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J. Qiao et al.
NLRP3/IL-1β in the regulation of platelet integrin aIIbβ3 outside-in signaling.
Inhibition of NLRP3 impairs clot retraction in human platelets
Using a selective and direct NLRP3 inhibitor (CY-09),30 we evaluated the role of NLRP3 in integrin aIIbβ3 signaling
transduction in human platelets. We found significantly reduced human platelet aggregation in response to low doses but not high doses of collagen and ADP following treatment with CY-09 (Figure 5A). Interestingly, CY-09 treatment did not affect platelet aggregation in response to CRP stimulation (Figure 5A). Using flow cytometry we found no differences in platelet degranulation (P-selectin
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Figure 5. Effect of NLRP3 inhibition on human platelet function. (A) Human platelet-rich plasma was incu- bated with CY-20 (20 μΜ) for 30 min at 37°C and then platelet aggregation in response to collagen (1 and 2 μg/mL), ADP (2.5 and 5 μΜ) or CRP (0.5 μg/mL) was measured in a light transmittance aggregometry (Helena Aggram, Helena Laboratories, Beaumont, USA). Maximum platelet aggregation (%) was recorded (mean ± SE, n = 3) (two-way ANOVA). (B) Platelet P-selectin expression and (C) aIIbβ3 activation in response to collagen, ADP or CRP stimulation was measured by flow cytometry using phycoerythrin-conjugated anti- P-selectin antibody and FITC-conjugated PAC-1 antibody, respectively, and represented as mean ± SE (n = 3) (one-way ANOVA). (D) Clot retraction was initiated using CY-09-treated washed human platelets stimulat- ed with 1 U/mL thrombin in the presence/absence of recombinant human IL-1β (10 ng/mL). Representative images at 30, 60, 90, and 120 min from three independent experiments are shown and data were quantified as the clot volume (%) (mean ± SD, n = 3) (two-way ANOVA). *P<0.05. Compared with vehicle, **P<0.01; ***P<0.001. Compared with CY-09, #P<0.05.
Figure 6. Role of NLRP3 in the regulation of platelet integrin aIIbβ3 outside-in signaling. Engagement of G protein coupled receptors (GPCR) by thrombin induces platelet intra- cellular reactive oxygen species (ROS) pro- duction (1), which activates NLRP3, leading to assembly of the NLRP3 inflammasome and subsequent activation of caspase-1, which processes immature pro-IL-1β into mature IL-1β. Once released, IL-1β binds to IL-1 receptor (IL-1R) and initiates IL-1R intra- cellular signaling transduction, resulting in phosphorylation of c-Src and Syk, which reg- ulates platelet spreading and clot retraction. Meanwhile, ligation of GPCR also induces ATP release (2), which can activate NLRP3 through binding to P2XR. LRR: Leucine-rich repeat; NACHT: NACHT, NAIP, CIITA, HET-E and TP1; PYD: Pyrin domain; ASC: Apoptosis-associated speck-like protein con- taining a CARD.
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haematologica | 2018; 103(9)