The asialoglycoprotein receptor minor subunit gene (ASGR2) contributes to pharmacokinetics of factor VIII concentrates in Hemophilia A

Background The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its N-linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in Hemophilia A (HA) is unknown. Objective To investigate the variability in FVIII PK outcomes in relation to genetic variation in the ASGR2, encoding the ASGPR2 subunit. Patients/Methods Thirty‐two HA patients with FVIII:C ≤ 2 IU/dL underwent 66 single dose FVIII PKs. PK parameters were evaluated in relation to ASGR2 5’ untranslated region (5’UTR) polymorphisms, that were investigated by recombinant and white blood cell RT-PCR approaches. Results The 5’UTR polymorphisms determine a frequent and conserved haplotype (HT1) in a regulatory region. The HT1 homozygotes may differ in the amounts of alternatively spliced mRNA transcripts and thus ASGPR2 isoforms. Compared to the other ASGR2 genotypes, the c.-95TT homozygotes (n=9), showed three-fold longer Alpha HL (3.60 h, 95% CI 1.44-5.76, p=0.006), and the c.-95TC heterozygotes (n=17) showed 25% shorter MRT (18.5 h, 15.0-22.0, p=0.038) and 32% shorter Beta HL (13.5 h, 10.9-16.0, p=0.016). These differences were confirmed in patients (n=27) undergoing PKs (n=54) with full-length FVIII only. In different linear regression models, the contribution of the ASGR2 genotypes remained significant after adjustment by ABO genotypes and VWF:Ag levels, and explained 14% (MRT), 15-18% (Beta HL) and 22% (Alpha HL) of parameter variability. Conclusions Infused FVIII distribution was modulated by frequent ASGR2 genotypes, independently from and together with ABO and VWF antigen levels, which has potential implications for genetically tailored substitutive treatment in HA.

Asialoglycoprotein Receptor-Targeted Superparamagnetic Perfluorooctylbromide Nanoparticles

Background. The decrease in asialoglycoprotein receptor (ASGPR) levels is observed in patients with chronic liver disease and liver tumor. The aim of our study was to develop ASGPR-targeted superparamagnetic perfluorooctylbromide nanoparticles (M-PFONP) and wonder whether this composite agent could target buffalo rat liver (BRL) cells in vitro and could improve R2 ∗ value of the rat liver parenchyma after its injection in vivo. Methods. GalPLL, a ligand of ASGPR, was synthesized by reductive amination. ASGPR-targeted M-PFOBNP was prepared by a film hydration method coupled with sonication. Several analytical methods were used to investigate the characterization and safety of the contrast agent in vitro. The in vivo MR T2 ∗ mapping was performed to evaluate the enhancement effect in rat liver. Results. The optimum concentration of Fe3O4 nanoparticles inclusion in GalPLL/M-PFOBNP was about 52.79 µg/mL, and the mean size was 285.6 ± 4.6 nm. The specificity of GalPLL/M-PFOBNP for ASGPR was confirmed by incubation experiment with fluorescence microscopy. The methyl thiazolyl tetrazolium (MTT) test showed that there was no significant difference in the optical density (OD) of cells incubated with all GalPLL/M-PFOBNP concentrations. Compared with M-PFOBNP, the increase in R2 ∗ value of the rat liver parenchyma after GalPLL/M-PFOBNP injection was higher. Conclusions. GalPLL/M-PFOBNP may potentially serve as a liver-targeted contrast agent for MR receptor imaging.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR that is shed by Furin

ABSTRACTThe hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote-carriers of ASGR1-deletions exhibited ~34% lower risk of coronary artery disease and ~10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, we examined the regulation of LDLR and/or PCSK9 by ASGR1. We investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality in naïve HepG2 and HepG2-PCSK9-knockout cells by Western-blot and immunofluorescence.ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. The lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in naïve HepG2 cells significantly increased total (~1.2-fold) and cell-surface (~4-fold) LDLR protein. In HepG2-PCSK9-knockout cells ASGR1-silencing led to ~2-fold higher levels of LDLR protein and DiI-LDL uptake associated with ~4-fold increased cell-surface LDLR. Overexpression of WT-ASGR1 reduced primarily the immature non-O-glycosylated LDLR (~110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR (~150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103↓ into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.