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NLRP3 regulates platelet function and thrombosis
from wild-type but not NLRP3-/- platelets (Figure 3E-i). Consistently, western blot analysis showed significantly reduced IL-1β expression after thrombin stimulation (Figure 3E-ii). A preliminary analysis suggested that the expression of active caspase-1, which is responsible for processing IL-1β, was significantly lower in NLRP3-/- platelets than in wild-type platelets after thrombin stimu- lation (Figure 3F). Supporting a role for IL-1β in platelet spreading, anti-IL-1β antibody treatment significantly impaired wild-type platelet spreading (Figure 3A) and when NLRP3-/- platelets were pre-treated with recombi- nant mouse IL-1β, platelet spreading was restored (Figure 3A). Since IL-1β also regulates platelet activation,29 we hypothesized that defective spreading of NLRP3-/- platelets might be due to decreased secretion of IL-1β. To test this, we measured IL-1β release from platelets after spreading for 90 min and found a small but significant decrease in IL- 1β secretion from NLRP3-/- platelets (59.0 ± 2.4 pg/mL) compared to that from wild-type platelets (52.5 ± 2.5 pg/mL) (mean ± SD; P=0.03;), suggesting that efficient spreading requires mature IL-1β. To then determine whether IL-1β exerted its effect on platelet spreading through IL-1 receptor (IL-1R) engagement, we pre-treated platelets with recombinant IL-1R antagonist (IL-1RA) and found defective spreading of wild-type platelets (Online Supplementary Figure S3). Furthermore, IL-1RA abolished IL-1β-mediated rescue of spreading of NLRP3-/- platelets
(Online Supplementary Figure S3), suggesting that IL-1β affects platelet spreading via IL-1R.
Consistent with the ablated platelet spreading, NLRP3-/- platelets showed a significant impairment of clot retraction (Figure 4A) which was recovered by IL-1β addition, sug- gesting that NLRP3 regulates clot retraction via an IL-1β- dependent mechanism. Impaired clot retraction was also evident in wild-type platelets treated with anti-IL-1β anti- body (Figure 4A). Furthermore, IL-1RA treatment impaired clot retraction of normal platelets and abolished the effect of IL-1β on the recovery of clot retraction in NLRP3-/- platelets (Online Supplementary Figure S4). As acti- vation of aIIbβ3 outside-in signaling leads to phosphory- lation of c-Src, Syk, and PLCγ2, and clot retraction,5,6 we measured the phosphorylation status of these signaling proteins. Consistent with the defective platelet spreading and clot retraction, NLRP3-/- platelets exhibited signifi- cantly reduced phosphorylation of c-Src (Figure 4B), Syk (Figure 4C), and PLCγ2 (Figure 4D) following thrombin stimulation compared with that of wild-type platelets. However, treatment with recombinant IL-1β reversed the decreased phosphorylation of signaling proteins (Figure 4B-D). Interestingly, robust phosphorylation of c-Src, Syk and PLCγ2 in response to CRP/GPVI engagement, which does not require aIIbβ3 signaling, was achieved in NLRP3-/- platelets (Online Supplementary Figure S5). Together, these findings support a specific role for
B
C
D
A
Figure 4. Impaired clot retraction and phosphorylation of c-Src, Syk and PLCγ2 in platelets from NLRP3-/- mice after thrombin stimulation. (A) Clot retraction was studied using washed platelets treated with 1 U/mL thrombin in the presence/absence of 10 ng/mL recombinant mouse IL-1β or 0.5 μg/mL anti-IL-1β antibody at 37°C. Representative images at 15, 30, 45, 60, 75, 90, 105 and 120 min from three independent experiments are shown. Data were quantified as the clot volume (%) and are presented as mean values (two-way ANOVA). Western blots of total and phosphorylated (B) c-Src (Tyr-416), (C) Syk (Tyr-525) and (D) PLCγ2 (Tyr-1217) in platelets treated with 1 U/mL thrombin in the presence of 2 mM Ca2+ and 0.5 mg/mL fibrinogen with or without 10 ng/mL recombinant mouse IL-1β pre-treatment at different time points were quantified (as a ratio of phosphorylated to total protein level) using Image J software and analyzed by two-way ANOVA for comparison (mean ± SD, n = 3). Images are representative of three independent western blot experiments. Compared with wild-type (WT): *P<0.05; **P<0.01; ***P<0.001.
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