scholarly journals Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

2021 ◽  
Author(s):  
Sokratis A. Apostolidis ◽  
Amrita Sarkar ◽  
Heather M. Giannini ◽  
Rishi R. Goel ◽  
Divij Mathew ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Activation ◽  
Clinical Manifestations ◽  
Vascular Complications ◽  
Surface Expression ◽  
Microfluidic Chamber ◽  
Mediators Of Inflammation ◽  
Syk Inhibitor

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection.

Platelets in patients with acute ischemic stroke are exhausted and refractory to thrombin, due to cleavage of the seven-transmembrane thrombin receptor (PAR-1)

2004 ◽  
Vol 91 (02) ◽  
pp. 334-344 ◽  
Author(s):  
Kerstin Jurk ◽  
Uli-Rüdiger Jahn ◽  
Hugo Van Aken ◽  
Carsten Schriek ◽  
Dirk Droste ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Platelet Activation ◽  
Surface Expression ◽  
Thrombin Receptor ◽  
Acute Cerebral Ischemia ◽  
High Concentrations

SummaryPlatelet activation is involved in the pathogenesis of cerebrovascular ischemia, but the major agonist involved has yet to be identified. To investigate the role of thrombin in platelet activation in patients with acute ischemic stroke, and while thrombin is the most likely candidate for activation of the thrombin receptor PAR-1 in vivo, we assessed its cleavage and internalization using the antibodies SPAN12, binding to uncleaved PAR-1, and WEDE15, recognizing cleaved and uncleaved, but not internalized PAR-1. In contrast to healthy age-matched controls, platelets from stroke patients exhibited significant cleavage and internalization of PAR-1 (P<0.001) and failed to respond to thrombin in vitro. Enhanced surface expression of CD62P, CD63, TSP-1 and less mepacrine uptake showed platelet degranulation during stroke. Platelets from patients with acute cerebral ischemia are exhausted and desensitized to thrombin through cleavage of PAR-1, indicating that high concentrations of thrombin occur with acute cerebrovascular ischemic events in vivo.

scholarly journals Mast Cells in Cardiovascular Disease: From Bench to Bedside

2019 ◽  
Vol 20 (14) ◽  
pp. 3395 ◽  
Author(s):  
Hermans ◽  
Lennep ◽  
van Daele ◽  
Bot
Keyword(s):  
Cardiovascular Disease ◽  
Mast Cells ◽  
Animal Studies ◽  
Intraplaque Hemorrhage ◽  
Hematological Disease ◽  
Plaque Instability ◽  
Clonal Proliferation ◽  
Progression Of Atherosclerosis

Mast cells are pluripotent leukocytes that reside in the mucosa and connective tissue. Recent studies show an increased prevalence of cardiovascular disease among patients with mastocytosis, which is a hematological disease that is characterized by the accumulation of mast cells due to clonal proliferation. This association suggests an important role for mast cells in cardiovascular disease. Indeed, the evidence establishing the contribution of mast cells to the development and progression of atherosclerosis is continually increasing. Mast cells may contribute to plaque formation by stimulating the formation of foam cells and causing a pro-inflammatory micro-environment. In addition, these cells are able to promote plaque instability by neo-vessel formation and also by inducing intraplaque hemorrhage. Furthermore, mast cells appear to stimulate the formation of fibrosis after a cardiac infarction. In this review, the available data on the role of mast cells in cardiovascular disease are summarized, containing both in vitro research and animal studies, followed by a discussion of human data on the association between cardiovascular morbidity and diseases in which mast cells are important: Kounis syndrome, mastocytosis and allergy.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

Platelets, inflammation and anti-inflammatory effects of antiplatelet drugs in ACS and CAD

2015 ◽  
Vol 114 (09) ◽  
pp. 498-518 ◽  
Author(s):  
Karin Müller ◽  
Madhumita Chatterjee ◽  
Dominik Rath ◽  
Tobias Geisler
Keyword(s):  
Cardiovascular Disease ◽  
Chronic Inflammation ◽  
Platelet Activation ◽  
Inflammatory Marker ◽  
Antiplatelet Agents ◽  
Prognostic Impact ◽  
Chronic Inflammatory Condition ◽  
Anti Inflammatory ◽  
Coronary Events

