NET-(works) in arterial and venous thrombo-occlusive diseases

Formation of Neutrophil Extracellular Traps (NETosis), accompanied by the release of extracellular decondensed chromatin and pro-inflammatory as well as pro-thrombotic factors, is a pivotal element in the development and progression of thrombo-occlusive diseases. While the process of NETosis is based on complex intracellular signalling mechanisms, it impacts a wide variety of cells including platelets, leukocytes and endothelial cells. Consequently, although initially mainly associated with venous thromboembolism, NETs also affect and mediate atherothrombosis and its acute complications in the coronary, cerebral and peripheral arterial vasculature. In this context, besides deep vein thrombosis and pulmonary embolism, NETs in atherosclerosis and especially its acute complications such as myocardial infarction and ischemic stroke gained a lot of attention in the cardiovascular research field in the last decade. Thus, since the effect of NETosis on platelets and thrombosis in general is extensively discussed in other review articles, this review focusses on the translational and clinical relevance of NETosis research in cardiovascular thrombo-occlusive diseases. Consequently, after a brief summary of the neutrophil physiology and the cellular and molecular mechanisms underlying NETosis are presented, the role of NETosis in atherosclerotic and venous thrombo-occlusive diseases in chronic and acute settings are discussed. Finally, potential prevention and treatment strategies of NET-associated thrombo-occlusive diseases are considered

Inflammasome activation in neutrophils of patients with severe COVID-19

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aymonnier, K., Ng, J., Fredenburgh, L. E., Zambrano-Vera, K., Muenzer, P., Gutch, S., Fukui, S., Desjardins, M., Subramaniam, M., Baron, R. M., Raby, B. A., Perrella, M. A., Lederer, J. A., & Wagner, D. D. Inflammasome activation in neutrophils of patients with severe COVID-19. Blood Advances, 6(7), (2022): 2001–2013, https://doi.org/10.1182/bloodadvances.2021005949.Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.P.M. received an Individual Marie Skłodowska-Curie Actions fellowship by the European Commission (796365 - COAGULANT). This work was supported by the National Institutes of Health (NIH)/Research Program Award grant R35 HL135765 (D.W.), by the NIH/National Heart, Lung, and Blood Institute grant T32 HL007633-35 (J.N.), and by the NIH/National Institute of Allergy and Infectious Diseases grant U01AI138318 (J.L and M.P); by the Massachusetts Consortium on Pathogen Readiness (MassCPR) Evergrande COVID‐19 Response Fund Award to B.R.; and by a generous gift to D.W. from the Steven Berzin family

NLRP3 inflammasome assembly in neutrophils is supported by PAD4 and promotes NETosis under sterile conditions

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Muenzer, P., Negro, R., Fukui, S., di Meglio, L., Aymonnier, K., Chu, L., Cherpokova, D., Gutch, S., Sorvillo, N., Shi, L., Magupalli, V. G., Weber, A. N. R., Scharf, R. E., Waterman, C. M., Wu, H., & Wagner, D. D. NLRP3 inflammasome assembly in neutrophils is supported by PAD4 and promotes NETosis under sterile conditions. Frontiers in Immunology, 12, (2021): 683803, https://doi.org/10.3389/fimmu.2021.683803.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.This work was supported by a grant from National Heart, Lung, and Blood Institute of the National Institutes of Health (grant R35 HL135765) and a Steven Berzin family support to DDW, an Individual Erwin Deutsch fellowship by the German, Austrian and Swiss Society of Thrombosis and Hemostasis Research to RES, a Whitman fellowship (MBL) to DDW, and an Individual Marie Skłodowska-Curie Actions fellowship by the European Commission (796365 - COAGULANT) to PM. ANRW was funded by the Deutsche Forschungsgemeinschaft (TRR156/2 –246807620) and a research grant (We-4195/15-19). CMW was supported by the Division of Intramural Research, NHLBI, NIH

CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-) physiology has remained elusive. The present study explored the impact of the CK2 regulatory b-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/-) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-Triphosphate receptor-dependent intracellular calcium (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

CRACking the Molecular Regulatory Mechanism of SOCE during Platelet Activation in Thrombo-Occlusive Diseases

Thrombo-occlusive diseases such as myocardial infarction, ischemic stroke and deep vein thrombosis with subsequent pulmonary embolism still represent a major health burden worldwide. Besides the cells of the vasculature or other hematopoietic cells, platelets are primarily responsible for the development and progression of an occluding thrombus. The activation and function of platelets crucially depend on free cytosolic calcium (Ca(2+)) as second messenger, which modulates platelet secretion, aggregation and thrombus formation. Ca(2+) is elevated upon platelet activation by release of Ca(2+) from intracellular stores thus triggering of the subsequent store-operated Ca(2+) entry (SOCE), which is facilitated by Ca(2+) release-activated channels (CRACs). In general, CRACs are assembled by the pore-forming unit Orai in the plasma membrane and the Ca(2+)-sensing stromal interaction molecule (STIM) in the endoplasmic reticulum after the depletion of internal Ca(2+) stores. In the last few years, there is a growing body of the literature demonstrating the importance of STIM and Orai-mediated mechanism in thrombo-occlusive disorders. Thus, this review provides an overview of the recent understanding of STIM and Orai signaling in platelet function and its implication in the development and progression of ischemic thrombo-occlusive disorders. Moreover, potential pharmacological implications of STIM and Orai signaling in platelets are anticipated and discussed in the end