Figure 2.

HepG2 cells ingest long-term refrigerated human platelets in vitro. (a) Ingestion of CM-Orange-labeled platelets is detected by flow cytometry as an increase in hepatocyte associated orange fluorescence (CM-Orange, y-axis). (b) Ingestion of fresh (22 °C), short-term (0 °C) or long-term refrigerated platelets (4 °C) by HepG2 cells. Long-term refrigeration increases the number of hepatocytes ingesting platelets by 4-5 fold. The inset shows a dose dependent inhibition by cytochalasin D of platelet uptake by HepG2 cells. Values are compared to room temperature platelet uptake. Mean ± s.e.m. of 3 experiments is shown. (c) HepG2 cells express both the ASGR1 and ASGR2 subunits of the Ashwell-Morell receptor but do not express α_Mβ₂ (CD11b/CD18) receptors. Representative flow cytometry histograms are shown. (d, e) Long-term refrigerated platelets are cleared predominantly by macrophage independent mechanisms. Survival of fresh room temperature (22 °C), short-term cooled (0 °C), long-term refrigerated (4 °C) or galactosylated long-term refrigerated (4 °C + UDP-Gal) platelets injected into recipient macrophages depleted (d) mice or in mock treated mice (e) are shown. The percentage of fresh platelets at 5 min in WT mice depleted of macrophages was set at 100%. Each time point is the mean of data from seven mice ± s.e.m.