The role of neuraminidase 1 and 2 in glycoprotein Ibα-mediated integrin αIIbβ3 activation

Upon vascular injury, platelets adhere to von Willebrand Factor (VWF) via glycoprotein Ib alpha (GPIb alpha). GPIb alpha 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 NEW and NEU2 in platelet signalling. Membrane association of platelet attached glycans, 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-didehydro-N-acetylneuraminic acid. Cellular localisation of NEU1 and NEU2 was examined by fluorescence microscopy. Desialylation occurred following GPIb alpha-dustering by VWE Basal levels of membrane NEU1 were low; glycoprotein Ib alpha-clustering induced a four-fold increase (n=3, P<0.05). Inhibition of alpha(IIb)beta(3)-integrin prevented the increase in NEU1 membrane-association by similar to 60%. Membrane associated NEU2 increased two-fold (n=3, NO.05) upon VWF-binding, while inhibition/removal of GPIb alpha reduced the majority of membrane associated NEU1 and NEW (n=3, P<0.05). High shear and addition of fibrinogen increased membrane NEU1 and NEU2. NEU-inhibitior prevented VWF-induced alpha IIb beta 3-integrin activation by 50% (n=3, P<0.05), however, promoted VWF-mediated agglutination, indicating a negative feedback mechanism for NEU activity. NEU1 or NEW were partially co-localised with mitochondria and a-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 GPIb alpha-mediated and alpha(IIb)beta(3)-integrin signalling.