Page 239 - Haematologica April 2020

Platelet Biology & its Disorders
The role of neuraminidase 1 and 2 in glycoprotein Ibα-mediated integrin αIIbβ3 activation
Dianne E. van der Wal,1 April M. Davis,1 Melanie Mach,1 Denese C. Marks1,2
1Australian Red Cross Lifeblood (formerly known as Blood Service) and 2Sydney Medical School, Uinversity of Sydney, Sydney, NSW, Australia
ABSTRACT
Upon vascular injury, platelets adhere to von Willebrand Factor (VWF) via glycoprotein Ibα (GPIbα). GPIbα contains many glycans, capped by sialic acid. Sialic acid cleavage (desialylation) triggers clearance of platelets. Neuraminidases (NEU) are responsible for desialylation and so far, NEU1-4 have been identified. However, the role of NEU in healthy platelets is currently unknown. Aim of the study was to study the role of NEU1 and NEU2 in platelet signalling. Membrane association of platelet attached gly- cans, NEU1 and NEU2 was measured following activation with agonists using flow cytometry. Adhesion on fibrinogen, aggregation and fibrinogen- binding were assessed with/without the NEU-inhibitor, 2-deoxy-2-3-dide- hydro-N-acetylneuraminic acid. Cellular localisation of NEU1 and NEU2 was examined by fluorescence microscopy. Desialylation occurred follow- ing GPIbα-clustering by VWF. Basal levels of membrane NEU1 were low; glycoprotein Ibα-clustering induced a four-fold increase (n=3, P<0.05). Inhibition of αIIbβ3-integrin prevented the increase in NEU1 membrane-asso- ciation by ~60%. Membrane associated NEU2 increased two-fold (n=3, P<0.05) upon VWF-binding, while inhibition/removal of GPIbα reduced the majority of membrane associated NEU1 and NEU2 (n=3, P<0.05). High shear and addition of fibrinogen increased membrane NEU1 and NEU2. NEU-inhibitior prevented VWF-induced αIIbβ3-integrin activation by 50% (n=3, P<0.05), however, promoted VWF-mediated agglutination, indicating a negative feedback mechanism for NEU activity. NEU1 or NEU2 were par- tially co-localised with mitochondria and α-granules respectively. Neither NEU1 nor NEU2 co-localised with lysosomal-associated membrane protein 1. These findings demonstrate a previously unrecognised role for NEU1 and NEU2 in GPIbα–mediated and αIIbβ3-integrin signalling.
Introduction
Glyocoprotein Ibα (GPIbα), part of the GPIb-V-IX-complex, binds to von Willebrand Factor (VWF), initiating platelet adhesion to the endothelium following vascular injury. GPIbα is heavily glycosyslated,1 with both O-2 and N-linked gly- cans.3 When fully assembled, N-linked glycans are complex, branched carbohy- drates, capped by sialic acid.3 The majority of O-linked structures on GPIbα are core 2 and also capped by sialic acid.4
Sialic acid can be cleaved from platelet glycoproteins under various conditions, known as desialylation. Desialylation is important for the clearance of senescent platelets.5 Desialylation also occurs following cold-storage of platelets, which also triggers GPIbα-clustering, resulting in rapid platelet clearance by liver phago- cytes.6Additionally, desialylation is linked to platelet activation7 and intrinsic apop- tosis.8,9 In the bleeding disorder immune thrombocytopenia (ITP), platelets are also desialylated and hyper-activated, resulting in clearance by the liver.10,11
Desialylation is mediated by neuraminidases (NEU), of which four have been described in mammalian cells,12 NEU1, NEU2, NEU3 and NEU4. NEU desialylate their substrates at the α2,6 and/or the α2,3 glycan-linkages.13 NEU differ in their intracellular location as well as their substrate specificity. NEU1 is typically located
Ferrata Storti Foundation
Haematologica 2019 Volume 105(4):1081-1094
Correspondence:
DIANNE E. VAN DER WAL
divanderwal@redcrossblood.org.au
Received: January 1, 2019. Accepted: July 3, 2019. Pre-published: July 4, 2019.
doi:10.3324/haematol.2019.215830
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/4/1081
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