Platelet-endothelial Interactions Research Articles

Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1

BackgroundDiabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells. ObjectivesWe investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia. MethodsImmunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA. ResultsPlatelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H2O2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors. ConclusionEndothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics.

Utility of Global Hemostatic Assays in Patients with Bleeding Disorders of Unknown Cause.

Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after exhaustive evaluation of plasmatic coagulation and platelet function. This review explores the utility of global hemostatic assays as confirmatory tests and in elucidating the pathophysiology of BDUC. Unlike traditional hemostatic tests that focus on coagulation factors, global assays are conducted both in plasma and also whole blood. These assays provide a more comprehensive understanding of the cell-based model of coagulation, aid in the identification of plasmatic factor abnormalities that may reduce hemostatic capacity, and allow for the assessment of impaired platelet-endothelial interactions under shear stress, as well as hyperfibrinolytic states. While clinical tests such as skin bleeding time and global assays such as PFA-100 exhibit limited diagnostic capacity, the role of viscoelastic testing in identifying hemostatic dysfunction in patients with BDUC remains unclear. Thrombin generation assays have shown variable results in BDUC patients; some studies demonstrate differences compared with healthy controls or reference values, whereas others question its clinical utility. Fibrinolysis assessment in vitro remains challenging, with studies employing euglobulin clot lysis time, plasma clot lysis time, and fluorogenic plasmin generation yielding inconclusive or conflicting results. Notably, recent studies suggest that microfluidic analysis unveils shear-dependent platelet function defects in BDUC patients, undetected by conventional platelet function assays. Overall, global assays might be helpful for exploring underlying hemostatic impairments, when conventional hemostatic laboratory tests yield no results. However, due to limited data and/or discrepant results, further research is needed to evaluate the utility of global assays as screening tools.

Isaridin E Protects against Sepsis by Inhibiting Von Willebrand Factor-Induced Endothelial Hyperpermeability and Platelet-Endothelium Interaction.

Endothelial hyperpermeability is pivotal in sepsis-associated multi-organ dysfunction. Increased von Willebrand factor (vWF) plasma levels, stemming from activated platelets and endothelium injury during sepsis, can bind to integrin αvβ3, exacerbating endothelial permeability. Hence, targeting this pathway presents a potential therapeutic avenue for sepsis. Recently, we identified isaridin E (ISE), a marine-derived fungal cyclohexadepsipeptide, as a promising antiplatelet and antithrombotic agent with a low bleeding risk. ISE's influence on septic mortality and sepsis-induced lung injury in a mouse model of sepsis, induced by caecal ligation and puncture, is investigated in this study. ISE dose-dependently improved survival rates, mitigating lung injury, thrombocytopenia, pulmonary endothelial permeability, and vascular inflammation in the mouse model. ISE markedly curtailed vWF release from activated platelets in septic mice by suppressing vesicle-associated membrane protein 8 and soluble N-ethylmaleide-sensitive factor attachment protein 23 overexpression. Moreover, ISE inhibited healthy human platelet adhesion to cultured lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), thereby significantly decreasing vWF secretion and endothelial hyperpermeability. Using cilengitide, a selective integrin αvβ3 inhibitor, it was found that ISE can improve endothelial hyperpermeability by inhibiting vWF binding to αvβ3. Activation of the integrin αvβ3-FAK/Src pathway likely underlies vWF-induced endothelial dysfunction in sepsis. In conclusion, ISE protects against sepsis by inhibiting endothelial hyperpermeability and platelet-endothelium interactions.

Research into New Molecular Mechanisms in Thrombotic Diseases Paves the Way for Innovative Therapeutic Approaches.

Thrombosis is a multifaceted process involving various molecular components, including the coagulation cascade, platelet activation, platelet-endothelial interaction, anticoagulant signaling pathways, inflammatory mediators, genetic factors and the involvement of various cells such as endothelial cells, platelets and leukocytes. A comprehensive understanding of the molecular signaling pathways and cell interactions that play a role in thrombosis is essential for the development of precise therapeutic strategies for the treatment and prevention of thrombotic diseases. Ongoing research in this field is constantly uncovering new molecular players and pathways that offer opportunities for more precise interventions in the clinical setting. These molecular insights into thrombosis form the basis for the development of targeted therapeutic approaches for the treatment and prevention of thrombotic disease. The aim of this review is to provide an overview of the pathogenesis of thrombosis and to explore new therapeutic options.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

Protein disulphide isomerase A1 (PDI) supports platelet-endothelium interaction in hyperglycaemia: impacts on endothelial membrane biophysics and secretion of adhesion-related proteins

Protein disulphide isomerase A1 (PDI) supports platelet-endothelium interaction in hyperglycaemia: impacts on endothelial membrane biophysics and secretion of adhesion-related proteins

Effect of far-infrared radiation therapy on von Willebrand factor in patients with chronic kidney disease.

