Dexamethasone Modulates the Cytokine Response but Not COVID-19-Induced Coagulopathy in Critically Ill.

Severe forms of coronavirus 2019 (COVID-19) disease are caused by an exaggerated systemic inflammatory response and subsequent inflammation-related coagulopathy. Anti-inflammatory treatment with low dose dexamethasone has been shown to reduce mortality in COVID-19 patients requiring oxygen therapy. However, the mechanisms of action of corticosteroids have not been extensively studied in critically ill patients in the context of COVID-19. Plasma biomarkers of inflammatory and immune responses, endothelial and platelet activation, neutrophil extracellular trap formation, and coagulopathy were compared between patients treated or not by systemic dexamethasone for severe forms of COVID-19. Dexamethasone treatment significantly reduced the inflammatory and lymphoid immune response in critical COVID-19 patients but had little effect on the myeloid immune response and no effect on endothelial activation, platelet activation, neutrophil extracellular trap formation, and coagulopathy. The benefits of low dose dexamethasone on outcome in critical COVID-19 can be partially explained by a modulation of the inflammatory response but not by reduction of coagulopathy. Future studies should explore the impact of combining dexamethasone with other immunomodulatory or anticoagulant drugs in severe COVID-19.

Anti-GPVI (HY101) activates platelets in a multicolor flow cytometry panel.

The platelet transmembrane receptor GPVI can be assessed together with other platelet membrane markers in a whole blood multicolor flow cytometry panel. The advantage of combining multiple antibodies in a single tube is the possibility of distinguishing multiple platelet subgroups. In this short communication, we describe an activation problem encountered with anti-GPVI, clone HY101. Activation of platelets was seen after the addition of anti-GPVI in a flow cytometry panel, highlighted by the expression of the activation markers CD62P, PAC-1, CD63, and CD107a. This was also confirmed by platelet aggregation studies.

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Inflammation-Induced Coagulopathy Substantially Differs Between COVID-19 and Septic Shock: A Prospective Observational Study.

Critical COVID-19, like septic shock, is related to a dysregulated systemic inflammatory reaction and is associated with a high incidence of thrombosis and microthrombosis. Improving the understanding of the underlying pathophysiology of critical COVID-19 could help in finding new therapeutic targets already explored in the treatment of septic shock. The current study prospectively compared 48 patients with septic shock and 22 patients with critical COVID-19 regarding their clinical characteristics and outcomes, as well as key plasmatic soluble biomarkers of inflammation, coagulation, endothelial activation, platelet activation, and NETosis. Forty-eight patients with matched age, gender, and co-morbidities were used as controls. Critical COVID-19 patients exhibited less organ failure but a prolonged ICU length-of-stay due to a prolonged respiratory failure. Inflammatory reaction of critical COVID-19 was distinguished by very high levels of interleukin (IL)-1ß and T lymphocyte activation (including IL-7 and CD40L), whereas septic shock displays higher levels of IL-6, IL-8, and a more significant elevation of myeloid response biomarkers, including Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) and IL-1ra. Subsequent inflammation-induced coagulopathy of COVID-19 also differed from sepsis-induced coagulopathy (SIC) and was characterized by a marked increase in soluble tissue factor (TF) but less platelets, antithrombin, and fibrinogen consumption, and less fibrinolysis alteration. In conclusion, COVID-19 inflammation-induced coagulopathy substantially differs from SIC. Modulating TF release and activity should be evaluated in critical COVID-19 patients.

Platelet Acetyl-CoA Carboxylase Phosphorylation: A Risk Stratification Marker That Reveals Platelet-Lipid Interplay in Coronary Artery Disease Patients.

Adenosine monophosphate-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK-induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK-induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function.

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.