Shear stress-mediated proPLT extension is a cytoskeleton-driven process. (A) Approximately 100 µm diameter primary mouse MK squeezing through 2 µm gap. (B) Release of large MK fragment from primary mouse MK into the lower channel resulting in prePLT formation. White arrows indicate proPLT extension. (C) ProPLT extension rates vary at different positions along the shaft, predictive of a regulated cytoskeletal driven process. The hiPSC-derived MKs are shown. White arrows indicate proPLT extension, and yellow arrows indicate site of abscission event. (D) Individual release events (yellow arrow) are routinely captured by high-resolution live-cell microscopy at different positions along the proPLT shaft. White arrow denotes proPLT end. Primary mouse MKs are shown in an earlier bioreactor design in which gaps are spaced 45 µm apart. (E) PrePLTs form at new proPLT ends after each abscission event (yellow arrow). Primary mouse MKs are shown. (F) Increasing shear stress from 100 to 1000 seconds⁻¹ does not increase proPLT extension rate in primary mouse MKs. Data are represented as a box-and-whisker plot where light gray indicates the upper quartile and dark gray indicates the lower quartile. (G) Primary mouse MKs retrovirally transduced to express GFP-β1 tubulin show proPLT extensions are comprised of peripheral MTs that form coils at the PLT-sized ends. (H) Jasplakinolide, 5 µM ([Jas], actin stabilizer) and 1 mM erythro-9-(3-[2-hydroxynonyl]) ([EHNA], cytoplasmic dynein inhibitor) inhibit shear-induced proPLT production in primary mouse MKs. (I) Representative images of drug-induced inhibition of proPLT production under physiological shear stress (from H). Scale bars represent 50 µm.