Hemostatic abnormality has contributed to vascular access thrombosis in patients with chronic kidney disease (CKD). Previous studies have demonstrated that far-infrared radiation (FIR) therapy can maintain the patency and maturity of arteriovenous fistulas of patients undergoing hemodialysis (HD). However, prolonged access bleeding is observed once FIR is conducted at the end of dialysis. FIR can block the binding of platelet and von Willebrand factor (vWF), a predictor of hemostatic abnormality and vascular access thrombosis. However, clinical studies exploring FIR and vWF are sparse. We recruited 20 HD patients, 21 CKD patients, and 20 controls to examine the alteration of vWF and a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) following a single 40-min session of FIR therapy. In addition, the alteration of these factors in the HD group was examined following a 40-min FIR session thrice a week for 3 months. A decreasing trend in the vWF activity-antigen ratio of participants in all groups following a single FIR session was observed. In addition, the ratio in the HD group was significantly lower following 3 months of FIR therapy. The subgroup analysis revealed a consistent trend and multiple regression analysis showed that participants not taking hydroxymethylglutaryl-coenzyme A reductase inhibitor, diabetes mellitus, and higher hemoglobin levels were the significant factors. The alteration of the vWF activity-antigen ratio correlated moderately to that of ADAMTS13 antigen and activity. FIR may alter the ratio of ultra-large vWF multimers through ADAMTS13, contributing to inhibiting platelet-endothelium interactions of CKD patients.

Endothelial dysfunction and immunothrombosis in sepsis.

Sepsis is a life-threatening clinical syndrome characterized by multiorgan dysfunction caused by a dysregulated or over-reactive host response to infection. During sepsis, the coagulation cascade is triggered by activated cells of the innate immune system, such as neutrophils and monocytes, resulting in clot formation mainly in the microcirculation, a process known as immunothrombosis. Although this process aims to protect the host through inhibition of the pathogen's dissemination and survival, endothelial dysfunction and microthrombotic complications can rapidly lead to multiple organ dysfunction. The development of treatments targeting endothelial innate immune responses and immunothrombosis could be of great significance for reducing morbidity and mortality in patients with sepsis. Medications modifying cell-specific immune responses or inhibiting platelet-endothelial interaction or platelet activation have been proposed. Herein, we discuss the underlying mechanisms of organ-specific endothelial dysfunction and immunothrombosis in sepsis and its complications, while highlighting the recent advances in the development of new therapeutic approaches aiming at improving the short- or long-term prognosis in sepsis.

Platelets and ductus arteriosus closure in neonates

Platelet plug formation is critically involved in murine ductus arteriosus closure and thrombocytopenia in preterm infants seems to negatively affect spontaneous and pharmacologically induced ductal closure. Furthermore, platelet dysfunction may contribute to ductal patency, especially in extremely immature infants. Neonatal platelets likely have multifaceted roles during ductal closure, such as secretion of several signaling molecules and facilitation of specific cell-cell interactions. The only available randomized-controlled trial on platelet transfusions in preterm infants with patent ductus arteriosus demonstrated that a liberal transfusion regimen did not promote ductal closure, but was associated with an increased rate of intraventricular hemorrhage.Herein, we discuss the available mechanistic evidence on the role of platelets in ductus arteriosus closure and their potential clinical implications in preterm infants. We further briefly outline future research directions aimed at a better understanding of platelet-endothelial interactions in neonatal health and disease.

Peptides Derived from a Plant Protease Inhibitor of the Coagulation Contact System Decrease Arterial Thrombus Formation in a Murine Model, without Impairing Hemostatic Parameters.

