Regulation Of Factor VIII Clearance By Mannose-Binding Lectins
Notice bibliographique
Résumé
Abstract 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. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».