Formation Of Platelet-leukocyte Aggregates Research Articles

A peptide from the staphylococcal protein Efb binds P‐selectin and inhibits the interaction of platelets with leukocytes

AimsP‐selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N‐terminal domain of Staphylococcus aureus extracellular fibrinogen‐binding protein (Efb) binds to P‐selectin and interferes with platelet‐leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet‐leukocyte aggregates. Methods and ResultsUsing a library of synthetic peptides, we mapped the platelet‐binding site to a continuous 20 amino acid stretch. The peptide Efb68‐87 was able to bind to resting and, to a greater extent, thrombin‐stimulated platelets in the absence of fibrinogen. Dot blots, pull‐down assays and P‐selectin glycoprotein ligand‐1 (PSGL‐1) competitive binding experiments identified P‐selectin as the cellular docking site mediating Efb68‐87 platelet binding. Accordingly, Efb68‐87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68‐87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet‐leukocyte aggregates (PLAs). Efb68‐87 also interfered with the platelet‐dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. ConclusionsWe have identified Efb68‐87 as a novel selective platelet‐binding peptide. Efb68‐87 binds directly to P‐selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68‐87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Peripheral versus central venous blood sampling does not influence the assessment of platelet activation in cirrhosis

Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00–24.66] vs. C: 4.12 [0.00–19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis. Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor

Platelet-to-Lymphocyte Ratio as Marker of Platelet Activation in Patients on Potent P2Y12 Inhibitors.

A high platelet-to-lymphocyte ratio (PLR) has recently been associated with ischemic outcomes in cardiovascular disease. Increased platelet reactivity and leukocyte-platelet aggregate formation are directly involved in the progress of atherosclerosis and have been linked to ischemic events following percutaneous coronary intervention (PCI). In order to understand the relation of PLR with platelet reactivity, we assessed PLR as well as agonist-inducible platelet aggregation and neutrophil-platelet aggregate (NPA) formation in 182 acute coronary syndrome (ACS) patients on dual antiplatelet therapy with aspirin and prasugrel (n = 96) or ticagrelor (n = 86) 3 days after PCI. PLR was calculated from the blood count. Platelet aggregation was measured by multiple electrode aggregometry and NPA formation was determined by flow cytometry, both in response to ADP and SFLLRN. A PLR ≥91 was considered as high PLR based on previous data showing an association of this threshold with adverse ischemic outcomes. In the overall cohort and in prasugrel-treated patients, high PLR was associated with higher SFLLRN-inducible platelet aggregation (67 AU [50-85 AU] vs 59.5 AU [44.3-71.3 AU], P = .01, and 73 AU [50-85 AU] vs 61.5 AU [46-69 AU], P = .02, respectively). Further, prasugrel-treated patients with high PLR exhibited higher ADP- (15% [11%-23%] vs 10.9% [7.6%-15.9%], P = .007) and SFLLRN-inducible NPA formation (64.3% [55.4%-73.8%] vs 53.8% [44.1%-70.1%], P = .01) as compared to patients with low PLR. These differences were not seen in ticagrelor-treated patients. In conclusion, high PLR is associated with increased on-treatment platelet reactivity in prasugrel-treated patients, but not in patients on ticagrelor.

S119: PRELIMINARY RESULTS OF A PHASE 1 STUDY IN HEALTHY SUBJECTS ADMINISTERED INCLACUMAB, A FULLY HUMAN IGG4 ANTI-P-SELECTIN MONOCLONAL ANTIBODY IN DEVELOPMENT FOR TREATMENT OF SICKLE CELL DISEASE