Several plant protein inhibitors with anticoagulant properties have been studied and characterized, including the Delonix regia trypsin inhibitor (DrTI). This protein inhibits serine proteases (trypsin) and enzymes directly involved in coagulation, such as plasma kallikrein, factor XIIa, and factor XIa. In this study, we evaluated the effects of two new synthetic peptides derived from the primary sequence of DrTI in coagulation and thrombosis models to understand the mechanisms involved in the pathophysiology of thrombus formation as well as in the development of new antithrombotic therapies. Both peptides acted on in vitro hemostasis-related parameters, showing promising results, prolonging the Partially Activated Thromboplastin Time (aPTT) and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid. In murine models, for arterial thrombosis induced by photochemical injury, and platelet-endothelial interactions monitored by intravital microscopy, both peptides at doses of 0.5 mg/kg significantly extended the time of artery occlusion and modified the platelet adhesion and aggregation pattern with no changes in bleeding time, demonstrating the high biotechnological potential of both molecules.

Microvascular Thrombosis as a Critical Factor in Severe COVID-19.

Platelet-endothelial interactions have a critical role in microcirculatory function, which maintains tissue homeostasis. The subtle equilibrium between platelets and the vessel wall is disturbed by the coronavirus disease 2019 (COVID-19), which affects all three components of Virchow's triad (endothelial injury, stasis and a hypercoagulable state). Endotheliitis, vasculitis, glycocalyx degradation, alterations in blood flow and viscosity, neutrophil extracellular trap formation and microparticle shedding are only few pathomechanisms contributing to endothelial damage and microthrombosis resulting in capillary plugging and tissue ischemia. In the following opinion paper, we discuss major pathological processes leading to microvascular endothelial activation and thrombosis formation as a possible major adverse factor driving the deterioration of patient disease course in severe COVID-19.

Pro-Inflammatory Chemokines CCL5, CXCL12, and CX3CL1 Bind to and Activate Platelet Integrin αIIbβ3 in an Allosteric Manner.

Activation of platelet integrin αIIbβ3, a key event for hemostasis and thrombus formation, is known to be mediated exclusively by inside-out signaling. We showed that inflammatory chemokines CX3CL1 and CXCL12 in previous studies, and CCL5 in this study, bound to the allosteric binding site (site 2) of vascular integrin αvβ3, in addition to the classical ligand binding site (site 1), and allosterically activated integrins independent of inside-out signaling. Since αIIbβ3 is exposed to inflammatory chemokines at increased concentrations during inflammation (e.g., cytokine/chemokine storm) and platelet activation, we hypothesized that these chemokines bind to and activate αIIbβ3 in an allosteric activation mechanism. We found that these chemokines bound to αIIbβ3. Notably, they activated soluble αIIbβ3 in 1 mM Ca2+ by binding to site 2. They activated cell-surface αIIbβ3 on CHO cells, which lack machinery for inside-out signaling or chemokine receptors, quickly (<1 min) and at low concentrations (1–10 ng/mL) compared to activation of soluble αIIbβ3, probably because chemokines bind to cell surface proteoglycans. Furthermore, activation of αIIbβ3 by the chemokines was several times more potent than 1 mM Mn2+. We propose that CCL5 and CXCL12 (stored in platelet granules) may allosterically activate αIIbβ3 upon platelet activation and trigger platelet aggregation. Transmembrane CX3CL1 on activated endothelial cells may mediate platelet–endothelial interaction by binding to and activating αIIbβ3. Additionally, these chemokines in circulation over-produced during inflammation may trigger αIIbβ3 activation, which is a possible missing link between inflammation and thrombosis.

