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Effects of 2'MOE ASO on human platelets
of the concentrations (Figure 5C-D). The concentration- dependent effects of 120704 and ODN 2395 are shown in Figure 5E, F.
104838, 501861, 120704 and ODN 2395-induced formation of platelet-leukocyte aggregrates in unstimulated whole blood is blocked by SYK pretreatment
When whole blood was pretreated with a SYK inhibitor, collagen-induced platelet-leukocyte aggregates were markedly reduced, while TRAP-induced platelet-leuko- cyte aggregates were maintained, confirming the selectiv- ity of the SYK inhibitor (Figure 6A). SYK pre-treatment completely reversed the ASO-induced formation of platelet-leukocyte aggregates (Figure 5A). Confocal imag- ing of the aggregates using a leukocyte marker (CD45) and a platelet marker (CD41/61), confirmed the presence of platelet-leukocyte aggregates (Figure 6B) but showed greater platelet-platelet aggregation in the platelet-leuko- cyte aggregates in whole blood treated with the CpG ASO (which was more similar to TRAP-treated samples) com- pared to the 2’MOE ASO, which mainly involved single platelets bound to the leukocytes (Figure 6B).
ASO increase levels of P-selectin-enriched platelet-neutrophil and platelet-monocyte aggregates, through a SYK-dependent mechanism, correlating
to GPVI levels
Further investigation into the types of immune cells that were driving the ASO-induced formation of platelet- leukocyte aggregates revealed an increase in both platelet-neutrophil aggregates and platelet-monocyte aggregates (Figure 7A, B). The CpG ASO 120704 and ODN 2395 once again produced a more robust response than the 2’MOE ASO 104838 and 501861 (65±7 and 47±7 vs. 24±10 and 27±11 vs. 6±1 for vehicle). The platelet-neu- trophil and platelet-monocyte aggregates that formed in the ASO-treated samples were enriched in P-selectin (Figure 7C, D) and the greatest increase was observed in the samples treated with 120704 and ODN 2395. However, platelet-neutrophil aggregates and platelet- monocyte aggregates were not enriched in CD11b (Online Supplementary Figure S4C, D), implying that the initial formation of these aggregates was driven by acti- vated platelets (not activated leukocytes). Pearson correla- tion analysis between platelet GPVI levels and platelet neutrophil aggregates showed a strong positive correla- tion for all the ASO tested; higher platelet GPVI expres- sion was associated with a stronger platelet-neutrophil aggregate response (Figure 7E).
CpG ASO bind to GPVI with a higher affinity compared to 2’MOE ASO
Fluorescence polarization experiments revealed that all ASO bound to human GPVI, with equilibrium dissocia- tion constant (KD) values in the low micromolar range (Figure 7F): the smaller the KD, the greater the binding affinity. All the ASO bound to GPVI with stronger affinity than to control human serum albumin (Online Supplementary Figure S5). The 2’MOE 487660 showed stronger affinity (KD 12.9 mM) to human serum albumin than the other two 2’MOE ASO (104838 KD 24 mM and 501861 KD 26 mM). The CpG ASO displayed higher affin- ity (lower KD) for GPVI compared to the 2’MOE ASO (Figure 7F).
Discussion
The main findings from the current study are: (i) none of the ASO sequences investigated had an inhibitory effect on proplatelet production by either human- or mouse- derived megakaryocytes; (ii) all ASO demonstrated uptake into human platelets (2’MOE 487660 less than the others); (iii) a subset of 2’MOE ASO (104838 and 501861) and CpG ASO (120704 and ODN 2395) activated human platelets, triggering P-selectin and SDF1a release from platelet a-granules and platelet-leukocyte aggregate for- mation; (iv) the ASO-induced platelet activation and platelet-leukocyte aggregate formation were fully reversed by pre-treatment with a SYK inhibitor; (v) ASO-induced platelet effects appeared to be sequence-dependent, rather than 2’MOE-dependent, since the 2’MOE ASO 487660 did not affect platelet function; (vi) all the ASO were shown to bind to human GPVI (CpG ASO had the strongest affinity); (vii) only the CpG ASO had a concen- tration-dependent proinflammatory effect, triggering IL-8 and MCP-1 release in whole blood after incubation for 6 h; (viii) the responsiveness to the ASO-induced platelet- activating and platelet-leukocyte aggregate-forming effects varied between donors and showed a strong posi- tive correlation to individual platelet GPVI surface expres- sion.
The novelty of this study concerns the effect of 2’MOE ASO on human platelets. Our focus was the 2’MOE ASO 104838, which has been shown to be representative of a subset of ASO sequences that produce phenotype 1 reduc- tions in platelet count in monkeys and humans.8,10 We identified that the 2’MOE ASO 104838 binds to GPVI receptors on human platelets, increasing platelet surface P-selectin and prompting the formation of platelet-neu- trophil aggregates and platelet-monocyte aggregates, most likely through an interaction between platelet P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1).29 This appears to be a platelet-driven interaction (at least when tested in vitro), as there were no signs of leukocyte activation (CD11b or IL-8/MCP-1 release) even after 10 μM of the 2’MOE ASO 104838.
Mechanistically, we suggest that 2’MOE ASO 104838 lowers platelet count, producting a phenotype 1, by acti- vating platelets, an effect which triggers platelet-leukocyte aggregates and subsequent clearance of platelets by leuko- cytes. Maugeri et al. showed that platelet-neutrophil inter- actions might contribute to platelet clearance through active phagocytosis of P-selectin-positive platelets by neu- trophils.30 The internalization of platelets by neutrophils has been observed in patients with viral infection (associ- ated with reductions in platelet count).31 Increased levels of P-selectin can also enhance sequestration of platelets on vascular endothelial surfaces.32 When monkeys were treated with 2’MOE ASO 104838, there was increased platelet sequestration in the densely vascularized liver and spleen.10 However, in vitro incubation of platelet-rich plas- ma from treatment-naïve monkeys with 2’MOE ASO 104838 did not lead to platelet activation10 (as we observed in human platelet-rich plasma and whole blood). This may be because the sample size used (n=3) did not capture the variability seen in ASO responsiveness. Monkeys treated with 2’MOE ASO 104838 also had high- er plasma levels of IL-8 and MCP-1,10 which we did not see in our in vitro assay with 104838 in human blood (using doses up to 10 mM). This may reflect in vitro rather than in
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