SummaryPlatelets play a pivotal role in chronic inflammation leading to progression of atherosclerosis and acute coronary events. Recent discoveries on novel mechanisms and platelet-dependent inflammatory targets underpin the role of platelets to maintain a chronic inflammatory condition in cardiovascular disease. There is strong and clinically relevant crosslink between chronic inflammation and platelet activation. Antiplatelet therapy is a cornerstone in the prevention and treatment of acute cardiovascular events. The benefit of antiplatelet agents has mainly been attributed to their direct anti-aggregatory impact. Some anti-inflammatory off-target effects have also been described. However, it is unclear whether these effects are secondary due to inhibition of platelet activation or are caused by direct distinct mechanisms interfering with inflammatory pathways. This article will highlight novel platelet associated targets that contribute to inflammation in cardiovascular disease and elucidate mechanisms by which currently available antiplatelet agents evolve anti-inflammatory capacities, in particular by carving out the differential mechanisms directly or indirectly affecting platelet mediated inflammation. It will further illustrate the prognostic impact of antiplatelet therapies by reducing inflammatory marker release in recent cardiovascular trials.

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1535-1535
Author(s):  
Suzana M. Zorca ◽  
Emma C. Josefsson ◽  
Viktoria Rumjantseva ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Lectin Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation. The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

The Toll-Like Receptor-Like Molecule CD180 and Soluble CD14 Transmit Survival Signals in B-Cell Chronic Lymphocytic Leukemia Cells Presumably by Acting As Co-Receptors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3883-3883
Author(s):  
Martina Seiffert ◽  
Angela Schulz ◽  
Stephan Stilgenbauer ◽  
Peter Lichter
Keyword(s):  
Intracellular Signaling ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Toll Like Receptor ◽  
Soluble Cd14 ◽  
Soluble Factors ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Survival Signals

Abstract Abstract 3883 Survival and proliferation of B-cell chronic lymphocytic leukemia (CLL) cells strongly depend on external factors. When removed from their natural microenvironment CLL cells rapidly undergo spontaneous apoptosis in vitro unless cocultured with stromal cells or non-malignant leukocytes. Recently, we could show that monocytes effectively support long-term survival of CLL cells in vitro. Our results from cytokine antibody arrays and extensive transcriptome analyses of primary CLL cell cocultures suggested a functional role of several soluble factors as well as signaling pathways of innate immunity, like Toll-like receptor-, TREM1- and NRF2-mediated signaling. The most interesting soluble factors are currently quantified in serum samples of the german CLL8 study cohort of CLL patients by cytometric bead arrays. So far, our data show that two of these candidates, CCL2 and soluble CD14, are significantly increased in the serum of CLL patients. In vitro studies using recombinant soluble CD14 demonstrated that CLL cell survival was significantly increased in the presence of this factor. CD14 which is expressed in particular by monocytes and macrophages is an important mediator of innate immunity. Along with TLR-4, CD14 acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS). Alternatively, LPS can also bind to the toll-like receptor-like molecule CD180, which shows strong homology to TLR-4, but does not harbor an intracellular signaling domain. Since TLR-4 is not expressed in CLL cells, we investigated the potential role of CD180 in CD14-mediated cell survival. Flow cytometry analysis revealed an upregulation of CD180 surface expression in CLL cells under survival-inducing culture conditions. Stimulation of CD180 with a cross-linking antibody resulted in activation of CLL cells measured by increased cell size and upregulation of the activation marker CD86, and significantly increased survival rates of CLL cells. Both CD14- and CD180-mediated survival signals lead to an increase in NF-κB activity and up-regulation of its target gene BCL-2. Depletion of CD180 surface expression in CLL cells abolished the pro-survival effect of soluble CD14, suggesting that this factor mediates its signals via binding to CD180. In summary, our data demonstrate that both, soluble CD14 and the toll-like receptor-like molecule CD180 transmit pro-survival signals in CLL cells, most likely by acting as co-receptors. Currently, we characterize the intracellular signaling machinery which is involved in these processes. Disclosures: No relevant conflicts of interest to declare.

