Page 242 - Haematologica April 2020

D.E. van der Wal et al.
strating that in platelets from healthy individuals, a pro- portion of NEU1 and NEU2 is membrane expressed. Secondary antibody-only controls showed little non-spe- cific fluorescence (Figure 2A-B). Blockade of GPIbα-clus- tering with GlcNAc28 prevented the increase in NEU1 and NEU2 membrane association.
In addition, removal of the 45 kDa N-terminal domain of GPIbα (VWF-binding domain) with OSGE, to mimic GPIbα-deficient platelets,29,30 prevented the ristocetin- induced increase in NEU1 (Figure 2A-B), suggesting that NEU1 expression is highly dependent on VWF-binding to GPIbα. Furthermore, when fibrinogen binding to αIIbβ3- integrin was blocked using RGDS peptide, the increase in membrane NEU1 was also significantly reduced by 50% (Figure 2A), and there was also a trend towards decreased NEU2 expression (Figure 2B). Similarly, the proportion of platelets that were positive for NEU1 and NEU2 increased in PRP stimulated with ristocetin, but these changes were not significantly different when compared to unstimula- ted platelets or those treated with inhibitors (Figure 2C).
Desialylation in washed platelets
Washed platelets were used to prevent interference by plasma proteins such as fibrinogen. Cleavage of the extra- cellular domain of GPIbα by OSGE to mimic GPIbα-defi- cient platelets,31 reduced binding of all lectins by over 50% and below unstimulated control (Online Supplementary
Figure S1A), demonstrating the importance of the VWF- binding domain in NEU mediated desialylation. When only N-linked glycans were cleaved by PNGase F, binding to most lectins was also reduced, albeit to a lesser degree, indicating desialylation of N-linked glycans (Online Supplementary Figure S1A).
Recombinant NEU (recNEU), which desialylates sialic acid at α2,3, α2,6, or α2,8-linkages was used as a positive control to achieve complete desialylation. RecNeu alone significantly increased binding of MAL-1, ECL and PNA (not shown). RecNEU treatment further increased RCA-1 binding (Online Supplementary Figure S1B), when compared to VWF/risto alone (Figure 4D), albeit to a lesser extent than ECL-binding. PNA-binding was significantly increased (Online Supplementary Figure S1B), demonstrat- ing potential desialylation of VWF itself. SNA-binding however was insensitive to VWF/risto+recNEU, indicat- ing different and more extensive desialylation by recNEU (Online Supplementary Figure S1A). As a negative control, washed platelets incubated with ristocetin only, which showed no increase in membrane NEU1 and NEU2 (Online Supplementary Figure S1C).
To further confirm the role of GPIbα-clustering in translocation of NEU to the membrane, washed platelets were activated with various agonists (VWF/risto, collagen, thrombin, AA, ADP and U46619). Only clustering of GPIbα by VWF/risto, and to a lesser extent, arachidonic
Figure 1. Platelet desialylation is induced by von Willebrand factor-mediated glycoprotein Ibα clustering. Platelets in platelet rich plasma (PRP) (200 x 106/mL) were stimulated with ristocetin (3 mg/mL) and ADP (200 μM), then stained with fluorescein-conjugated lectins (A) RCA-1 or (B) WGA; 10,000 single platelets (doublets and small aggregates were excluded from analysis) were measured by flow cytometry (n=3). Washed platelets were stimulated (VWF/risto: 10 μg/mL VWF/ 1.2 mg/mL ristocetin), and (C) binding to fluorescein-conjugated MAL-1, ECL, PNA, SNA and RCA-1 lectins was measured by flow cytometry (n=3). Mean fluorescent inten- sities (MFI) ± standard error of mean (SEM). *P<0.05 significant when compared to unstimulated controls (unstim) using a one-way ANOVA. (D) Lectin binding in unstimulated platelets was set at ~1% positive and samples were analysed following risto or ADP-addition. Data ± SEM. *P<0.05 significant when compared to unstimulated controls (t-test). VWF: von Willebrand factor; GPIbα: glycoprotein Ibα.
A
B
CD
1084
haematologica | 2020; 105(4)