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A. Dupont et al.
severe life-threatening bleedings. Identification of the causes of thrombocytopenia is crucial for the appropriate management of patients. A new and original concept relates to platelet desialylation a process in which terminal sialic acids are cleaved on the platelet surface and leads to accelerate platelet clearance and thrombocytopenia.
Sialic acids are terminal sugar components of glycopro- tein oligosaccharide chains. Platelet desialylation is involved in physiological platelet aging in vivo. Indeed, the removal of platelet sialic acid exposes β-galactose residues (considered to be senescence antigens), and facilitates platelet uptake by Kupffer cells in co-operation with hepa- tocytes via the hepatic asialoglycoprotein receptor.6,7 Over the past few decades, it has been shown that platelet desialylation is responsible for platelet clearance in many contexts, such as immune thrombocytopenia.8,9 Recently, Deng et al. described a new mechanism for platelet clear- ance mediated by active vWF bound to GPIba.10 More specifically, the researchers demonstrated that a vWF/botrocetin complex and vWF from a patient carrying a p.V1316M mutation led to β-galactose exposure in vitro. On the basis of these observations, Deng et al. predicted that this β-galactose exposure might be responsible (at least in part) for thrombocytopenia in type 2B vWD. However, this hypothesis had not previously been tested in vivo.
Methods
Patients
A total of 36 patients with type 2B vWD (from 17 unrelated families) and 35 healthy age- and sex-matched controls were enrolled. In accordance with the tenets of the Declaration of Helsinki, the study participants were informed about the anony- mous use of their personal data, and gave their written, informed consent. The study was approved by the local investigational review board (Lille University Medical Center, Lille, France). The French National Reference Centre for von Willebrand Disease database and biological resource center (plasma and DNA) were registered with the French National Data Protection Authority (reference: CNIL 1245379). Platelet counts and mean platelet vol- ume (MPV) were measured with an automated analyzer (XN-10, Sysmex France).
Mice
The type 2B vWD knock-in mouse model (p.V1316M) has been described elsewhere.11 Mice homozygous for the p.V1316M muta- tion are referred to hereafter as “2B mice”, and their control litter- mates are referred as wild-type (WT) mice. Platelet counts were determined with an automated analyzer (Scil Vet ABC Plus, Horiba Medical). Male and female mice were used indifferently. The study was approved by the local animal care and use commit- tee (reference: APAFIS#1294-2015072816482568).
Flow cytometry
platelets (5 μL) were incubated with lectins for 30 minutes (min) at room temperature in a final volume of 100 μL. The reaction was stopped with PBS (400 μL). In some experiments, washed murine platelets (100 μL, 105/mL) were treated with 10,000 U/mL of Peptide:N-glycosidase F (PNGase F) for 18 hours (h) at 37°C or treated with 100 μg/mL O-sialoglycoprotein endopeptidase (OSGE) for 30 min at 37°C. The platelets were washed and then stained with a lectin or an FITC-conjugated antibody against mouse GPIba, GPVI or aIIbβ3. Surface neuraminidase-1 (NEU1) expression was measured with rabbit anti-NEU1 antibody (4 μg/mL) and detected with Alexa Fluor 488 secondary antibody (6 μg/mL). Lectin or antibody binding was determined using a flow cytometer (a BD Accuri system for mouse samples, and a Beckman Coulter Navios system for human samples).
Statistical analysis
Statistical analyses were performed using Prism 6 for Mac soft- ware (version 6; GraphPad, Inc., San Diego, CA, USA). If only two groups were compared, a Student’s t-test was used. For three or more groups, a one-way analysis of variance (ANOVA) and Dunnett’s post-test were used. Before performing these tests, a D’Agostino-Person normality test was used to determine whether data were normally distributed. Equality of variance was tested with an F test prior to Student’s t-test or with Bartlett’s test prior to an ANOVA. Correlations were assessed by calculating Pearson’s coefficient r.
Results
Platelet desialylation in human and murine type 2B von Willebrand disease in vivo
To examine the putative link between platelet desialyla- tion and thrombocytopenia in type 2B vWD, we assessed β-galactose exposure at the platelet surface (i.e. a marker of sialic acid removal from glycoproteins). We first ana- lyzed the platelet count and the extent of platelet β-galac- tose exposure (by measuring RCA binding) in 36 patients with type 2B vWD and 35 healthy controls. The mean±Standard Deviation (SD) platelet count was signifi- cantly lower in the patient group (217±70x109/L) than in the control group (256±47x109/L; P=0.012). The amount of β-galactose [measured as the mean±SD fluorescence intensity (MFI) for RCA] was significantly higher in the patient group (7.0±2.6) than in the control group (5.5±2.3; P=0.011). The individual platelet counts were weakly cor- related with levels of surface-exposed β-galactose in patients with type 2B vWD (r2=0.113; P=0.048) but not correlated in controls (r2=0.095; P=0.092) (Figure 1A and B). Patients bearing the p.R1341Q mutation displayed a significantly lower mean platelet count and a significantly greater RCA MFI (Figure 1C and D). In contrast, the platelet count and RCA binding in patients bearing the p.R1306Q mutation did not differ significantly from the values observed for controls. Interestingly, patients carry- ing the severe p.V1316M mutation exhibited the lowest platelet count and the highest amount of β-galactose (2.1- fold more than controls) (Figure 1C and D). To take platelet size into account, we also measured the ratio between the RCA MFI and the MPV. The elevated level of RCA binding (relative to controls) was no longer observed for patients with the p.R1341Q mutation but was still observed for those bearing the p.V1316M mutation (Figure 1E).
Given that the p.V1316M mutation in vWF was associ-
Platelet surface β-galactose exposure was determined by using FITC-conjugated Ricinus communis agglutinin I (RCA for platelet- rich plasma 12.5 μg/mL, for washed mouse platelets 5 μg/mL) and Erythrina cristagalli lectin (ECL, 10 μg/mL). Samples in which RCA or ECL was incubated with β-lactose12 (200 mM) were used as corresponding negative controls. Platelet surface a-2,3-sialyla- tion on O-glycans was determined by using biotinylated Maackia amurensis lectin II7,13 (MALII, 10 μg/mL) and phycoerythrin (PE)- streptavidin (10 μg/mL). Briefly, platelet-rich plasma or washed
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