear stress of MEG-01 cells (n = 6, ±SEM; *+#, P < .05; **++, P < .005; +, Significant from DMSO culture; #, significant from blebbistatin culture; *, significant from previous shear stress condition). The data are fit with a combination of a 1-phase exponential decay and a 1-site–specific binding model. DMSO fit: y = [2+4.0*exp(–0.62*x)]*[0.10+(0.66x/(0.65+x))], R² = 0.97). Bleb fit: y = [2+4.0*exp(–0.62*x)]*[0.26+(1.5x/(0.94+x))], R² = 0.98). (D) Activation of cone and plate generate PLP cell suspension from CD34⁺-derived MKs (middle) or MEG-01 (lower). Sheared samples are stimulated by 100 μg/mL collagen-I and 1 mM CaCl₂, and activation is determined by a shift in side scatter and Annexin-V⁺ CD41⁺ 7AAD^– (n = 3, ±SEM). (E) Considering the increased number of PLPs generated from MEG-01 shear (as in C), shear results in an ∼8× net increase in functional PLPs compared with unsheared, untreated MEG-01 culture (n = 5, ±SEM, *+, P < .05; #, significant from untreated condition; *, significant from previous shear stress condition).