Page 240 - Haematologica April 2020

D.E. van der Wal et al.
within lysosomes and cleaves oligosaccharides and gly- copeptides. NEU2 and NEU3 are specific for gangliosides and located in the cytosol and on the plasma membrane respectively,14,15 whereas NEU4 is located within mito- chondria16 and cleaves all aforementioned substrates. Sialic acid is also present in mitochondria.17
Within secretory lysosomes NEU1 is complexed with other degradation enzymes including sulphate 6-sul- phatase, β-galactosidase and cathepsin A.13 NEU are involved in many cell signalling processes: NEU1 binds Toll-like receptors,18 negatively regulates lysosomal exocy- tosis19 and suppresses cell adhesion by interfering with integrin phosphorylation, ERK1/2 and matrix metallopro- teinase-7 signalling.20 NEU3 also interacts with α6β4-inte- grin, inducing ERK signalling.21
Earlier studies have shown upregulation of membrane- associated NEU1 in platelets following cold-storage,22 and in ITP-patients where anti-GPIbα antibodies were pres- ent.10 An association between sialic acid and platelet acti- vation has been previously observed,23 whereby surface sialic acid increases following stimulation of platelets by ADP, thrombin and collagen. Additionally, sialic acid is cleaved off the platelet membrane following GPIbα-clus- tering after binding its ligand VWF.24 Addition of a NEU inhibitor, 2-deoxy-2-3-didehydro-N-acetylneuraminic acid (DANA), prevents clustering, indicating a potential role for desialylation in GPIbα-mediated platelet activa- tion. While low levels of NEU activity have been demon- strated in platelets,25 the role of NEU1-4 in the haemostatic function of platelets has not been studied. The aim of this study was therefore to investigate the role of both NEU1 and NEU2 in general platelet function under physiological conditions.
Methods
Ethics
Ethics approval was obtained from Australian Red Cross Blood Service Ethics Committee prior to conducting this study.
Platelet incubations
See the Online Supplementary Material and Methods (reagent sources, concentrations and further experimental details). Platelet rich plasma (PRP)/platelets were isolated10 from whole blood, collected in sodium citrate, diluted (200x109/L with non- autologous apheresis-derived plasma/HEPES-Tyrode’s buffer [Tyrode’s] respectively), stimulated with agonists ± inhibitors (Table 1).7
Glycan binding lectins
Washed platelets/PRP/apheresis platelets were incubated with fluorescein-conjugated lectins (5 μg/mL), Ricinus Communis Agglutinin-1 (RCA-1, 1/500) and Wheat Germ Agglutinin (WGA, 1/1000) to assess galactose/sialic acid exposure respec- tively by flow cytometry (20 min, 21°C, BD FACSCAnto II, FACS Diva software, San Diego, CA, USA.).
Membrane NEU expression
PRP/washed platelets (± agonists/inhibitors) were diluted 1/2, stained with anti-NEU1, anti-NEU2 or anti-NEU4 (1/60, 30 min at 21°C) followed by anti-goat A488 or anti-rabbit A647 (1/60, 30 min, 21°C) antibodies respectively. Platelets were fixed (1% paraformaldehyde [PFA] prior to flow cytometry. Single platelets were gated; doublets and small aggregates were excluded.
Platelet activation markers
To assess αIIbβ3-integrin activation, PAC-1-FITC (neat, 2x106 platelets), anti-fibrinogen-FITC was added (1/50) to 50 μL of 2x106/L platelets (15 min, 21°C). Washed platelets were stimu- lated (VWF+ristocetin; VWF/risto), stained with anti-lysosomal- associated membrane protein 1 (LAMP-1, 1/50, 45 min, 21°C) and anti-mouse A488 or P-selectin-PE (45 min, 21°C).
Aggregation of washed platelets
Aggregation or agglutination (VWF/risto) was performed with indicated agonists using an AggRAM aggregometer (Helena Laboratories, Beaumont, TX, USA), stirring at 600 rpm.
NEU-activity
Activity of NEU in apheresis plasma (1/8 and 1/32 diluted in MQ H20) was measured using an (adapted) protocol provided by C.A. Foote (Dalton Cardiovascular Research Center and26, Online Supplementary Materials and Methods). Activity of recombinant NEU was measured by Amplex Red Sialidase kit (recNEU, 2.5- 40 mU/mL, diluted in reaction buffer) ± fibrinogen, collagen and D-dimer (500 μg/mL). Fluorescence was measured after 30 min, (excitation λex=530nm, emission λem=590nm).
Adhesion of platelets to fibrinogen
Glass coverslips were coated (100 μg/mL fibrinogen 2 hours [h], 21°C) and pre-blocked (1% BSA/PBS, 1 h, 21°C). PRP±DANA (1 mM, 15 min, 37°C), prior to adhesion (30x109/L; 30 min, 37°C). Platelets were fixed (2% PFA, 20 min, 21°C), per- meabilised (0.5% Triton X-100) and blocked (5% BSA) prior to phalloidin-CF594 (1/42; 20 min, 21°C) staining. Fields of view (FOV) were analysed (ImagePro Premier v9.2, Media Cybernetics, Rockville, MD, USA).
Intracellular localisation of NEU1 and NEU2
Washed platelets (±VWF/risto, 5 min, 37°C) were fixed (1% PFA, 15 min, 21°C), unreacted aldehyde was neutralised (20 mM NH4Cl-Tris) and adhered (200x109/L) to pre-coated microchan- nel slides (25 μg/mL laminin, 1 h, 37°C). Platelets were perme- abilised (0.1% Triton X-100, 10 min, 21°C), blocked (1% BSA, 1 h, 21°C) prior to antibody staining (Online Supplementary Materials and Methods). Secondary anti-mouse A488 (1/2000), anti-rabbit A647 (1/800, 1 h, 21°C) and non-fading mounting media were added. Images (FITC and Texas Red filters) were captured with 60X water objective, additional 1.6X optical zoom (10X ocular; 960X total magnification) on Olympus IX71 (Olympus Corporation, Tokyo, Japan) inverted microscope (DP71 CCD camera). All images were taken with the same exposure time across treatments, with exception of NEU2 (1/2 exposure time), due to high fluorescence post-stimulation.
Statistics
Data were analysed using one-way ANOVA or paired t-tests using (GraphPad Software, Inc version 7.05.). A P-value of <0.05 was considered to be significant.
Results
Desialylation in platelets in presence of plasma
Platelets were stimulated with different agonists to investigate desialylation and cleavage of other glycans, using glycan-binding lectins and flow cytometry. Activation of GPIbα only by VWF (risto), but not ADP, increased desialylation by more than two-fold compared to unstimulated controls, as deduced from RCA-1-bind-
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