FV⁺MKs release functional FV⁺platelets in vivo. (A) Day 11/12 MKs were incubated with 200 nM FV–Alexa 488 for 1 hour at 37°C prior to infusion into NSG mice. Representative flow plots showing the sizes of CD42a⁺CD42b⁺ FV⁺ (red) and FV⁻ (black) human platelet events detected in the circulation of mice at the specified time points after the infusion of FV-labeled MKs. The sizes of mouse platelets are shown in gray. (B) Left: relative sizes of endogenous mouse platelets and infused human donor platelets. Platelets were considered large if their FSC-As were larger than 90% of the mouse platelets’ FSC-As. Right: percentage of FV⁺ (red) and FV⁻ (black) platelets that were large at the various time points postinfusion. Mean ± 1 SEM is shown. (n = 4) (C) Day 11 MKs double-labeled with calcein red-orange (red) and FV–Alexa 488 (green) were infused into NSG mice 30 minutes prior to the induction of the first cremaster arteriole laser injury. Representative confocal images of clots formed after laser injury are shown. Mouse platelets are labeled with CD41–Alexa 647 (blue). All human platelets derived from infused MKs are calcein-labeled (red). FV-labeled (green) human platelets appear yellow in the overlay. Scale bars shown are 10 µm. (D) Representative flow plots showing the percentage of FV-labeled MKs that were infused (left) and FV-labeled human platelets circulating in mice postinfusion (right). (E) Quantification of the percentages of FV-labeled MKs that were infused with 5 independent studies, FV-labeled human platelets detected in the circulation postinfusion (n = 4), and FV-labeled platelets detected in the clot (n = 25). *P < .05, **P < .01, ***P < .005 for all statistical analyses.