Page 241 - Haematologica April 2020

The role of neuraminidases in platelet function
ing (to underlying galactose-residues), (Figure 1A). WGA- binding (to sialic acid and GlcNAc-residues), was decreased by 25% following ristocetin addition, also indi- cating some desialylation (Figure 1B). Similarly, the pro- portion of platelets binding RCA-1 increased significantly upon ristocetin stimulation (Figure 1, Table 1).
Additionally, binding to other lectins MAL-1 and ECL (bind to exposed β-galactosidase residues),27 PNA (binds only to underlying GalNAc-residues on the T-antigen of VWF) and SNA (binds sialic acid) was examined in washed platelets (Figure 1C). MAL-1 and ECL-binding were increased following VWF/risto (Figure 1C), which was consistent with RCA-1 binding, although the
increase was smaller (1.5-fold) when compared to over a two-fold increase on platelets in PRP (Figure 1A).
NEU1 and NEU2 are expressed on the platelet membrane
In order for desialylation to occur, it was hypothesised that the enzymes responsible, NEU1 and/or NEU2 were most likely associated with the plasma membrane. Since clustering of GPIbα specifically induced platelet desialyla- tion, we investigated membrane association of both enzymes following addition of ristocetin. Following risto- cetin-stimulation, there was a significant increase in mem- brane associated NEU1 and NEU2 (Figure 2A-B), demon-
Table 1. Concentrations of platelet agonists and inhibitors.
Platelet rich plasma Compound
Ristocetin
Adenosine diphosphate Arachidonic acid O-sialoglycoprotein endopeptidase Arg-Gly-Asp-Ser peptide N-acetylglucosamine
Fibrinogen
2-deoxy-2-3-didehydro-
N-acetylneuraminic acid Calcium chloride Collagen
Washed platelets Compound
Ristocetin + von Wilebrand factor
Adenosine diphosphate Arachidonic acid O-sialoglycoprotein endopeptidase Peptide:N-glycosidase F
2-deoxy-2-3-didehydro-
N-acetylneuraminic acid
Collagen
(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)- 3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1] heptan-6-yl]hept-5-enoic acid
Abbreviation
Risto
ADP AA OSGE RGDS GlcNAc Fg DANA
CaCl2 coll
Abbreviation
Risto + VWF
ADP
AA
OSGE PNGAse F
DANA
coll U46619
thr
BAPTA-AM indo
30 μM CaCl2
Fg
GM3
recNEU
Function
facilitates/potentiates vWF binding to GPIbα
platelet agonist through binding to P2Y1 and P2Y12 platelet agonist through formation of TXA2
cleaves all O-linked glycans & GPIbα VWF-binding domain inhibits fibrinogen binding to GPIIb/IIIa integrin
inhibits GPIbα clustering
binds GPIIb/IIIa integrin
neuraminidase inhibitor
recalcification
platelet agonist through binding to GPVI
Function
Platelet stimulation through activation of GPIbα
Platelet agonist through binding to P2Y1 and P2Y12 Platelet agonist through formation of TXA2
Cleaves all O-linked glycans & GPIbα VWF-binding domain Cleaves all N-linked glycans
Neuraminidase-inhibitor
Platelet agonist through binding to GPVI TxA2 mimetic
Platelet stimulation through activation of GPIbα (low-dose only)
Calcium chelator
Blocks TXA2 formation;
ADP inhibitor: hydrolyses ADP
recalcification
binds GPIIb/IIIa integrin
ganglioside; NEU2 substrate and involved in GPIbα clustering desialylation enzyme
Final concentration
3 mg/mL
200 μM 800 μM 80 μg/mL 200 μM 100 mM 500 μg/mL 1mM
1mM
10 μg/mL
Final concentration
1.2 mg/mL + 10 μg/mL
20 μM
50 μM
80 μg/mL
10,000 U/mL +
1/10 reaction buffer 1mM
10 μg/mL 1 μM
0.1 U/mL
10 μM
0.1 U/mL 1 mM
500 μg/mL 10 μM
0.2 U/mL
α-thrombin 1,2-Bis(2-aminophenoxy)ethane-
N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)
Indomethacin Cyclo-oxygenase-1 inhibitor Apyrase
Calcium chloride
Fibrinogen Monosialodihexosylganglioside
Recombinant neuraminidase (Clostridium)
All agonists and metabolic inhibitors used for stimulation or inhibition of platelet rich plasma (PRP) or washed platelets respectively were used at final concentrations as indi- cated. More details and abbreviations are described in the Online Supplementary Materials and Methods.
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