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Effects of 2'MOE ASO on human platelets
coefficients: 104838 r=0.97, 501861 r=0.96, 120704 r=0.85, ODN 2395 r=0.86) between donors’ platelet GPVI levels and their platelet activation in response ASO treatment, with the highest ASO responders also having the highest platelet GPVI levels (Figure 3 C, D).
2’MOE ASO (104838 and 501861) and CpG ASO trigger SDF1a release from whole blood
As an additional marker of a-granule release from acti-
vated platelets, we measured secreted plasma levels of stromal cell derived factor 1a (SDF1a) in ASO-treated platelet-rich plasma and whole blood. TRAP stimulated SDF1a release in both platelet-rich plasma and whole blood (Figure 3 E, F). Consistent with the pattern of the P- selectin effects, the CpG ASO 120704 and ODN 2395 trig- gered robust release of SDF1a in platelet-rich plasma and whole blood in a concentration-dependent manner: thus the effect could be seen with doses of 5 and 10 mM, but
AB
CD
EF
Figure 4. Platelet aggregation in human platelet-rich plasma and whole blood treated with antisense oligonucleotides. (A, B) Ninety-six-well platelet aggregometry was used to generate full concentration-response curves to the platelet agonist collagen after incubating platelet-rich plasma (PRP) with vehicle or (A) 5 mM of the 2’MOE antisense oligonucleotides (ASO) 487660, 104838 and 501861 or (B) the CpG ASO 120704 or ODN 2395. *P<0.05 by two-way analysis of variance (ANOVA) with Bonferroni post-test, compared to vehicle (HEPES, 10 mM), n=6-8 human donors. (C) Platelet aggregation was also assessed using traditional light transmission aggregometry (LTA) by incubating PRP (n=8 human donors) with the ASO for 30 min at 1200 rpm stirring speed without stimulation (to detect spontaneous aggre- gation) or after stimulation with the platelet agonist thrombin receptor activating peptide (TRAP, 25 mM). (D) Impedance aggregometry was used to analyze platelet aggregation by incubating whole blood (WB) (n=7 human donors) with the ASO (at 1 and 5 mM) for 30 min at 1200 rpm stirring speed, without stimulation, or TRAP (25 mM) stimulated aggregation. (E) Platelet-platelet aggregates in WB (n=7 human donors) treated with vehicle (HEPES, 10 mM), collagen (20 mg/mL), TRAP (25 mM) or ASO (5 mM) were analyzed using flow cytometry. (F) Platelet-leukocyte aggregates (platelet marker CD41/61+ leukocyte marker CD14+) were analyzed in WB (n=6 human donors) treated with vehicle (HEPES, 10 mM), collagen (20 mg/mL), TRAP (25 mM), or ASO (5 mM), using flow cytometry. *P<0.05 compared to vehi- cle by one-way ANOVA, Dunnett post-test (C, D, E, F).
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