Page 250 - Haematologica April 2020

D.E. van der Wal et al.
PNGase digestion did not affect SNA-binding, demon- strating that some sialic acid was still present on remain- ing O-linked glycans, potentially those on VWF, as shown by the small increase in PNA-binding to the VWF T-anti- gen. However, as SNA-binding was unchanged following VWF/risto-stimulation, these α2,3-linked glycans are not NEU1 and NEU2 substrates. In this study, we did not investigate whether VWF or GPIbα originated from a for- merly internalised pool and was re-expressed on the membrane.
NEU membrane association is highly dependent on VWF-binding to GPIbα, as GPIbα removal by OSGE or inhibition by GlcNAc prevented membrane association. α2,3-linked sialic acid has been earlier described to be insensitive to OSGE-cleavage, indicating these structures might be attached to VWF or other platelet glycopro- teins.42 Control experiments with recNEU, which cleaves α2,3, α2,6 and α2,8-linked sialic acid, showed more bind- ing to MAL-1, ECL and RCA-1 when compared to VWF/risto alone, demonstrating that more pronounced desialylation had occurred.
In general, platelet granule contents are not released fol- lowing stimulation of GPIbα by VWF/risto without shear. However, this study showed that VWF-stimulation trig- gers P-selectin release as well as increased LAMP-1 mem- brane association, indicating release of α, δ-granule and lysosome content. This is consistent with the co-localisa- tion of NEU2 with P-selectin. However, this can also be partly attributed to some degree of pre-activation due to the long incubation times required for NEU staining, as LAMP-1 membrane-association is dependent on platelet activation.36
This study demonstrates that a negative feedback loop exists between the activity of NEU and platelet agglutina- tion, as inhibition of NEU activity by DANA potentiates platelet VWF-mediated agglutination. Following desialyla- tion, the underlying glycans on GPIbα, including βGlcNAc-residues, are more prone to further cleavage, which was previously found to reduce VWF-binding to platelets.3 NEU inhibition is described to block both desia- lylation and consequent degalactosylation by β-galactosi- dase, which was described as the first step in GPIbα-clus- tering.32 Interestingly, NEU1 activity was inversely corre- lated with integrin-mediated adhesion to laminin.20 Recent findings showed that pneumococcal NEUA induced platelet hyperactivity through desialylation, which was dependent on ADP-secretion.46
GM are glycosphingolipid-containing glycans involved in cell-cell recognition, adhesion and signal transduction, and may be another substrate for NEU1. GM3 blocks GPIbα-clustering by preventing linking with lipid rafts24 and GM blocks the second wave of aggregation by ADP.35 Our results demonstrate that GM might be important for NEU2 membrane association, and are potentially involved in the negative feedback loop between NEU activity and VWF-induced agglutination.
The results presented here also indicate that calcium inhibits NEU2 membrane association but promotes its activity, as treatment with DANA prevented the calcium- potentiated increase in PAC-1-binding. NEU activity is also important for integrin activation, as there was no fur- ther inhibition of fibrinogen binding by DANA in the presence of RGDS. Previous studies have shown that chelation of cations by EDTA generally inhibited the enzyme important for sialic acid metabolism, sialyl-trans-
ferase, as does ADP, and similar mechanisms might be important for NEU activity.25 Also, full activity of NEU (Vibrio cholera) was induced by calcium (1 mM47), which is in line with our findings, whereby DANA only inhibited PAC-1 in the presence of calcium. DANA was able to inhibit fibrinogen-binding and consequent activation of αIIbβ3, although this inhibition was only partial. This could be due to DANA being more specific for NEU1 and less effective in NEU2 inhibition, as another NEU inhibitor, Zanamivir, is more specific for NEU2.48 However, it is not known which NEU-inhibitor is most effective in blocking NEU activity in platelets. Also, NEU3 and NEU4 might also play a role in healthy platelets, further contributing to desialylation.
Following GPIbα-clustering, fibrinogen binds to αIIbβ3- integrin, a crucial step for platelet-platelet interactions and aggregation. Interestingly, in the presence of plasma, at least 50% of membrane expressed NEU1 was dependent on fibrinogen binding, as demonstrated by the RGDS blockade, while NEU2 was unaffected. Both NEU become highly membrane-bound when high concentrations of fib- rinogen are present. Of interest, the amino acid sequence of NEU2 contains a RGD-motif, which could potentially interfere with fibrinogen-binding. Additionally, calcium signalling plays an important role as its chelation by BAPTA-AM enhanced membrane association of NEU2. The need for a fibrinogen binding conformation of αIIbβ3 has also been demonstrated, as PAC-1-binding following VWF/risto-stimulation is low in the absence of calcium.27 Following VWF/risto-stimulation in the presence of satu- rating levels of fibrinogen, NEU1 and NEU2 became highly associated with the plasma membrane as shown by flow cytometry, potentially through their trans-membrane domain(s).49 Similar results were found by microscopy for NEU2, which was more highly expressed on the platelet surface following VWF/risto-stimulation, even without additionalfibrinogen.
Notably, washed platelets had a significantly higher MFI for RCA-1 binding in comparison to platelets from PRP, both pre- and post-stimulation with VWF/risto, correlating with higher fibrinogen binding due to washing and longer incubation times. Fibrinogen also increased recNEU activi- ty. Earlier findings showed that two-thirds of asialo-VWF binds to GPIbα, while the remainder binds to αIIbβ3 in the presence of fibrinogen. Without fibrinogen, asialo-VWF binds exclusively to αIIbβ3.50 When NEU becomes mem- brane-bound, it could potentially cleave platelet-bound VWF, thus enhancing its binding to GPIbα. In addition to cleavage of GPIbα itself, desialylation of glycosylated VWF and/or fibrinogen cannot be excluded, as desialylation also affects their platelet binding properties. Further to this, desialylated fibrinogen has a higher affinity for αIIbβ351,52 and platelet aggregation in response to asialo-VWF is approxi- mately 60% lower than native VWF.53 However, other studies have demonstrated spontaneous binding of asialo- VWF to GPIbα, in which was able to potentiate aggrega- tion in the presence of fibrinogen.54 These studies have established that the presence of plasma proteins including fibrinogen affect platelet desialylation and thereby also the VWF-binding potential. Whether NEU membrane expres- sion is important for VWF clearance is currently unknown, however a recent study demonstrated a link between VWF-desialylation (terminal α(2-6)-linked sialic acid) and its clearance in low-VWF patients,55 however no significant changes in SNA were observed.
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