The extracellular domain of GPIbα is required for hepatic TPO generation. (A) Representative diagram of the IL4Rα/GPIbα-tg receptor. ELISA-determined serum (B) and plasma (C) TPO levels (1:2 dilution) in WT and IL4Rα/GPIbα mice (C57BL/6J background, n = 5). TPO mRNA levels were measured via RT-qPCR (D) and TPO protein concentrations were measured by ELISA (E) (n = 6) in WT and IL4Rα/GPIbα-tg mice hepatic tissue. Plasma TPO levels were measured by ELISA at the indicated time points following transfusion of 2.5 × 108 IL4Rα/GPIbα platelets into WT or GPIbα−/− mice (F) or 2.5 × 108 WT or GPIbα−/− platelets into IL4Rα/GPIbα-tg mice (G). Values are normalized to TPO levels on day 0 (100%) (n = 3). (H) Platelets from IL4Rα/GPIbα mice were incubated with FL83B cells for 24 hours after which FL83B cellular TPO mRNA expression was measured by RT-qPCR. In some instances, platelets were desialylated with neuraminidase. (I) Flow cytometry analysis of hepatocyte-associated platelets (WT, GPIbα, and IL4Rα/GPIbα) quantified as CMFDA-stained platelet–positive hepatocytes. (J) TPO mRNA expression in hepatocytes was measured after incubation with recombinant GPIbα-coupled beads compared with control beads and recombinant GPIbα alone. *P < .05, **P < .01, ***P < .001, ****P < .0001. NS, not significant.
