Mature MKs take up FV into their α granules. (A) Surface expression of LRP1 receptor on day 14 MKs. Left: representative flow plot of LRP1 surface expression on LG and HG/CD42b⁺ MKs. Right: graph quantifying median fluorescence intensity of LRP1 expression over isotype for LG and HG/CD42b⁺ MKs. Mean ± 1 SEM is shown (n = 5). (B) Day 14 MKs were pulse-labeled with 200 nM FV–Alexa 488 or Alexa 647 for 1 hour at 37°C. Left: representative flow plot of FV uptake by CD42b⁺ MKs of different ploidy classes. Right: graph quantifying median FV fluorescence over background associated with MKs of different ploidy classes (n = 4). (C) Day 11 or day 15 MKs pulse-labeled with FV as in panel B. Left: representative flow plot of FV uptake by LG and HG/CD42b⁺ MK populations is shown. Right: graph quantifying median FV fluorescence over background associated with LG and HG/CD42b⁺ MKs on days 11 and 15. (D) Day 11 or 14 MKs labeled with FV and CD42b were sorted for gene expression analysis by quantitative polymerase chain reaction (qPCR). Messenger RNA (mRNA) expression of late MK markers relative to housekeeping gene TATA box binding protein (TBP) in sorted CD42b^low, CD42b^high FV⁻, and CD42b^high FV⁺ populations is shown (n = 4). (E) Immunostaining of CD42b and VWF was performed on day 14 FV-labeled MKs that were adhered to fibronectin-coated glass coverslips. Confocal images of the MKs show FV colocalizing with VWF in α-granules. Images were acquired using a DMi8 microscope (Leica Biosystems) equipped with a 63× Plan Apochromat objective (1.4 NA) and a Hamamatsu Photonics ORCA-Flash4.0 sCMOS digital camera. (F) Day 11 MKs were pulse-labeled with FV as in panel B. Excess FV was removed by washing, and MKs were followed for 48 hours. Representative flow plot showing that the intensity of FV fluorescence associated with HG/CD42b⁺ MKs 1 hour postlabeling is the same as that associated with HG/CD42b⁻ MKs 48 hours later, indicating that HG/CD42b⁺ MKs give rise to HG/CD42b⁻ MKs. *P < .05, **P < .01, ***P < .005, ****P < .001 for all statistical analyses. DAPI, 4′,6-diamidino-2-phenylindole; Max, maximum.