The Ashwell receptor mitigates the lethal coagulopathy of sepsis

Abstract

The Ashwell receptor, the major lectin of hepatocytes, rapidly clears from blood circulation glycoproteins bearing glycan ligands that include galactose and N-acetylgalactosamine. This asialoglycoprotein receptor activity remains a key factor in the development and administration of glycoprotein pharmaceuticals, yet a biological purpose of the Ashwell receptor has remained elusive. We have identified endogenous ligands of the Ashwell receptor as glycoproteins and regulatory components in blood coagulation and thrombosis that include von Willebrand factor (vWF) and platelets. The Ashwell receptor normally modulates vWF homeostasis and is responsible for thrombocytopenia during systemic Streptococcus pneumoniae infection by eliminating platelets desialylated by the bacterium's neuraminidase. Hemostatic adaptation by the Ashwell receptor moderates the onset and severity of disseminated intravascular coagulation during sepsis and improves the probability of host survival.

Figure 1

Ashwell receptors of hepatocytes modulate vWF homeostasis and blood coagulation. (a–c) Liver sections from 8-week-old WT C57BL/6NHsd and Asgr-deficient mice imaged by phase-contrast (Ph) and fluorescent deconvolution microscopy using antibodies to vWF (green) and Asgpr-1 or Asgr-2 (red); DNA is stained by DAPI (blue). The percentage of vWF colocalized (yellow) with Asgr-1 (a) or Asgr-2 (b) in WT hepatocytes is indicated. Magnified views of the boxed regions are shown. Staining with Texas Red–conjugated secondary antibody to goat IgG (2°) alone is also shown. vWF abundance in Asgr-1–deficient hepatocytes compared to WT or Asgr-2–deficient hepatocytes is quantified (c). The percentage of vWF colocalized in Asgr-1–deficient or Asgr-2–deficient hepatocytes is indicated. The micrographs shown are representative of ten fields of view obtained from three mice of each genotype. All scale bars denote 5 μm unless otherwise indicated. (d) Plasma vWF abundance in mice lacking either Asgr-1 or Asgr-2. Horizontal bars indicate median vWF abundance, and vertical bars denote the interquartile range. (e) Coagulation factor measurements in mice lacking either Asgr-1 or Asgr-2 compared with WT littermates, ***P < 0.001. (f) Half-life of biotinylated plasma vWF from WT mice transfused into either WT or Asgr-1–deficient recipients. Plots represent data from eight WT and nine Asgr-1–deficient recipients. (g,h) Bleeding times (g) and bleeding times and plasma vWF abundance (h) in mice of the indicated genotypes. Mice homozygous for a null (deletion) mutation in the St3gal4 gene are denoted as St3gal4^Δ/Δ. Histograms include data from 20–25 mice of each genotype. All values are means ± s.e.m.; ***P < 0.001; *P < 0.05.

Figure 2

The Ashwell receptor and the ST3Gal-IV sialyltransferase participate in platelet clearance. (a–c) Platelet abundance (a), mean platelet volume (b), and reticulated platelet frequencies (c) in mice of indicated genotypes. Histograms include data from 20–25 mice of each genotype. (d) Platelet half-life analyses in mice of indicated genotypes. Each curve includes data from six mice. Three separate experiments were performed. (e) Platelet antigens (CD41, left images) were detected in perfused liver sections of WT and St3gal4^Δ/Δ mice, and hepatocytes were further visualized with DAPI and the actin-binding protein phalloidin (right images). Kupffer cells were detected by antibodies to CD68 (green). The images shown are representative of multiple fields of view from three mice of each genotype, comprising a total of 200 separate CD41 signals. (f) Sections of perfused liver were analyzed from WT recipients of biotinylated platelet-rich plasma from donors of the indicated genotypes to detect platelets (CD41, green) and Asgr-1 (red). Images are representative of multiple fields of view from three mice of each genotype, comprising a total of 46 separate CD41 signals. Scale bars denote 5 μm unless otherwise indicated. ***P < 0.001. All values are means ± s.e.m.

