TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Abstract

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3 −/−) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3 −/− mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

Platelets in Inflammation and Resolution

“…Aside from pathogen-related inflammation, platelets are also of relevance in sterile inflammation. With regard to the aforementioned NLRP3 inflammasome, an upregulation of platelet NLRP3 could be observed in a model of hindlimb ischemia in a TLR4dependent manner, ultimately triggering platelet aggregation (96). In another sterile inflammatory condition, in nonalcoholic steatohepatitis, platelet-dependent liver damage was observed (97).…”

Innate immune receptors in platelets and platelet-leukocyte interactions

“…Inflammasome assembly and cytokine processing in nucleated cells require 2 signals: the first to induce transcription of the cytokines and inflammasome components and the second to trigger inflammasome activation 83 . Recent reports using different models have shown that platelet inflammasome activation requires TLR4‐mediated signaling, 15,86,87 suggesting a similar 2‐signals response in platelets. The involvement of ATP from dense‐granules in platelet NLRP3 activation is shown to mediate platelet prothrombotic responses through IL‐1β secretion 88 .…”

“…During dengue infection, platelet inflammasome activation requires reactive oxygen species generated in mitochondria, but not ATP release from granules 50,83 (see Section 6.1). Hence, other evidence regarding NLRP3 and IL‐1β participation in platelet prothrombotic responses have emerged 15,86–90 …”

“…TLR4 and NLRP3 signaling in platelets participate in muscle ischemia‐reperfusion in mice 86 . Increased caspase‐1 activation and IL‐1β processing in platelets from ischemic mice depend on NLRP3 activation, and are inhibited in mice whose platelets lack TLR4, indicating TLR4‐mediated inflammasome activation 86 .…”

“…Increased caspase‐1 activation and IL‐1β processing in platelets from ischemic mice depend on NLRP3 activation, and are inhibited in mice whose platelets lack TLR4, indicating TLR4‐mediated inflammasome activation 86 . NLRP3 or platelet TLR4 deficiency impairs platelet hyperaggregability and improves ischemic muscle reperfusion over time, 86 suggesting involvement of these receptors in prothrombotic and pro‐angiogenic responses. Accordingly, platelet NLRP3 and IL‐1β participate in arterial thrombosis 17,88 .…”