Functional Alterations Involved in Increased Bleeding in Hereditary Hemorrhagic Telangiectasia Mouse Models.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal-dominant genetic disorder involving defects in two predominant genes known as endoglin (ENG; HHT-1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT-2). It is characterized by mucocutaneous telangiectases that, due to their fragility, frequently break causing recurrent epistaxis and gastrointestinal bleeding. Because of the severity of hemorrhages, the study of the hemostasis involved in these vascular ruptures is critical to find therapies for this disease. Our results demonstrate that HHT patients with high bleeding, as determined by a high Epistaxis Severity Score (ESS), do not have prolonged clotting times or alterations in clotting factors. Considering that coagulation is only one of the processes involved in hemostasis, the main objective of this study was to investigate the overall mechanisms of hemostasis in HHT-1 (Eng+/−) and HHT-2 (Alk1+/−) mouse models, which do not show HHT vascular phenotypes in the meaning of spontaneous bleeding. In Eng+/− mice, the results of in vivo and in vitro assays suggest deficient platelet-endothelium interactions that impair a robust and stable thrombus formation. Consequently, the thrombus could be torn off and dragged by the mechanical force exerted by the bloodstream, leading to the reappearance of hemorrhages. In Alk1+/− mice, an overactivation of the fibrinolysis system was observed. These results support the idea that endoglin and Alk1 haploinsufficiency leads to a common phenotype of impaired hemostasis, but through different mechanisms. This contribution opens new therapeutic approaches to HHT patients' epistaxis.

Emerging Role of Platelet-Endothelium Interactions in the Pathogenesis of Severe SARS-CoV-2 Infection-Associated Myocardial Injury.

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2–induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2–driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.

The role of platelets in the pathogenesis of atherosclerosis

The participation of platelets in the development of atherothrombosis in acute forms of cardiovascular diseases is generally recognized and well studied, while their significance in the pathogenesis of vascular atherosclerosis, starting from the earliest stages, requires further study. This article analyzes the role of platelets in the pathogenesis of atherosclerosis. Modern views on the pathogenesis of atherosclerosis, platelet-endothelial interactions, molecular mechanisms of platelet activation, platelet immune function from the point of view of the initial stages of atherogenesis, studies of the effect of antiplatelet therapy in primary and secondary prevention of atherosclerosis are considered. The leading role of platelets in the inflammatory and immune reactions of the human body is indicated, which is realized due to a complex of immune receptors, adhesion molecules and mediators.

Platelets amplify endotheliopathy in COVID-19.

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y12 represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Abstract 109: Platelets Amplify Endotheliopathy In Covid-19

In addition to their pivotal role in thrombosis and hemostasis, platelets participate in inflammatory responses and endothelial cell activation - hallmarks in the pathogenesis of coronavirus disease 2019 (COVID-19). Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). To explore this interaction, ECs were treated with platelet releasate from patients with and without COVID-19, and EC mRNA sequencing performed. We demonstrate that platelet released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. Investigation of the COVID-19 platelet transcriptome identified pathways related to organelle/granule release, metabolism, and immune effector function in addition to upregulation of S100A8 and S100A9 mRNA. Incubation of primary megakaryocytes with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also induced upregulation of S100A8 and S100A9 mRNA. Consistent with increased gene expression, the heterodimer protein product of S100A8 / A9 , myeloid-related protein (MRP)8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates microvascular endothelial cells, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to thromboinflammation and poor clinical outcomes in COVID-19 patients. Finally, we present evidence that therapeutic targeting of platelet P2Y 12 represents a promising candidate to reduce proinflammatory and prothrombotic platelet-endothelial interactions. Altogether, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Arterial Platelet Adhesion in Atherosclerosis-Prone Arteries of Obese, Insulin-Resistant Nonhuman Primates.

BackgroundPlatelet–endothelial interactions are thought to contribute to early atherogenesis. These interactions are potentiated by oxidative stress. We used in vivo molecular imaging to test the hypothesis that platelet–endothelial interactions occur at early stages of plaque development in obese, insulin‐resistant nonhuman primates, and are suppressed by NADPH‐oxidase‐2 inhibition.Methods and ResultsSix adult rhesus macaques fed a Western‐style diet for a median of 4.0 years were studied at baseline and after 8 weeks of therapy with the NADPH‐oxidase‐2‐inhibitor apocynin (50 mg/kg per day). Six lean control animals were also studied. Measurements included intravenous glucose tolerance test, body composition by dual‐energy X‐ray absorptiometry, carotid intimal medial thickness, carotid artery contrast ultrasound molecular imaging for platelet GPIbα (glycoprotein‐ Ibα) and vascular cell adhesion molecule‐1, and blood oxidative markers on mass spectrometry. Compared with lean controls, animals on a Western‐style diet were obese (median body mass: 16.0 versus 8.7 kg, P=0.003; median truncal fat: 49% versus 20%, P=0.002), were insulin resistant (4‐fold higher insulin–glucose area under the curve on intravenous glucose tolerance test, P=0.002), had 40% larger carotid intimal medial thickness (P=0.004), and exhibited oxidative signatures on proteomics. In obese but not lean animals, signal enhancement on molecular imaging was significantly elevated for GPIbα and vascular cell adhesion molecule‐1. The signal correlated modestly with intimal medial thickness but not with the degree of insulin resistance. Apocynin significantly (P<0.01) reduced median signal for GPIbα by >80% and vascular cell adhesion molecule‐1 signal by 75%, but did not affect intimal medial thickness, body mass, or intravenous glucose tolerance test results.ConclusionIn nonhuman primates, diet‐induced obesity and insulin resistance leads to platelet–endothelial adhesion at early atherosclerotic lesion sites, which is associated with the expression of pro‐inflammatory adhesion molecules. These responses appear to be mediated, in part, through oxidative pathways.

