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

Approche in vivo de la fonction de deux protéines de l'hémostase (facteur von Willebrand et facteur X)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Cellular expression and in vivo clearance of O-glycosylation mutants.

<p><i>Panel A:</i> pNUT vectors containing WT-m<i>Vwf</i> cDNA, T1255A/T1256A or the Del-O-Gly mutant were transfected in COS-7 cells by electroporation. 96 hours later, cell supernatants were collected and cells were lysed. VWF antigen levels were measured by ELISA in the supernatants (grey bars) and lysates (white bars) for each mutant and the WT mVWF. n = 7–8 individual transfections, * p = 0.022 using unpaired t-test when comparing to WT. <i>Panel B:</i> After injection with NHS-biotin, residual biotinylated VWF was determined at indicated time points. Data present the percentage of residual biotinylated VWF measured at t = 0, which was set at 100% for each mouse. Curves indicate the best fit for an exponential decay.</p

List of primers used for generation of O-linked glycosylation mutants.

<p>Mutated nucleotides are written in bold.</p

Ex vivo thrombus formation on collagen.

<p><i>Panel A:</i> Blood was collected from mice 4 days after hydrodynamic injection with pLIVE encoding WT VWF or one of the following mutants: T1255A, T1256A, T1255A/T1256A, S1486A. As a control, mice were treated with an empty pLIVE plasmid (empty pLIVE). Anticoagulated whole blood was incubated with rhodamine 6G to fluorescently label platelets and perfused over collagen-coated glass coverslips (flow rate 2500 s⁻¹) for a period of 1 min. Unbound platelets were removed by subsequent perfusion with Hepes-Tyrode buffer. Thrombus formation was then visualized via image acquisition using Metamorph software. Shown are representative images for WT VWF, each mutant and the negative control (empty pLIVE). <i>Panel B:</i> Thrombus formation was quantified using ImageJ software in order to calculate percentages of platelet surface coverage. Data represent the mean±SEM of three independent perfusions, with 3–8 images being analyzed for each perfusion. **: p = 0.006 using unpaired t-test when compared to WT. ***: p<0.0001 using unpaired t-test when compared to WT.</p

VWF∶Ag expression levels following hydrodynamic gene delivery.

<p>VWF-deficient mice were injected with 100 µg of pLIVE-m<i>Vwf</i>, WT or the various O-glycosylation mutants. Plasma was collected 96 hours later and VWF∶Ag levels were measured by ELISA. Data are represented as mean plus or minus SEM. Normal pooled plasma from 20 C57BL/6 mice was used as a reference and set at 100%. Results are expressed as a percentage of a normal murine level. n = 22 for WT cDNA and 4–5 for the various mutants. * p<0.05 calculated with unpaired t-test when comparing the mutant with WT cDNA.</p

Detection of T antigen on VWF in murine plasma.

<p>Microtiter wells coated with polyclonal anti-VWF antibodies (3 µg/ml) were incubated with serial dilutions of murine plasma previously adjusted for VWF concentration. After catching VWF, wells were incubated with neuraminidase (5 mU/ml) in PBS/1 mM CaCl₂ overnight at 37°C (neuraminidase incubation was omitted for data presented in panels B and C). Subsequently, wells were blocked with the avidin/biotin blocking kit and incubated for 2 hours at 37°C with btPNA (panel A), btECL (panel B) or btWGA (panel C). After washing, HRP-conjugated streptavidin was added to the wells and bound lectin was detected by measuring HRP activity using OPD as a substrate. <i>Panel A:</i> btPNA binding to pooled plasma of C57Bl6J mice (Pool), plasma of WT-VWF expressing mice (WT), plasma of Del-O-Gly expressing mice (Del-O-Gly) and plasma of VWF-deficient mice (VWF−/−); <i>Panel B:</i> btECL binding to Pool and WT; <i>Panel C:</i> btWGA binding to Pool and WT. Plotted is the relative response (% of maximum binding) versus concentration of VWF in diluted samples. The relative response is defined as binding being relative to binding of the lectins to WT VWF (5 µg/ml for btPNA and 0.125 µg/ml for btECL and btWGA), which was arbitrarily set at 100%. Data points represent the mean±SD of 3–6 measurements.</p

Multimeric analysis of O-glycosylation mutants.

<p>VWF-deficient mice were injected with 100 µg of pLIVE-m<i>Vwf</i>, WT or the various O-glycosylation mutants. Plasma was collected 96 hours later and analysis of plasma samples was performed by SDS/2% agarose gel electrophoresis.</p

Conservation of residues known to carry O-glycosylations on human VWF in a number of animal species.

<p>In bold are residues that are not conserved and in italics are residues that are modified but could still carry O-glycosylations (change of a threonine to serine). Residues are conserved when nothing is indicated.</p