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M.H. Lundberg Slingsby et al.
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Figure 7. The effect of antisense oligonucleotides on platelet-neutrophil, platelet-monocyte and glycoprotein VI interactions. Whole blood (WB) was incubated with vehicle (10 mM HEPES), thrombin receptor activating peptide (TRAP, 25 mM), or the 2’MOE antisense oligonucleotides (ASO): 104838 or 501861, or the CpG ASO: 120704 or ODN 2395 (all at 5 mM) and analyzed by flow cytometry for (A) platelet-neutrophil aggregates (platelet marker CD41/61+, neutrophil marker CD66b+), (B) platelet-monocyte aggregates (platelet marker CD41/61+, monocyte marker CD14+). (C) Surface P-selectin-positive (CD62p+) platelet-neutrophil aggregates (CD41/61+, CD66b+, CD62p+) (median fluorescence intensity, MFI). (D) Surface P-selectin-positive platelet-monocyte aggregates (CD41/61+, CD14+, CD62p+) (MFI) in WB from five to nine human donors. (E) Individual donor platelet glycoprotein (GP)VI levels were correlated to platelet-neutrophil aggregate formation after treat- ment with the ASO in six human donors. *P<0.05 by Pearson correlation analysis. (F) A fluorescence polarization assay was used to measure binding affinity of Alexa647-labeled ASO to human GPVI. Bmax is the total density of receptors in a sample and KD is the equilibrium dissociation constant. The smaller the KD, the greater the binding affinity of the ASO to human GPVI.
cytes and neutrophils, we assessed whether ASO increased surface expression of CD11b, a broad immune cell activation marker. Lipopolysaccharide was included as a positive control to increase expression of CD11b on the surface of neutrophils (Online Supplementary Figure S4A) or monocytes (Online Supplementary Figure S4B). SYK is also involved in leukocyte intracellular signal- ing,27,28 and there was an inhibitory effect on CD11b sur- face expression in SYK-treated samples exposed to vehi- cle and lipopolysaccharide (Online Supplementary Figure S4 A, B). None of the ASO tested had any effect on CD11b expression on either neutrophils or monocytes (after 30 min incubation); hence the ASO did not appear to acti- vate these cells directly within this timeframe (Online
Supplementary Figure S4A,B). In support of these data, the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were not released from whole blood incubated with ASO (1, 5 and 10 mM) for 30 min (Figure 5A, B).
TreatmentwithCpGASO(butnot2’MOEASO)leads to IL-8 and MCP-1 release
CpG motifs have been shown to be immunostimulato- ry15 and proinflammatory effects of the CpG ASO 120704 and ODN 2395 were apparent after 6 h of incubation of whole blood, with 5 and 10 mM (but not 1 mM) resulting in robust IL-8 and MCP-1 release (Figure 5C, D). None of the 2’MOE ASO evoked a proinflammatory effect at any
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haematologica | 2022; 107(2)