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NLRP3 regulates platelet function and thrombosis
expression) and aIIbβ3 activation (PAC-1 binding) in colla- gen, ADP or CRP-stimulated human platelets after CY-09 treatment (Figure 5B), consistent with minimal effects of NLRP3 depletion on other platelet activation pathways. Thrombin-initiated clot retraction was significantly impaired in CY-09-treated human platelets (Figure 5C). Addition of recombinant human IL-1β rescued the impaired clot retraction of CY-09-treated platelets, suggesting that NLRP3 might also be involved in the regulation of human platelet integrin aIIbβ3 outside-in signaling. Collectively, our data support a role for NLRP3 in regulating platelet aIIbβ3 outside-in signaling in human and murine platelets.
Discussion
Systemic inflammation has been demonstrated to be a potent prothrombotic stimulus, with mechanisms including upregulation of procoagulant factors, inhibition of natural anticoagulants and fibrinolytic activity as well as increased platelet reactivity.31,32 However, the relationship between inflammation and platelet function remains poorly under- stood. In this study, we identified a specific contribution of the NLRP3 inflammasome to aIIbβ3 outside-in signaling, and hemostasis and arterial thrombosis in vivo.
Inflammasomes are multiprotein complexes that respond to various inflammatory stimuli by controlling secretion of the pro-inflammatory cytokine, IL-1β.14 The NLRP3 inflammasome is one of the largest and most studied cytosolic inflammasomes. It undergoes oligomer- ization upon stimulation, leading to activation of caspase- 1, which mediates the maturation of IL-1β.14 Along with immune cells, platelets express NLRP3 inflammasomes. Platelet NLRP3 has been shown to be involved in the reg- ulation of endothelial permeability after dengue infection by processing pro-IL-1β and releasing mature IL-1β.13 Additionally, NLRP3 contributes to platelet activation, aggregation and thrombus formation in vitro.15 Consistent with this, we showed reduced platelet aggregation in response to threshold concentrations of collagen, throm- bin and ADP in NLRP3-deficient platelets. However, degranulation (P-selectin expression) and aIIbβ3 activa- tion were normal in NLRP3-/- platelets. As platelet NLRP3 deficiency significantly impaired hemostasis and arterial thrombosis in vivo, we suggest that NLRP3 is a novel molecular link between inflammation and thrombosis.
Platelet spreading and clot retraction, regulated by aIIbβ3 outside-in signaling, play important roles in stabiliz- ing thrombus formation.26 To investigate the role of NLRP3 in aIIbβ3 outside-in signaling, we measured platelet spreading and clot retraction and showed that NLRP3 defi- ciency significantly decreased platelet spreading on immo- bilized fibrinogen and impaired clot retraction. As IL-1β has been demonstrated to increase platelet adhesion to col- lagen and fibrinogen,29 we hypothesized that the effect of NLRP3 on aIIbβ3 signaling might be through IL-1β. To test
that, we measured IL-1β secretion from thrombin-stimu- lated platelets and found significantly reduced IL-1β release from NLRP3-deficient platelets, indicating that NLRP3 is responsible for processing IL-1β in platelets. Moreover, addition of IL-1β rescued impaired platelet spreading and clot retraction, supporting the role of NLRP3/IL-1β in platelet integrin aIIbβ3 signaling. Through the use of a selective and direct NLRP3 inhibitor, CY-09,30 we also eval- uated whether NLRP3 exerts the same functional effects on human platelets and showed reduced platelet aggrega- tion in response to threshold doses of collagen and ADP without differences in degranulation and aIIbβ3 activation. However, inhibition of NLRP3 by CY-09 significantly ablated clot retraction in human platelets. Importantly, addition of recombinant human IL-1β rescued impaired clot retraction of CY-09-treated human platelets, indicating that NLRP3 might also participate in the regulation of aIIbβ3 outside-in signaling in human platelets, which is consistent with findings in mouse platelets.
A potential limitation of our findings is that we cannot exclude the interesting possibility that NLRP3 depletion in mice affects platelet hemostatic/thrombotic function through as yet unknown indirect pathways. Further studies are required to explore the exact mechanisms of NLRP3/IL- 1β signaling and the specific role of this inflammasome complex in other thrombotic models and human disease.
In conclusion, our results demonstrate that NLRP3 regu- lates platelet spreading and clot retraction by a mechanism involving IL-1β. Moreover, impaired hemostasis and arteri- al thrombosis were observed in vivo in mice with NLRP3-/- platelets. Furthermore, inhibition of NLRP3 impairs clot retraction in human platelets. These data identify a unique role for NLRP3 in the regulation of platelet function and thrombus formation (Figure 6), and provide a novel molec- ular link between thrombosis and inflammation.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (grant n. 81400082, 81370602, 81570096, 81671584, 81641151 and 81700178), the Natural Science Foundation of Jiangsu Province (grant n. BK20141138 BK20140219), funding for the Distinguished Professorship Program of Jiangsu Province, the Shuangchuang Project of Jiangsu Province, the National Health and Medical Research Council of Australia, the Six Talent Peaks Project of Jiangsu Province (WSN-133), the 333 projects of Jiangsu Province (BRA2017542), the Key University Science Research Project of Jiangsu Province (17KJA320008), Jiangsu Province’s Key Provincial Talents Program (ZDRCA2016054), the Colleges’ Science Foundation of Jiangsu Province (16KJB320013), Postgraduate Research Innovation Project of Jiangsu Province (KYCX18_2186), and Key University Science Research Project of Jiangsu Province (18KJA320010).
We thank Prof. Rongbin Zhou (University of Science and Technology of China, Hefei, China) for kindly providing the NLRP3 inhibitor CY-09.
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