The pathophysiology of COVID-19–associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation ...system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA–dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.
•Severe COVID-19 is associated with increased antibody-mediated procoagulant platelets.•Procoagulant platelets and platelet apoptosis in severe COVID-19 is correlated with D-dimer and higher incidence of thromboembolisms.
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Both qualitative and quantitative platelet abnormalities are common in patients with coronavirus disease 2019 (COVID-19) and they correlate with clinical severity and mortality. Activated platelets ...contribute to the prothrombotic state in COVID-19 patients. Several groups have shown immune-mediated activation of platelets in critically ill COVID-19 patients. Vaccine-induced immune thrombotic thrombocytopenia is an autoimmune condition characterized by thrombocytopenia and life-threatening thrombotic events in the arterial and venous circulation. Although the initial trigger has yet to be determined, activation of platelets by immune complexes through Fc gamma RIIA results in platelet consumption and thrombosis. A better understanding of platelet activation in COVID-19 as well as in vaccine-induced thrombotic complications will have therapeutic implications. In this review, we focused on the role of immune-mediated platelet activation in thrombotic complications during COVID-19 infection and vaccine-induced immune thrombotic thrombocytopenia.
Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation ...affects platelet life span in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from immune thrombocytopenic patients which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleave of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment in production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.
The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently ...underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.
Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies reactive to complexes of platelet factor 4 and heparin. Platelets (PLTs) and their ...interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient antibodies (Abs) induce a new PLT population that is characterized by increased P-Selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-gamma-RIIA by HIT Abs and resulted in significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLTs propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLTs intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-Selectin and PS was dissected. While inhibition of P-Selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLTs are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.
The proper communication between gut and brain is pivotal for the maintenance of health and, dysregulation of the gut-brain axis can lead to several clinical disorders. In Parkinson's disease (PD) ...85% of all patients experienced constipation many years before showing any signs of motor phenotypes. For differential diagnosis and preventive treatment, there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperone protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 influences the risk of PD remains incompletely understood. In the present study, we provide evidence that DJ-1 is implicated in shaping the gut microbiome including; their metabolite production, inflammation and innate immune cells (ILCs) development. We revealed that deficiency of DJ-1 leads to a significant increase in two specific genera/species, namely Alistipes and Rikenella. In DJ-1 knock-out (DJ-1
) mice the production of fecal calprotectin and MCP-1 inflammatory proteins were elevated. Fecal and serum metabolic profile showed that malonate which influences the immune system was significantly more abundant in DJ-1
mice. DJ-1 appeared also to be involved in ILCs development. Further, inflammatory genes related to PD were augmented in the midbrain of DJ-1
mice. Our data suggest that metabolites and inflammation produced in the gut could be used as biomarkers for PD detection. Perhaps, these metabolites and inflammatory mediators could be involved in triggering inflammation resulting in PD pathology.
Cold storage of platelets has been suggested as an alternative approach to reduce the risk of bacterial contamination and to improve the cell quality as well as functionality compared to room ...temperature storage. However, cold-stored platelets (CSPs) are rapidly cleared from the circulation. Among several possible mechanisms, apoptosis has been recently proposed to be responsible for the short half-life of refrigerated platelets. In the present study, we investigated the impact of apoptosis inhibition on the hemostatic functions and survival of CSPs. We found that blocking the transduction of the apoptotic signal induced by glycoprotein Ib (GPIb)-alpha clustering or the activation of caspase 9 does not impair CSPs functionality. In fact, the inhibition of GPIb-alpha clustering mediated-apoptotic signal by a RhoA inhibitor better conserved delta granule release, platelet aggregation, adhesion and the ability to form stable clots, compared to untreated CSPs. In contrast, upregulation of the protein kinase A caused a drastic impairment of platelet functions and whole blood clot stability. More importantly, we observed a significant improvement of the half-life of CSPs upon inhibition of the intracellular signal induced by GPIb-alpha clustering. In conclusion, our study provides novel insights on the in vitro hemostatic functions and half-life of CSPs upon inhibition of the intracellular cold-induced apoptotic pathway. Our data suggest that the combination of cold storage and apoptosis inhibition might be a promising strategy to prolong the storage time without impairing hemostatic functions or survival of refrigerated platelets.
ABSTRACT
The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up‐regulated in several clinical disorders, including dehydration. NFAT5‐sensitive genes include serum and ...glucocorticoid‐inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca2+ channel accomplishing store‐operated Ca2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)‐5 influences Ca2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG‐01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild‐type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT‐PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca2+ concentration (Ca2+i) by fura‐2‐fluorescence. SOCE was estimated from the increase of Ca2+i following readdition of extracellular Ca2+ after store depletion with thapsigargin (1 mM). Platelet degranulation was estimated from P‐selectin abundance and integrin activation from αIIbb3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG‐01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IkB inhibitor BMS 345541 (5 mM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 mM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen‐related peptide. In summary, NFAT5 is a powerful regulator of Orai1‐expression and SOCE in megakaryocytes.—Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al‐Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5‐sensitive Orai1 expression and store‐operated Ca2+ entry in megakaryocytes. FASEB J. 31, 3439–3448 (2017). www.fasebj.org
Medial vascular calcification, a major pathophysiological process associated with cardiovascular disease and mortality, involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle ...cells (VSMCs). In chronic kidney disease (CKD), osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification is mainly driven by hyperphosphatemia, resulting from impaired elimination of phosphate by the diseased kidneys. Hyperphosphatemia with subsequent vascular calcification is a hallmark of klotho-hypomorphic mice, which are characterized by rapid development of multiple age-related disorders and early death. In those animals, hyperphosphatemia results from unrestrained formation of 1,25(OH)
D
with subsequent retention of calcium and phosphate. Analysis of klotho-hypomorphic mice and mice with vitamin D
overload uncovered several pathophysiological mechanisms participating in the orchestration of vascular calcification and several therapeutic opportunities to delay or even halt vascular calcification. The present brief review addresses the beneficial effects of bicarbonate, carbonic anhydrase inhibition, magnesium supplementation, mineralocorticoid receptor (MR) blockage, and ammonium salts. The case is made that bicarbonate is mainly effective by decreasing intestinal phosphate absorption, and that carbonic anhydrase inhibition leads to metabolic acidosis, which counteracts calcium-phosphate precipitation and VSMC transdifferentiation. Magnesium supplementation, MR blockage and ammonium salts are mainly effective by interference with osteo-/chondrogenic signaling in VSMCs. It should be pointed out that the, by far, most efficient substances are ammonium salts, which may virtually prevent vascular calcification. Future research will probably uncover further therapeutic options and, most importantly, reveal whether these observations in mice can be translated into treatment of patients suffering from vascular calcification, such as patients with CKD.
Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be ...reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na
/K
-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na
/K
pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na
/K
pump capacity was estimated from K
-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na
/K
pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (V
) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on V
was virtually abrogated by ouabain. Na
/K
α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na
/K
pump capacity.
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