In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor

The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments

Study of the clearance of proteins of hemostasis

Le but de ce projet de thèse a été d'étudier la clairance de glycoprotéines jouant un rôle clé dans l'hémostase, le facteur X (FX) et le couple facteur Willebrand (FW)/facteur VIII (FVIII) qui circule dans le plasma sous forme de complexe non covalent. Dans la première partie de nos travaux, nous avons étudié l'implication des N-glycosylations du FX sur sa clairance. Il avait été préalablement établi par l'équipe que ces N-glycans influençaient la cinétique d'élimination du FX. Nous avons désormais établi qu'ils influencent également la biodistribution et les interactions cellulaires de la protéine. Ainsi, comparé au FX, un variant de FX N-déglycosylé interagit avec des types cellulaires différents au niveau du foie qui est l'organe principal de biodistribution de cette protéine. Le FX N-déglycosylé se lie et est dégradé par les hépatocytes. Quant au FX, il se lie aux cellules de Kupffer. Cette liaison du FX aux cellules de Kupffer semble d'ailleurs faire partie d'un mécanisme original protégeant le FX d'une clairance accélérée. Le FW et le FVIII sont des glycoprotéines dont les glycans sont fortement sialylés. Dans la deuxième partie de cette thèse, le rôle du récepteur Sialic acid-binding Ig-like lectin 5 (Siglec-5) dans la clairance du complexe FVIII/FW a été étudié. Siglec-5 est un récepteur présent à la surface des macrophages, type cellulaire dominant dans la clairance du complexe FVIII/FW. Nous avons montré que le FVIII et le FW peuvent se lier au Siglec-5. De plus, une surexpression de Siglec-5 in vivo est associée à une diminution des taux endogènes de FW et de FVIII. Ces résultats suggèrent que Siglec-5 joue un rôle dans le catabolisme du complexe FVIII/FW.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Kamus istilah akuntansi : lebih dari 2.500 istilah mutahir di definisikam dan dijabarkan dengan gamblang

*Buku ini hanya bisa dibaca & fotocopy di dalam perpustakaan / tidak dipinjamkan.**Silahkan hubungi petugas untuk mengetahui informasi lebih lanjut.---Mencakup akutansi keuangan, pemeriksaan, analisis laporan keuangan, pajak disertai gambar, grafik & dafta

Coagulation factor X interaction with macrophages through its N-glycans protects it from a rapid clearance.

Factor X (FX), a plasma glycoprotein playing a central role in coagulation has a long circulatory half-life compared to closely related coagulation factors. The activation peptide of FX has been shown to influence its clearance with two N-glycans as key determinants of FX's relatively long survival. To decipher FX clearance mechanism, organ biodistribution and cellular interactions of human plasma FX (pd-FX), recombinant FX (rFX), N-deglycosylated FX (N-degly-FX) and recombinant FX mutated at both N-glycosylation sites (rFX(N181A-N191A)) were evaluated. Biodistribution analysis of (125)I-labelled FX proteins after administration to mice revealed liver as major target organ for all FX variants. Liver tissue sections analysis showed an interaction of pd-FX and N-degly-FX to different cell types. These findings were confirmed in cell binding studies revealing that FX and FX without N-glycans interact with macrophages and hepatocytes, respectively. N-degly-FX appeared to be degraded in hepatocytes while interestingly pd-FX was not by macrophages. Furthermore, the chemical inactivation of macrophages by gadolinium chloride resulted in a significant decrease of circulating pd-FX into mice and not of N-degly-FX. Altogether our data lead to the conclusion that FX interaction with macrophages through its N-glycans protects it from a rapid clearance explaining its relatively long circulatory half-life

Investigation of ¹²⁵I-pd-FX and ¹²⁵I-N-degly-FX degradation by differentiated THP-1 cells and HepG2 cells, respectively.

<p>(A) ¹²⁵I-pd-FX and ¹²⁵I-N-degly-FX were added to differentiated THP-1 cells and HepG2 cells, respectively, for a 1-hour period at 4°C. Cells were then washed, and incubation was continued at 37°C to initiate endocytosis. At indicated time points, cell lysates and cell supernatants were taken to determine the amount of degraded material. Degraded material is defined as the radioactivity that is soluble in 10% trichloroacetic acid. In all experiments, controls were included to determine the amount of nonspecific degradation in the absence of cells, which routinely was less than 10% of degradation in the presence of cells. Data represent mean ± S.D. of 3 experiments. (B) ¹²⁵I-pd-FX was incubated with differentiated THP-1 cells at indicated time points (1h at 4°C, then for 15′, 30′ and 60′ at 37°C). Cell lysate and supernatant were migrated on SDS-PAGE and results were visualized by autoradiography using PharosFX™ Plus Molecular Imager (BioRad, Hercules, CA, USA).</p

Identification of cells as main target for FX variants in liver.

<p>Liver sections from mice injected with 20 µg/mouse of pd-FX (C, F), N-degly-FX (B, E) or with PBS as control (A, D). Livers were collected 10 minutes after injection. In a first set of sections (A, B, and C) FX variants are stained with rabbit anti-human FX (Eurogentec followed by highly sensitive polymer-based detection reagent (HRP) (Dako, Trappes, France) and then with diaminobenzamidine. These tissue sections were slightly counterstained with diluted hematoxylin (magnification 200). One representative experiment out of six performed with different mice is shown. (D, E, and F) Merged images of liver sections stained with rabbit anti-human FX followed by Alexa Fluor 488 F(ab’)₂ fragment (Invitrogen) of goat anti-rabbit IgG (H+L) as secondary antibody (green) and with monoclonal rat anti-mouse CD68 (AbD Serotec, Oxford, UK) followed by TRITC-conjugated goat anti-rat immunoglobulins (Southern Biotech, Birmingham, AL, USA) (red). Original magnification 200. One representative experiment out of four performed with different mice is shown.</p