Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: Blood was collected from 115 consecutive COVID-19 patients presenting non-severe (n=71) and severe (n=44) respiratory symptoms. We document the presence of SARS-CoV-2 RNA associated with platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both non-severe and severe COVID-19 patients, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both non-severe and severe forms of COVID-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in non-severe, but not in severe cases of COVID-19. Levels of D-dimers, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVID-19 subjects presenting non-severe and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: Taken together, the data suggest that platelets are at the frontline of COVID-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVID-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVID-19.

Extracellular vesicles (EVs) are a means of cell-to-cell communication and can facilitate the exchange of a broad array of molecules between adjacent or distant cells. Platelets are anucleate cells derived from megakaryocytes and are primarily known for their role in maintaining hemostasis and vascular integrity. Upon activation by a variety of agonists, platelets readily generate EVs, which were initially identified as procoagulant particles. However, as both platelets and their EVs are abundant in blood, the role of platelet EVs in hemostasis may be redundant. Moreover, findings have challenged the significance of platelet-derived EVs in coagulation. Looking beyond hemostasis, platelet EV cargo is incredibly diverse and can include lipids, proteins, nucleic acids, and organelles involved in numerous other biological processes. Furthermore, while platelets cannot cross tissue barriers, their EVs can enter lymph, bone marrow, and synovial fluid. This allows for the transfer of platelet-derived content to cellular recipients and organs inaccessible to platelets. This review highlights the importance of platelet-derived EVs in physiological and pathological conditions beyond hemostasis.

Rationale: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. Objective: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. Methods and Results: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. Conclusions: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

Accumulation of MDA epitopes plays a major role during diet-induced hepatic inflammation and can be ameliorated by administration of an anti-MDA antibody. (Hepatology 2017;65:1181-1195).

In addition to preserving homeostasis and contributing to immunity, platelets may exhibit a dysregulated state 1,2 in acute respiratory distress syndrome (ARDS) and potentially exacerbate lung injury. [3][4][5] Although thrombocytopenia has been associated with increased mortality in ARDS, 6 and particularly in patients infected with H1N1 influenza, 7 analysis of quantitative changes in platelet activation levels in ARDS and of how these may differ between the various etiologies of ARDS is limited. 3 Coronavirus disease 2019 (COVID-19) is clinically associated with a high incidence of thrombosis [8][9][10] and with a high burden of pulmonary and systemic platelet-rich microthrombi. 11,12 Interestingly, in contrast to a cohort of patients with H1N1-influenza infection with ARDS of equal severity, patients with COVID-19 ARDS displayed a 9 times higher burden of alveolar capillary platelet-rich microthrombi. 11 Patients with COVID-19 who were autopsied demonstrated an abundance of platelet-rich thrombi in the pulmonary and systemic microvasculature despite having received anticoagulation therapy. 12 Clinically, thromboelastography (TEG) studies have also demonstrated a hypercoagulable profile in patients with severe COVID-19 9,13 despite prophylactic anticoagulation. TEG profiles remained hypercoagulable, with parameters suggesting a significant hypercoagulable effect from platelet activity and fibrin. 13 Multiple study groups [14][15][16] including ours 17 have reported dramatically increased platelet activation, platelet reactivity, and platelet-leukocyte aggregates in COVID-19 compared with those in healthy blood donors (controls). In one study, 15 patients with severe COVID-19 harbored a higher degree of platelet activation and platelet-monocyte aggregation compared with patients with COVID-19 that was not severe. In addition, plasma from patients with severe COVID-19 increased the ex vivo activation of platelets and monocytes from healthy controls, a phenomenon inhibited by abciximab, a platelet aggregation inhibitor. Platelet activation correlated with COVID-19 severity in that study. 15 A second study demonstrated an 89-fold increase in platelet hyperreactivity with low-dose stimulation by thromboxane A 2 (TXA 2 ) receptor agonist compared with that in controls. 16 A third study 14 demonstrated increases in platelet activation, platelet reactivity, and platelet-leukocyte aggregates in patients with COVID-19 compared with healthy controls. These studies demonstrate that platelet reactivity is enhanced in patients with COVID-19 and posit that platelet hyperreactivity may be a primary driver of thrombosis in patients with severe COVID-19, contributing to organ failure and death. Whether this degree of platelet hyperreactivity is unique to COVID-19 or is also encountered during ARDS not related to COVID-19 was addressed in our study.COVID-19 patients without (n 5 11) and with ARDS (n 5 9) and patients with ARDS unrelated to COVID-19 etiology (n 5 8) who were admitted to the Cheikh Zaid Hospital of Abulcasis Unive...

Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: We document the presence of SARS-CoV-2 RNA in platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in COVID-19, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents and phosphatidylserine-exposing extracellular vesicles. Functionally, platelets were hyperactivated in COVID-19 subjects, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: These data suggest that platelets could participate in the dissemination of SARS-CoV-2 and in the overwhelming thrombo-inflammation observed in COVID-19. Thus, blockade of platelet activation pathways may improve outcomes in this disease.

Platelets are hyper-activated in COVID-19. However, the mechanisms promoting platelet activation by SARS-CoV-2 are not well understood. This may be due to inherent challenges at discriminating the contribution of viral versus host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles, as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself, or components derived from SARS-CoV-2-infected human lung epithelial cells, could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions and the release of extracellular vesicles. We find that neither SARS-CoV-2 nor purified Spike activate platelets. In contrast, TF produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and extracellular vesicles were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with virus or extracellular vesicles, which were indistinguishable. Increased TF mRNA expression and activity were also found in lungs in a murine model of COVID-19 and in plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere in platelet activation and coagulation that is characteristic of COVID-19.