The coagulation factors von Willebrand factor (FVIII) and factor VIII (FVIII) circulate in the plasma in a non-covalent complex. VWF acts as a carrier for FVIII and protects it from proteolysis and accelerated clearance. However, in the absence of VWF, the molecular mechanisms that regulate FVIII clearance are largely uncharacterized.
The glycosylation of FVIII may regulate its interaction with lectin clearance receptors such as the asialoglycoprotein receptor and siglec-5. The FVIII polypeptide is modified by the addition of 24 N-linked glycans and 7 O-linked glycans. The majority of N-linked glycans on FVIII are complex type and predominantly localized to the B-domain. However, two high mannose glycans are found on the A1 and C1 domains. In these studies, we characterized the ability of mannose binding lectins to interact with FVIII and mediate its clearance from the plasma.
We have previously reported that the endothelial mannose binding lectin CLEC4M is a novel clearance receptor for FVIII. Removal of N-linked glycans, or pre-incubation of recombinant CLEC4M with the mannose polymer mannan attenuated binding of CLEC4M to FVIII. Additionally, the pre-incubation of CLEC4M-expressing cells with mannan significantly decreased binding and internalization of FVIII.
In these studies, high mannose glycans on recombinant human FVIII were removed using the endoglycosidase endoH. Effective removal of FVIII high mannose glycans by endoH was confirmed via a lectin binding assay to Galanthus Nivalis lectin (50%, p=0.006). The binding of recombinant CLEC4M-Fc to endoH-treated FVIII was measured using a solid phase binding assay. Removal of high mannose glycans attenuated the interaction between FVIII and CLEC4M-Fc by 41% (p=0.005). A 49% decrease in the internalization of endoH-treated FVIII was observed on CLEC4M-expressing cells relative to control (p=0.046).
We next sought to confirm the contribution of mannose binding lectins to the clearance of FVIII in a murine model. Although there is no murine ortholog to CLEC4M, mice express a series of CLEC4M homologs termed SIGNRs. Similar to CLEC4M, SIGNR1 is expressed on liver sinusoidal endothelial cells. While significant structural differences exist between CLEC4M and SIGNR1, they retain 59% amino acid identity between their carbohydrate recognition domains, and SIGNR1 has been shown to bind mannosylated as well as fucosylated glycoproteins.
We assessed the contribution of SIGNR1 to VWF and FVIII clearance. Plasma levels of VWF:Ag, and FVIII:C were quantified in SIGNR1 deficient mice by ELISA and chromogenic assay respectively. Endogenous VWF (88% of wild type, p=0.233, n=35) and FVIII (110% of wild type, p=0.435, n=35) in SIGNR1 deficient mice were not different compared to wild type mice. SIGNR1/VWF deficient mice were bred on a C57Bl/6 background; mice received tail vein infusions of recombinant human FVIII (200 U/kg) or plasma derived VWF-FVIII complex (200 U/kg). Blood was sampled via retro-orbital puncture. VWF:Ag and FVIII:Ag levels were measured with ELISA and elimination half life was calculated using one phase exponential decay. Half-lives of infused recombinant human FVIII (VWF-/- t½=8.74 minutes, SIGNR1-/-/VWF-/- t½=10.03 minutes, p=0.898) and human plasma-derived VWF-FVIII complex (VWF-/- t½=46.09 minutes, SIGNR1-/-/VWF-/- t½=49.83 minutes, p=0.934) were not significantly different.
We finally sought to characterize the contribution of other mannose-binding lectins to FVIII clearance in mice. VWF deficient mice received tail vein infusions of mannan (6 mg/kg) or saline followed after 3 minutes by tail vein infusions of recombinant human FVIII (200 U/kg). Pre-infusion of mannan into VWF deficient mice did not modify the half-life of FVIII relative to the control (control t½= 10.56 minutes, mannan t½= 9.38 minutes. p=0.33). We also assessed the clearance of endoH-treated FVIII in VWF deficient mice, and did not observe a change relative to the control (control t½=12.28 minutes, endoH FVIII t½=11.23 minutes, p=0.812).
These studies suggest that CLEC4M interacts with FVIII in part through its high mannose glycans in vitro. However, our in vivo studies were unable to reveal a significant role for mannose-binding lectins in FVIII clearance in mouse models.
James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.
Asterisk with author names denotes non-ASH members.