Dual roles for hepatic lectin receptors in the clearance of chilled platelets

Abstract

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage beta(2) integrin binding to beta-N-acetylglucosamine (beta-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the beta-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Figure 1

Hepatocytes clear long-term refrigerated platelets. (a) Comparison of fresh (22 °C) or 48 h refrigerated (4 °C) biotinylated platelet survival in wild type (WT) mice. Data are from 5 mice for each condition. Insert. Platelet biotinylation is stable: fresh (red line); 24 h (green line) or 48 h (blue line) refrigerated platelet histograms. (b) ¹¹¹Indium-labeled refrigerated platelets were injected into WT mice and tissues were harvested after 30 min. The survival of ¹¹¹Indium-labeled platelets is also shown (Inset). Data are expressed as percent of total radioactivity CPM g⁻¹ tissue. Each bar depicts the mean values for 4 animals ± s.e.m. (c) Biotinylated fresh (22 °C, left panel) or 48 h refrigerated (4° C, right panel) platelets were injected into WT mice. Livers (upper panels) and spleens (lower panels) were harvested 5 min after platelet transfusion and the distribution of platelet-derived biotin determined. Abundant biotin (arrows) is detected in hepatocytes when 4 °C, but not when fresh, 22 °C platelets were transfused. Images are representative of 10 randomly selected fields in organs harvested from 3 different mice. (d) Biotin-positive hepatocytes or macrophages were quantified in sections of organs harvested 5, 15 and 30 min after platelet transfusion (* P < 0.05, ***P < 0.001). Spleen 22 °C (□) and 4 °C (■); Liver 22 °C (○) and 4 °C (●). Data from 3 mice at each time point ± s.e.m.

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.

Figure 3

The Ashwell-Morell receptor mediates the hepatic recognition and clearance of long-term refrigerated platelets. (a) βGalactose exposure on glycoproteins is detected with RCA I and ECA lectins. Lectin binding to fresh (22 °C), short-term cooled (0 °C) or long-term refrigerated (4 °C) platelets are compared. Exposure of βGlcNAc residues on platelet glycoprotein is detected with the sWGA lectin. The mean fluorescence detected on fresh platelets (22 °C) is defined as 1. Histograms report the mean ± s.e.m. for 3 separate experiments. (b) Long-term refrigerated platelets were co-injected with asialofetuin (ASF/4 °C), a competitive binding inhibitor of the Ashwell-Morell receptor, or fetuin as control (Fet/4 °C) (**P < 0.01, ***P < 0.001). Fresh control platelets were also transfused (22 °C). Data are compared to platelet recoveries and survivals of fresh platelets. (c) Short-term cooled platelets were co-injected with asialofetuin (ASF/0 °C), or fetuin (Fet/0 °C) (**P < 0.01, ***P < 0.001). Fresh platelets were transfused as a control (22 °C). Data are compared to platelet recoveries and survivals of fresh platelets. (d) Survival of transfused long-term refrigerated (4 °C) or fresh platelets (22 °C) in mice of indicated genotypes. Data on 22 °C platelet clearance: mean of 6 mice ± s.e.m.; Data on 4 °C platelet clearance: mean of 3-5 mice ± s.e.m.

Figure 4

Asialoglycoproteins on long-term refrigerated human platelets are targets for the Ashwell-Morell receptor on HepG2 cells. (a) Platelet concentrates were stored at 4 °C for up to 10 d. βGal or βGlcNAc exposure on human platelets was measured using RCA I and ECA lectin or sWGA lectin, respectively. MFI values for each lectin measured at 22 °C were set as 1. Each point is the mean ± s.e.m. of 4 independent experiments. NS > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. All values are compared to lectin binding values to 22 °C platelets. (b) Effect of soluble galactose, or βGlcNAc, on the ingestion of long-term refrigerated platelets (4 °C) by HepG2 cells. All values are compared to HepG2 cells incubated with 22 °C platelets. (c) Asialofetuin (ASF), but not fetuin (Fet), inhibits the ingestion of long-term refrigerated platelets by HepG2 cells. ASF or fetuin was added to HepG2 cells and platelets at the indicated concentrations. Each point is the mean ± s.e.m. of 3 independent experiments. All values are compared to HepG2 cells incubated with 22 °C platelets. **P < 0.01, ***P < 0.001 (d) Effect of Asialofetuin (100 μg ml⁻¹, ASF) or fetuin (100 μgml⁻¹, Fet) on the ingestion of human platelets refrigerated for up to 10 days by HepG2 cells. All values are compared to HepG2 cells incubated with fresh RT and 100 μg ml⁻¹ fetuin. NS > 0.05 *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

The extracellular domain of GPIbα is required for recognition of platelets by the Ashwell receptor. (a) Survivals of fresh (22 °C) or long-term refrigerated mouse platelets (4 °C) stripped of GPIbα 45 kDa domain by enzymatic treatment with O-sialoglycoprotein endopeptidase (+ OSP). Mean ± s.e.m. for 5-6 recipient mice. Inset. Flow cytometric analysis of GPIbα on untreated (− OSP) and treated (+ OSP) platelets. (b) Effect of the GPIbα N-Terminus removal by mocarhagin (Moc, inset) on the ingestion of fresh (22 °C) or long-term refrigerated human platelets (4 °C) by HepG2 cells. ***P < 0.001 (c) GPIbα immunoprecipitates (IP GPIbα) from fresh (22 °C) or long-term refrigerated mouse platelets (4 °C) were subjected to immunoblotting using RCA I or sWGA lectins. n = 3. (d) GPIbα immunoprecipitates from control platelets (−) or following treatment with OSP (+) were subjected to immunoblotting using anti GPIbα antibodies or RCA I lectin. GPIbα’s C-Terminus is indicated (*). (e) Immunoblotts of total platelet lysates from fresh (22 °C) or long-term refrigerated platelets (4 °C) using sWGA, RCA I lectins or antibodies to mouse vWf or GPIbα. GPIbα (*) and VWf (**) are indicated. n = 3. (f-h) GPIbα aggregates on human cold stored platelets. GPIbα is visualized by electron microscopy. Surface of a (f) fresh platelet (22 °C) and a (g) long-term refrigerated platelet (4 °C). (h) Quantification of gold aggregate size in control and stored platelets. A plot of L(r) - r vs. r visualizes clustering. Values <-1 indicate dispersal whereas values >1 indicate significant clustering.