Page 245 - Haematologica April 2020

The role of neuraminidases in platelet function
S2A-B). RecNEU significantly enhanced LAMP-1 surface expression, P-selectin and PAC-1 binding relative to unstimulated platelets (Online Supplementary Figure S2A- C). Fibrinogen also potentiated LAMP-1 (Online Supplementary Figure S2A) when compared to VWF/risto- stimulation alone, while PAC-1 binding (Online Supplementary Figure S2C) was slightly decreased, as expected. As anticipated, addition of calcium increased PAC-1 and P-selectin (Online Supplementary Figure S2B-C), whereas OSGE abolished the VWF-induced increase in LAMP-1 and PAC-1-binding (Online Supplementary Figure S2A, C) indicating that dense granule/lysosome secretion and fibrinogen-binding do not occur when GPIbα is removed.
In summary, these findings demonstrate that NEU translocate to the plasma membrane following clustering of GPIbα by VWF, and NEU2 membrane expression is negatively controlled by high calcium concentrations. More importantly, fibrinogen-binding following platelet activation by VWF potentiated the NEU-translocation.
AB
The role of NEU-activity in αIIbβ3-integrin activation The data presented thus far demonstrate that NEU1 and NEU2 are specifically translocated to the membrane fol- lowing VWF-mediated GPIbα-clustering, and is down- stream of secondary signalling, leading to desialylation. To further investigate a potential role for NEU activity in platelet activation, a NEU-inhibitor DANA10,22 was used. DANA inhibited desialylation, as RCA-1-binding was sig- nificantly decreased (approximately two-fold decrease), but not completely inhibited, following recNEU-treat-
ment (Online Supplementary Figure S3).
Since our findings indicated that fibrinogen binding
potentiated the membrane association of NEU1 in partic- ular, the role of NEU activity in αIIbβ3-integrin activation was further studied using PAC-1 and fibrinogen antibod- ies. Incubation of PRP with DANA prior to addition of ris- tocetin significantly reduced fibrinogen binding (Figure 4A), suggesting NEU activity plays a role in fibrinogen binding. Furthermore, the partial inhibition of fibrinogen binding by RGDS was not observed in the presence of
CD
Figure 4. Neuramidase-inhibition reduces fibrinogen binding. Platelets in platelet rich plasma (PRP) were pre-incubated with neuramidase (NEU) inhibitor 2-deoxy- 2-3-didehydro-N-acetylneuraminic acid (DANA) and/or +/- αIIbβ3 integrin inhibitor RGDS prior to stimulation with ristocetin (3 mg/mL) and then stained with (A) fib- rinogen-FITC or (B) FITC-conjugated PAC-1 antibodies respectively. 10,000 single platelets were measured by flow cytometry. Data represents the mean fluorescent intensities (MFI) ± standard error of mean (SEM), n=3. *P<0.05, *: significant against unstimulated (unstim), §: significant against risto + calcium (Ca) (one-way ANOVA). (C) Unstimulated washed platelets (wash plt) or PRP were stained with fibrinogen-FITC antibody. (D) PRP was treated with RGDS, or fibrinogen, prior to recNEU-treatment (Table 1), then stained with fluorescein-conjugated RCA-1 (n=5). Data represents mean ± standard error of mean (SEM). *P<0.05 significant against unstimulated (unstim), †: significant against risto using a one-way ANOVA.
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