GPIbα^−/−platelets exhibit impaired binding to hepatocytes and do not significantly induce hepatic TPO generation in vitro. FL83B cells (murine hepatocytes) were cocultured with CMFDA-labeled (A-B) or nonlabeled (C-E) WT or GPIbα^−/− platelets (1:20 ratio) for 24 hours. (A) Representative immunofluorescence images of CMFDA-stained platelets bound to hepatocytes. Blue = 4′,6-diamidino-2-phenylindole (DAPI; nuclear counterstain), green = CMFDA-stained platelets, red = albumin (hepatocyte marker). Scale bars, 20 µm. Quantification of CMFDA-stained platelet–hepatocyte adherence presented as platelet mean fluorescence intensity (MFI) per field, analyzed from 3 individual experiments of 3 randomly selected fields. (B) Flow cytometry analysis of hepatocyte-associated CMFDA-stained platelets, quantified as CMFDA-stained platelet–positive hepatocytes. Alexa Fluor 647–conjugated anti-CD61 antibody was subsequently used to stain the hepatocyte surface-bound platelets (CD61⁺ population). TPO protein concentration was quantified by ELISA in hepatic cell lysates (C) and culture medium (D). (E) TPO mRNA expression in hepatocytes was measured by RT-qPCR (n = 3; 3 individual experiments in triplicate). (F-H) Platelets from the indicated strains (WT and GPIbα^−/−) were incubated with FL83B cells for 24 hours. In some instances, platelets were desialylated with neuraminidase. (F-G) FL83B cellular TPO mRNA expression was measured by RT-qPCR. (G-H) Asialofetuin, a competitive blocker of AMR, was added as indicated. (H) The CMFDA-platelet–positive population was gated and quantified by flow cytometry to evaluate platelet binding or uptake by hepatocytes following desialylation. *P < .05, **P < .01, ***P < .001, ****P < .0001 vs platelet-negative control, ^#P < .05 vs asialofetuin-positive platelet-negative control. NS, not significant.