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

2005 ◽  
Vol 98 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
Antonino Coppola ◽  
Ludovico Coppola ◽  
Liliana dalla Mora ◽  
Francesco M. Limongelli ◽  
Antonio Grassia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
B Lymphocytes ◽  
Platelet Activation ◽  
Critical Role ◽  
Strenuous Exercise ◽  
Platelet Glycoprotein ◽  
Vigorous Exercise ◽  
Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3464-3472 ◽  
Author(s):  
Frauke May ◽  
Ina Hagedorn ◽  
Irina Pleines ◽  
Markus Bender ◽  
Timo Vögtle ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Antibody Treatment ◽  
Cellular Activation ◽  
Primary Hemostasis ◽  
Arterial Thrombus ◽  
Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

scholarly journals The possible role of mutated endothelial cells in myeloproliferative neoplasms

Haematologica ◽  
2021 ◽  
Author(s):  
Mirko Farina ◽  
Domenico Russo ◽  
Ronald Hoffman
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Hematopoietic Cells ◽  
Clinical Manifestations ◽  
Vascular Complications ◽  
Hematologic Malignancies ◽  
Driver Mutations ◽  
Vascular Events ◽  
Hematopoietic Stem ◽  
High Incidence

Myeloproliferative neoplasms (MPN) are chronic, clonal hematologic malignancies characterized by myeloproliferation and a high incidence of vascular complications (thrombotic and bleeding). Although MPN-specific driver mutations have been identified, the underlying events that culminate in these clinical manifestations require further clarification. We reviewed the numerous studies performed during the last decade identifying endothelial cell (EC) dysregulation as a factor contributing to MPN disease development. The JAK2V617F MPN mutation and other myeloid-associated mutations have been detected not only in hematopoietic cells but also in EC and their precursors in MPN patients, suggesting a link between mutated EC and the high incidence of vascular events. To date, however, the role of EC in MPN continues to be questioned by some investigators. In order to further clarify the role of EC in MPN, we first describe the experimental strategies used to study EC biology and then analyze the available evidence generated using these assays which implicate mutated EC in MPN-associated abnormalities. Mutated EC have been reported to possess a pro-adhesive phenotype as a result of increased endothelial Pselectin exposure, secondary to degranulation of Weibel-Palade bodies, which is further accentuated by exposure to pro-inflammatory cytokines. Additional evidence indicates that MPN myeloproliferation requires JAK2V617F expression by both hematopoietic stem cells and EC. Furthermore, the reports of JAK2V617F and other myeloid malignancy- associated mutations in both hematopoietic cells and EC in MPN patients support the hypothesis that MPN driver mutations may first appear in a common precursor cell for both EC and hematopoietic cells.

scholarly journals HEMOSTASIS BERLANDASKAN SEL HIDUP (IN VIVO)

2016 ◽  
Vol 19 (3) ◽  
pp. 204
Author(s):  
Liong Boy Kurniawan ◽  
Mansyur Arif
Keyword(s):  
Tissue Factor ◽  
Platelet Activation ◽  
Bleeding Risk ◽  
Coagulation Factors ◽  
Cascade Theory ◽  
Classic Theory

Understanding of hemostasis has developed substantially in the last century from stasis in vitro to in vivo concept. Hemostasis theory develops from classic theory, discovery of coagulation factors leading to cascade/waterfall theory, as well as to in vivo cell based theory which explains the limitations of cascade theory. Phases of cell based hemostasis theory include initiation, amplification, propagation and termination with the role of tissue factor, platelet activation and coagulation factors in thrombin and fibrin synthesis. Common hemostasis tests used nowadays are important in evaluating bleeding risk but this matter still can not explain cell based hemostasis theory comprehensively so we need to find new tests to evaluate in vivo hemostasis.

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