Page 169 - 2018_09-Mondo

NLRP3 regulates platelet function and thrombosis
a low- to a high-affinity state (inside-out signaling) and enable platelet aggregation and thrombus formation through binding of soluble fibrinogen and other aIIbβ3 ligands.3 Ligand binding to aIIbβ3 also triggers aIIbβ3 out- side-in signaling,4 leading to tyrosine phosphorylation of signaling proteins,5-7 including c-Src, spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2), and initiates down- stream platelet responses, such as granule secretion, platelet spreading and clot retraction.8,9
Platelets also have roles in the inflammatory response and in inflammatory pathology associated with athero- sclerosis, malarial or dengue infection and rheumatoid arthritis.10-13 Inflammasomes are multiprotein complexes that mediate responses to various inflammatory stimuli by controlling secretion of the pro-inflammatory cytokine, interleukin 1β (IL-1β).14 Upon stimulation, NLRP3 under- goes oligomerization, leading to conversion of pro-cas- pase-1 to active caspase-1, which then cleaves pro-IL-1β to mature IL-1β.14 For example, in dengue virus infection, platelet NLPR3 is activated, triggering IL-1β secretion.13 NLPR3 also contributes to platelet activation, aggregation and thrombus formation in vitro, as shown by caspase activity measurements and pharmacological inhibition or genetic ablation of the NLPR3-associated adaptor protein, Bruton tyrosine kinase (BTK).15 In this study using NLRP3- deficient platelets, we demonstrated a specific contribu- tion of NLRP3 to aIIbβ3 outside-in signaling, and hemo- stasis and arterial thrombosis in vivo.
Methods
Animals
NLRP3-/- C57BL/6 mice16 were purchased from Jackson Laboratories. All experimental procedures were approved by the Ethics Committee of Xuzhou Medical University.
Platelet preparation
Procedures involving collection of mouse and human blood were approved by the Medical Ethics Committee of Xuzhou Medical University. For mouse platelet studies, blood was collect- ed from the retro-orbital plexus using ACD (85 mM trisodium cit- rate, 83 mM dextrose, and 21 mM citric acid) as anticoagulant and diluted in modified Tyrode buffer (12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 2 mM MgCl2, 0.42 mM NaH2PO4, 10 mM HEPES, pH 7.4). Platelets were then pelleted by centrifugation at 180 g in the presence of PGE1 (0.1 μg/mL) and apyrase (1 U/mL) (Sigma-Aldrich), washed twice with CGS buffer (120 mM sodium chloride, 12.9 mM trisodium citrate, 30 mM D- glucose, pH 6.5) and re-suspended in modified Tyrode buffer. Isolated platelets were allowed to rest for 1 h at room temperature before use. For human platelet studies, venous human blood was collected and then the platelets were isolated as described previ- ously.18
Platelet analyses in vitro
Platelet receptor expression, activation, aggregation and
immunoblotting were studied as previously described.17,18 Antibodies against c-Src (anti-Tyr-416, Cell Signaling Technology; pan-c Src, Proteintech), Syk (anti-Tyr-525 and pan-Syk, Bioworld Technology) and PLCγ2 (anti-Tyr-1217 and pan-PLCγ2; Bioworld Technology), IL-1β (Cell Signaling Technology) and Caspase-1 (BioVision) were used.
Detailed methods of the electron microscopy of platelet spread- ing, and clot retraction are provided in the Online Supplement.
Quantitative real-time polymerase chain reaction
haematologica | 2018; 103(9)
The mRNA expression of GPIba, GPVI and IL-1β was measured by quantitative real-time polymerase chain reaction (PCR) as described previously.19,20 In brief, RNA was reversely transcribed into cDNA using oligo(dT) and M-MLV Reverse Transcriptase (Thermo Fisher Scientific) and PCR amplification was performed in triplicate on a LightCycler® R480 II (Roche Life Science) with a total volume of 20 μL, consisting of 10 μL SYBR Green qPCR Super Mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 5 μL cDNA and 4 μL sterile water. The primers for GPIba, GPVI and IL-1β were designed as follows: GPIba forward primer: 5’-AGTTCATACTACCCACTGGAGCC-3’, reverse primer: 5’-GTGGGTTTATGAGTTGGAGGC-3’; GPVI forward primer: 5’-AGGAGACCTTCCATCTTACCCA-3’, reverse primer: 5’-GAGCAAAACCAAATGGAGGG-3’; IL-1β forward primer: 5’- CCTGAACTCAACTGTGAAATGC-3’, reverse primer: 5’-GAT- GTGCTGCTGCGAGATT-3’. The relative mRNA expression of GPIba, GPVI and IL-1β was calculated using the 2-DDCt method and normalized to an internal control (β-actin).
Detailed methods on RNA extraction are provided in the Online Supplement.
Tail bleeding time
Tail bleeding assays were performed as previously described.18 In brief, a 10-mm segment of tail tip was cut off and the tail was then immersed in pre-warmed sterile saline solution (37°C). Tail bleed- ing time was calculated as the time taken for bleeding to stop.
FeCl3-induced thrombosis in vivo
Platelets isolated from wild-type and NLRP3-/- mice were labeled
with calcein and infused into wild-type mice via tail vein injection. Injury to mesenteric arterioles was induced by 0.62 M FeCl3 and thrombus formation was monitored by fluorescence microscopy (OlympusBX53).
Statistical analysis
Data are represented as mean ± standard deviation (SD) or stan- dard error (SE) where indicated and analyzed by the Student t-test, one-way or two-way ANOVA.
Results
NLRP3 deficiency in platelets impairs in vivo hemostasis and thrombosis
To evaluate whether NLRP3 deficiency affects platelet production or clearance, we measured platelet count, mean platelet volume, platelet distribution width and plateletcrit and found similar values in wild-type and NLRP3-/- mice (P>0.05) (Figure 1A). Platelet receptors GPIba, GPVI and integrin aIIbβ3 are critical for platelet function.21,22 Evaluation of these receptors by flow cytom- etry and reverse transcriptase PCR revealed equivalent mRNA and protein levels in wild-type and NLRP3-/- mice (P>0.05) (Figure 1B). Electron microscopy analysis indicat- ed that NLRP3 deficiency did not affect platelet ultrastruc- tural organization, or the number and size of a- and dense granules (Figure 1C). Together, these data suggest that NLRP3 does not affect platelet production, expression of platelet receptors or granules.
In order to investigate whether NLRP3 influences platelet function in vivo, we performed tail bleeding assays and monitored FeCl3-induced mesenteric arteriole throm- bus formation. As NLRP3-/- mice demonstrated decreased tail bleeding times (mean ± SD) of 33.33 ± 14.43 s (n = 6),
1569