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Effects of 2'MOE ASO on human platelets
A
B
Figure 6. SYK inhibition of the formation of platelet-leukocyte aggregates in whole blood treated with antisense oligonucleotides. Whole blood (WB) from four human donors was pretreated or not with a spleen tyrosine kinase (SYK) inhibitor (PRT-060318, 10 mM) followed by vehicle (10 mM HEPES), thrombin receptor acti- vating peptide (TRAP, 25 mM), collagen (20 mg/mL), or the 2’MOE non CpG 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-leukocyte aggregates (PLA, platelet marker CD41/61+, leukocyte marker CD14+). *P<0.05 com- pared to vehicle by one-way analysis of variance (ANOVA), Dunnett post-test, #P<0.05 paired Student t-test for the effect of the SYK inhibitor. (B) Representative con- focal images of platelet-leukocyte aggregates in fixed and fluorescently labeled WB. Red represents CD45 (leukocyte marker), green represents CD41/61 (platelet marker). The white boxes indicate the location of the zoomed-in part of image shown to the right. Scale bar = 10 mm.
the 30 min incubation (12±6 Ω vs. 0.4±0.3 Ω with vehicle) (Figure 4D). When platelet-platelet interactions were stud- ied in ASO-treated (but otherwise unstimulated) whole blood samples using flow cytometry, only the CpG ASO 120704 and ODN 2395 had significantly more platelet- platelet aggregates (13±6 and 14±6%, respectively, vs. 0.2±0.04% with vehicle) (Figure 4E), consistent with the impedance aggregometry results. After stimulation with TRAP, whole blood aggregation was potentiated to a sim- ilar level in both the 2’MOE ASO (104838 and 501861) and the CpG (120704 and ODN 2395)-treated samples at both 1 and 5 mM (Figure 4D).
2’MOE ASO (104838 and 501861) and CpG ASO trigger formation of platelet-leukocyte aggregates in unstimulated whole blood
The impedance aggregometry results led us to hypothe- size that the enhancement of whole blood aggregation
was perhaps not solely driven by homogenous platelet- platelet aggregates but could also contain heterogeneous platelet-leukocyte aggregates.26 We, therefore, analyzed ASO-treated (but otherwise unstimulated) whole blood for the formation of platelet-leukocyte aggregates by flow cytometry (Figure 4F). TRAP and collagen triggered sub- stantial formation of platelet-leukocyte aggregates, where- as the 2’MOE ASO 487660 evoked a similar response to that produced by the vehicle (Figure 4F). However, whole blood treated with 2’MOE ASO 104838 and 501861 and the CpG 120704 and ODN 2395 had more platelet-leuko- cyte aggregates (33±8, 37±9, 69±4, and 46±4%, respec- tively) than vehicle-treated whole blood (12±1%) (Figure 4F).
Brief ASO treatment does not induce neutrophil or monocyte activation
To further explore the interaction of ASO with mono-
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