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Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo .

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. Platelets derive from large precursor cells (megakaryocytes) in the bone marrow. Dütting et al . show that megakaryocyte polarization and platelet biogenesis in the bone-marrow sinusoids are directed by adhesion receptor GPIb signalling and resulting balanced antagonism between RhoA (stop-signal) and Cdc42 (go-signal).

Inflammation is a common denominator in chronic diseases of aging. Yet, how inflammation fuels these diseases remains unknown. Neutrophils are the primary leukocytes involved in the early phase of innate immunity and inflammation. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs have been shown to induce tissue injury and thrombosis. Here, we demonstrated that Sirt3, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, an enzyme linked to human longevity, was expressed in mouse neutrophils and platelets. Using Sirt3-/- mice as a model of accelerated aging, we investigated the effects of Sirt3 deficiency on NETosis and platelet function, aiming to detect enhancement of thrombosis. More mitochondrial reactive oxygen species (ROS) were generated in neutrophils and platelets of Sirt3-/- mice compared to WT, when stimulated with a low concentration of phorbol 12-myristate 13-acetate (PMA) and a high concentration of thrombin, respectively. There were no differences in in vitro NETosis, with or without stimulation. Platelet aggregation was mildly augmented in Sirt3-/- mice compared to WT mice, when stimulated with a low concentration of collagen. The effect of Sirt3 deficiency on platelet and neutrophil activation in vivo was examined by the venous thrombosis model of inferior vena cava stenosis. Elevation of plasma DNA concentration was observed after stenosis in both genotypes, but no difference was shown between the two genotypes. The systemic response to thrombosis was enhanced in Sirt3-/- mice with significantly elevated neutrophil count and reduced platelet count. However, no differences were observed in incidence of thrombus formation, thrombus weight and thrombin-antithrombin complex generation between WT and Sirt3-/- mice. We conclude that Sirt3 does not considerably impact NET formation, platelet function, or venous thrombosis in healthy young mice.

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The G ᵢ protein Gα ᵢ₂ mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα ᵢ₂ exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα ᵢ₂ in megakaryocytes and platelets ( Gnai2 ᶠˡ/ᶠˡ/PF4-Cre mice) and found bleeding defects comparable to those in global Gα ᵢ₂-deficient mice. To examine the impact of platelet Gα ᵢ₂ in postischemic thrombo-inflammatory infarct progression, Gnai2 ᶠˡ/ᶠˡ/PF4 -Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2 ᶠˡ/ᶠˡ/PF4 -Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2 ᶠˡ/ᶠˡ/PF4 -Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα ᵢ₂ not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo. Significance Platelet activation is crucial for hemostasis and thrombosis but also contributes to inflammation and progression of tissue damage following ischemia/reperfusion injury. Here we demonstrate that platelet activation through the G ᵢ protein Gα ᵢ₂ not only controls hemostatic responses but also thrombo-inflammatory tissue damage following cerebral and cardiac ischemia. Our report on a dual role of G ᵢ proteins in platelet function opens new options for pharmaco-therapeutic strategies fighting ischemic diseases such as heart attack and stroke.

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The G... protein Gα... mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα... exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα... in megakaryocytes and platelets (Gnai2.../PF4-Cre mice) and found bleeding defects comparable to those in global G...-deficient mice. To examine the impact of platelet Gα... in postischemic thrombo-inflammatory infarct progression, Gnai.../PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2.../PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2.../PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα... not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo. (ProQuest: ... denotes formulae/symbols omitted.)

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The Giprotein Gαi2mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gαi2exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gαi2in megakaryocytes and platelets (Gnai2fl/fl/PF4-Cremice) and found bleeding defects comparable to those in global Gαi2-deficient mice. To examine the impact of platelet Gαi2in postischemic thrombo-inflammatory infarct progression,Gnai2fl/fl/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion strokeGnai2fl/fl/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia,Gnai2fl/fl/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gαi2not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.

Platelet activation is crucial for hemostasis and thrombosis but also contributes to inflammation and progression of tissue damage following ischemia/reperfusion injury. Here we demonstrate that platelet activation through the G i protein Gα i2 not only controls hemostatic responses but also thrombo-inflammatory tissue damage following cerebral and cardiac ischemia. Our report on a dual role of G i proteins in platelet function opens new options for pharmaco-therapeutic strategies fighting ischemic diseases such as heart attack and stroke. Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The G i protein Gα i2 mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα i2 exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα i2 in megakaryocytes and platelets ( Gnai2 fl/fl /PF4-Cre mice) and found bleeding defects comparable to those in global Gα i2 -deficient mice. To examine the impact of platelet Gα i2 in postischemic thrombo-inflammatory infarct progression, Gnai2 fl/fl /PF4 -Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2 fl/fl /PF4 -Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2 fl/fl /PF4 -Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα i2 not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.