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D.E. van der Wal et al.
DANA, suggesting potential competition for the same binding site by NEU and RGDS, further linking αIIbβ3 acti- vation and NEU activity. Cations are essential for complete fibrinogen-binding to αIIbβ3.37 Ristocetin induced PAC-1 binding and was only sensitive to DANA-inhibition upon addition of calcium (Figure 4B). Similarly, PAC-1 binding was also further increased by calcium addition, confirming the importance of calcium in NEU activity (Figure 4B). In contrast, ADP-induced PAC-1 binding was unaffected by DANA treatment, demonstrating again that NEU activity is GPIbα-VWF specific (data not shown). Basal levels of fib- rinogen binding in washed platelets were much greater than in PRP (Figure 4C), indicating some activation had occurred that may have led to higher basal NEU1 and NEU2 membrane association. Remarkably, the recNEU induced RCA-1 binding was significantly reduced by addi- tional fibrinogen, due to saturation of αIIbβ3-integrin’s fib- rinogen binding site (Figure 4D); however fibrinogen-bind- ing kinetics will be different during aggregation/adhesion.
These findings emphasise the importance of calcium in modulating surface bound NEU expression following GPIbα-clustering. This facilitates αIIbβ3 activation, which in turn potentiates NEU activity and is downregulated again by fibrinogen-binding.
NEU-activity and platelet aggregation
To further examine GPIbα-mediated signalling without plasma in a buffered system, washed platelets were stim- ulated by VWF/risto and agglutination was measured. DANA treatment increased agglutination, which was fur- ther increased by addition of fibrinogen (Figure 5A). When binding of fibrinogen to platelets was blocked with RGDS, agglutination was only slightly reduced. These data indicate an inhibitory role of NEU activity in VWF- mediated agglutination. Additionally, DANA treatment increased fibrinogen-binding to αIIbβ3 (Figure 5B). To inves- tigate the role of NEU in other activation pathways, washed platelets were pre-incubated with DANA prior to addition of collagen and AA. DANA had no effect on platelet aggregation in response to these agonists (Figure 5C). Static adhesion and spreading of platelets to a fibrino- gen coated surface was also unaffected by DANA (Figure 5D). It is important to note that additional platelet adhe- sion receptors and mechanisms are involved in platelet adhesion when compared to platelets in suspension. As fibrinogen binding appeared to be linked with NEU-activ- ity, recNEU was incubated with fibrinogen and in line with the previous results; fibrinogen enhanced the activity of recNEU (Figure 5E). In contrast, when using control proteins of similar molecular weights, NEU-activity was completely abolished by collagen, while D-dimer showed inhibition by ~50% (Figure 5E). NEU activity in plasma (n=4) was 187.47±22.81 mU/mL (1/32 dilution), while only 84.28±11.26 mU/mL was found when a dilution of 1/8 was used, indicating an inhibitory effect by plasma factors. The maximum platelet activity of 80 mU/mL was reached following platelet permeabilisation by Triton X- 100: using 400x106/mL platelets. When NEU activity was measured without Triton X-100 (Amplex Red assay), only 35.45±3.51 mU/mL (1/8 dilution), which was ~40%.
Intracellular NEU localisation
It was hypothesised that in order for NEU to cleave their substrates, the enzymes would need to be localised on or within the platelet membrane. Previous findings in cold-
stored22 or platelets from ITP-patients10 showed that intra- cellular stores of NEU1 appear to be localised in ‘granule’- like organelles; however the actual location was not shown. Therefore, the intracellular origin of NEU was investigated. In permeabilised unstimulated platelets, NEU1 stained in a punctate pattern within the cytoplasm and on the cellular periphery, while NEU2 staining was mostly cytoplasmic and punctate (Figure 6A-B). NEU1 did not co-localise with the lysosomal/δ-granule markers LAMP-1 and β-galactosidase as initially anticipated, nor with an α-granule markers coagulation factor V (FV) (Figure 6A) and P-selectin (Figure 6C). Upon further inves- tigation, NEU1 did however appeared to co-localise with mitochondria to a limited extent (Figure 6A) in approxi- mately 20% of permeabilised platelets. Within these, 10- 100% of NEU1 co-localised with mitochondria, whereas the remaining NEU1 was sequestered in other locations. Mitochondria did not stain in unstimulated and stimulated non-permeabilised platelets (Online Supplementary Figure S4), while granule and lysosome contents were released following VWF/risto incubation (Online Supplementary Figure S2A), in line with the flow cytometry data. Although mitochondria are potentially releasing NEU1, the mitochondrial protein was not retained on the platelet membrane (Figure 6A). NEU2 staining was mostly cyto- plasmic and punctate (Figure 6B). Following GPIbα activa- tion (stimulated), NEU2 surface localisation was signifi- cantly enhanced. Difficulties were encountered visualising this localisation and due to the large increase in fluores- cence (Fig. 6B), the exposure time had to be halved. As with NEU1, NEU2 failed to co-stain with LAMP-1 (data not shown). In contrast to NEU1, NEU2 co-localised with P-selectin (Figure 6C), which is in line with results using DANA, whereby DANA partially reduced expression of P-selectin following ristocetin-stimulation (Figure 6D).
When using a general membrane dye (Figure 6E), some co-localisation was observed, although not 100%. As a control for non-specific staining, platelets were incubated with a secondary antibody only, and no fluorescence was observed (not shown). The overall findings from this study and a proposed model of NEU activity are presented in Figure 7.
Discussion
In this study, we have demonstrated a novel role for NEU1 and NEU2 in platelets, which is highly dependent on VWF-GPIbα and consequent αIIbβ3-integrin activation. Specifically, we have demonstrated that NEU1, NEU2 and NEU4 are present on the plasma membrane of unstimulat- ed platelets. Specific clustering of GPIbα by VWF triggers increased membrane association of NEU1 and NEU2, par- tially from their respective intracellular stores mitochon- dria and α-granules, which is even more pronounced under high shear conditions. Membrane association of NEU is highly regulated by mechanisms different for NEU1 and NEU2.
GPIbα is heavily glycosylated, containing N- and O- linked glycans, capped by sialic acid. Desialylation has been studied before in cold-stored platelets and ITP.6,10,38 The glycan changes in platelets under these conditions are similar to those observed following activation by VWF,8,9,27,32,39 as clustered GPIbα leads to various degrees of glycan cleavage (e.g. sialic acid and/or galactose). To date,
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