Figure 3

Thrombocytopenia caused by NanA-dependent platelet desialylation and Ashwell receptor function in S. pneumoniae infection. (a) Asialoglycoproteins were detected by Ricinus communis agglutinin (RCA) and Erythrina cristagalli agglutinin (ECA) lectin binding at the indicated times after infection with S. pneumoniae strain D39 (S. pneumoniae WT). PNA lectin binding was used to detect the Thomsen Friedenrich antigen. Mean fluorescence is enumerated at the indicated times after infection. (b) Detection of asialoglycoproteins on platelets and red blood cells after infection with the S. pneumoniae NanA⁻ mutant. Data in a and b are representative of three to five independent experiments comparing at least six mice per group. (c) Detection of asialoglycoproteins on platelets after infection with the S. pneumoniae NanA⁻ mutant complemented by a functional nanA gene in trans (NanA^T). Data are representative of four mice analyzed. (d) Platelet abundance in four to eight mice of the indicated genotypes after infection with S. pneumoniae WT, S. pneumoniae NanA⁻ or S. pneumoniae NanA⁻ complemented with NanA^T (NanA⁻ + NanA^T). Similar results were obtained after S. pneumoniae WT infection of Asgr-2–deficient mice (data not shown). SPN, S. pneumoniae. (e) Platelet turnover in uninfected WT mice and in WT or Asgr-1–deficient mice after S. pneumoniae WT infection. Each plot includes data from six mice. (f) Detection of asialo-vWF by RCA lectin binding and total vWF levels at 0 and 48 h after infection with S. pneumoniae WT when bacteremia was >1 × 10⁹ CFU/ml. Each histogram presents data from four to six mice. All comparative analyses were performed on mice with equivalent levels of bacteremia at each timepoint. ***P < 0.001 and **P < 0.01. All values are means ± s.e.m.

Figure 4

Extension of lifespan and reduction in coagulopathy by the Ashwell receptor in lethal S. pneumoniae infection. (a) Survival times of WT and Asgr–deficient mice after infection with a lethal dose of S. pneumoniae WT. (b) Survival times of WT mice infected with S. pneumoniae variants that retain or lack NanA sialidase activity. (c–e) Tissue histopathology in mice of the indicated genotypes 24 h after infection with S. pneumoniae WT. (c) Hemorrhage of the spleen with involvement of the lymphoid follicles occurred in all mice lacking Asgr-1. Data are representative of results from eight littermate pairs. (d) In the kidney, reduced vascular flow and increased deposition of fibrin (F) were evident in capillaries of the glomeruli and between the tubules. Rbc, red blood cell. (e) In the left four images, fibrin deposition was also markedly increased in the veins and sinusoids of the liver in Asgr-1 deficiency (left and center graphs). In the right four images, hepatocyte death was detected by the presence of pyknotic nuclei (right graph). Boxed regions are shown enlarged to the right. Scale bars denote 100 μm (c), 10 μm (d) and 50 μm (e). Images in c–e are representative of 20–30 randomly selected fields of view used for quantitation. These studies included six or more littermate comparisons of the indicated genotypes infected with equivalent doses of S. pneumoniae variants (2 × 10⁵ CFU administered i.p.). All comparisons were performed on mice with equivalent levels of bacteremia. ***P < 0.001 and **P < 0.01. All values are means ± s.e.m.

Figure 5

Increased severity of coagulopathy in sepsis after infection with the S. pneumoniae NanA⁻ mutant. Tissue histopathology in WT mice 24 h after infection with S. pneumoniae WT (D39) or S. pneumoniae NanA⁻. (a) Splenic hemorrhage occurred in all mice infected with the S. pneumoniae NanA⁻ strain. (b) Accumulation of fibrin (F, arrowheads) and impaired vascular flow are evident among capillaries of kidney glomeruli and between tubules. (c) Fibrin deposition in liver veins and pyknotic nuclei indicating cell death. Boxed regions are shown enlarged to the right. Scale bars denote 100 μm (a), 10 μm (b) and 50 μm (c). Images are representative of 18–24 randomly selected fields of view used to quantify results. These studies include three to six littermate comparisons of the indicated genotypes infected with equivalent doses of S. pneumoniae variants (2 × 10⁵ CFU administered i.p.). All comparisons were performed on mice with equivalent levels of bacteremia. ***P < 0.001 and **P < 0.01. All values plotted are means ± s.e.m.

Figure 6

The Ashwell receptor decreases mortality in S. pneumoniae sepsis. None of the 15 Asgr-1–deficient mice survived infection with a low dose of S. pneumoniae WT (D39) (1 × 10³ CFU administered i.p.). In contrast, 6 of 16 WT littermates survived. This finding was reproduced in three independent experiments with additional cohorts of mice.