Elevated P-Selectin in Severe Covid-19: Considerations for Therapeutic Options.

BackgroundCoronavirus disease 2019 (COVID-19) is mainly a respiratory tract disease and acute respiratory failure with diffuse microvascular pulmonary thrombosis are critical aspects of the morbidity and mortality of this new syndrome.PurposeThe aim of our study was to investigate, in severe COVID-19 hospitalized patients, the P-selectin plasma concentration as a biomarker of endothelial dysfunction and platelet activation.Methods46 patients with severe or critical SARS-CoV-2 infection were included in the study. Age-matched patients then were divided in those requiring admission to the intensive care unit (ICU, ICU cases) vs those not requiring ICU hospitalization (non-ICU cases). Blood samples of severe COVID-19 patients were collected at the time of hospital admission. The quantification of soluble P-selectin was performed by ELI, assay.ResultsOur study showed a higher P-selectin plasma concentration in patients with Covid-19, regardless of ICU admission, compared to the normal reference values and compared to ten contextually sampled healthy donors (HD); (COVID-19): median 65.2 (IQRs: 45.1–81.1) vs. HD: 40.3 (IQRs: 24.3–48.7), p=0023. Moreover, results showed a significant reduction of P-sele din after platelets removal in HD, in contrast, both ICU and non-ICU COVID-19 patients showed similar high levels of P-selectin with and without platelets.ConclusionElevation of P-selectin suggests a central role of platelet endothelium interaction as part of the multifaced pathogenic mechanism of COVID-19 leading to the local activation of hemostatic system forming pulmonary thrombi. Further work is necessary to determine the therapeutic role of antiplatelets agents or of the anti P-selectin antibody Crizanlizumab.

Impairment of platelet NO signalling in coronary artery spasm: role of hydrogen sulphide.

The pathophysiology of coronary artery spasm (CAS), with its associated ischaemic crises, is currently poorly understood and treatment is frequently ineffective. In view of increasing evidence that platelet-based defects may occur in CAS patients, we investigated platelet reactivity in CAS patients and whether symptomatic crises reflect activation of platelet-endothelial interactions. CAS patients were evaluated during acute and/or chronic symptomatic phases and compared with healthy control subjects. Inhibition of ADP-induced platelet aggregation by the NO donor sodium nitroprusside (SNP) and plasma concentrations of syndecan 1 (glycocalyx shedding marker), tryptase (mast cell activation marker) and platelet microparticles were measured. Inhibition of platelet aggregation by SNP was diminished in chronic CAS, with further (non-significant) deterioration during symptomatic crises, whereas plasma concentrations of syndecan 1, tryptase and platelet microparticles increased. Treatment of patients with high-dose N-acetylcysteine (NAC) plus glyceryl trinitrate rapidly increased platelet responsiveness to SNP and decreased plasma syndecan 1 concentrations. The effect of NAC on platelet responsiveness to SNP was confirmed in vitro and mimicked by the H2 S donor NaHS. Conversely, inhibition of enzymatic production of H2 S attenuated NAC effect. CAS is associated with substantial impairment of platelet NO signalling. During acute symptomatic exacerbations, platelet resistance to NO is aggravated, together with mast cell activation and damage to both vasculature and platelets. NAC, via release of H2 S, reverses platelet resistance to NO and terminates glycocalyx shedding during symptomatic crises: This suggests that H2 S donors may correct the pathophysiological anomalies underlying CAS.