Background: Inclacumab, a fully human IgG4 anti-P-selectin monoclonal antibody, is being developed for the reduction of vaso-occlusive crises (VOCs) in patients with sickle cell disease (SCD). P-selectin-mediated platelet-leukocyte aggregate (PLA) formation has been shown to contribute to vaso-occlusion. Safety and pharmacology of inclacumab have previously been well characterized in over 700 subjects (healthy volunteers and patients with cardiovascular disease), at doses up to 20 mg/kg every 4 weeks for up to 9 months. The current Phase 1 study was initiated to evaluate the safety and pharmacology of inclacumab at doses of 20 mg/kg and 40 mg/kg in healthy subjects in support of a target Phase 3 dose of 30 mg/kg administered every 12 weeks to patients with SCD. Methods: Healthy adult subjects over 18 years of age without significant current or prior health conditions received a single intravenous (IV) dose of 20 mg/kg inclacumab infused over approximately one hour (Cohort 1). Following a review of safety, a second cohort received a single IV dose of 40 mg/kg infused over approximately one hour (Cohort 2). The total study duration and sample collection period was 29 weeks. Final safety and preliminary pharmacokinetics (PK), anti-drug antibody (ADA), and ex vivo thrombin receptor-activating peptide (TRAP)-activated PLA formation data are reported. Results: Fifteen subjects received a single dose of inclacumab 20 mg/kg (n=6) or 40 mg/kg (n=9). Median age was 42 years (range 22–52 years); median body weight was 73.6 kg (range 63.7–89.3 kg). Through the prespecified 72-hour post-infusion safety assessment period in both cohorts, no treatment-emergent adverse events (AEs) > grade 1 (mild) nor dose-limiting toxicities were reported. Across the duration of the study, there were no serious AEs, infusion-related reactions, or hypersensitivity reactions. Additionally, no clinically significant changes in vital signs, laboratory findings, or ECGs were observed. The most common AEs were upper respiratory tract infections, headache, myalgia, back pain, and contact dermatitis. The only events assessed by the investigator as potentially related to inclacumab were headache and dizziness, which were experienced by one subject (20 mg/kg) and occurred 4 hours following the end of infusion. In healthy subjects, inclacumab demonstrated dose-proportional PK over the dose range tested; PK parameter estimates were consistent with those reported for monoclonal antibodies. Geometric mean Cmax following single doses of 20 and 40 mg/kg were 402 and 970 µg/mL, respectively. Mean TRAP-activated pre-dose PLA formation was 33–39% across cohorts and decreased to 9–14% at 2 hours following end of infusion. PLA inhibition was sustained up to 23 weeks for both the 20 and 40 mg/kg doses. Two subjects in the 40 mg/kg cohort were ADA-positive on Week 12 and thereafter; a preliminary analysis demonstrated no apparent impact on PK or safety in these subjects. Conclusions: Inclacumab displayed a well-tolerated safety profile for up to 29 weeks following a single dose of 20 or 40 mg/kg in healthy subjects. Durable inhibition of TRAP-activated PLA formation was observed up to 23 weeks. Overall, the results support a Phase 3 dose of 30 mg/kg every 12 weeks in patients with SCD-related VOCs. Funding: This study was supported by Global Blood Therapeutics.

Adverse Outcome in COVID-19 Is Associated With an Aggravating Hypo-Responsive Platelet Phenotype.

Thromboembolic complications are frequently observed in Coronavirus disease 2019 (COVID-19). While COVID-19 is linked to platelet dysregulation, the association between disease outcome and platelet function is less clear. We prospectively monitored platelet activation and reactivity in 97 patients during the first week of hospitalization and determined plasma markers of platelet degranulation and inflammation. Adverse outcome in COVID-19 was associated with increased basal platelet activation and diminished platelet responses, which aggravated over time. Especially GPIIb/IIIa responses were abrogated, pointing toward impeded platelet aggregation. Moreover, platelet-leukocyte aggregate formation was diminished, pointing toward abrogated platelet-mediated immune responses in COVID-19. No general increase in plasma levels of platelet-derived granule components could be detected, arguing against platelet exhaustion. However, studies on platelets from healthy donors showed that plasma components in COVID-19 patients with unfavorable outcome were at least partly responsible for diminished platelet responses.Taken together this study shows that unfavorable outcome in COVID-19 is associated with a hypo-responsive platelet phenotype that aggravates with disease progression and may impact platelet-mediated immunoregulation.

Preliminary Results of a Phase 1 Study in Healthy Subjects Administered Inclacumab, a Fully Human IgG4 Anti-P-Selectin Monoclonal Antibody in Development for Treatment of Sickle Cell Disease

Background: Inclacumab, a fully human IgG4 anti-P-selectin monoclonal antibody, is being developed for the reduction of vaso-occlusive crises (VOCs) in patients with sickle cell disease (SCD). P-selectin-mediated platelet-leukocyte aggregate (PLA) formation has been shown to contribute to vaso-occlusion. Safety and pharmacology of inclacumab have previously been well characterized in over 700 subjects (healthy volunteers and patients with cardiovascular disease), at doses up to 20 mg/kg every 4 weeks for up to 9 months. The current Phase 1 study was initiated to evaluate the safety and pharmacology of inclacumab at doses of 20 mg/kg and 40 mg/kg in healthy subjects in support of a target Phase 3 dose of 30 mg/kg administered every 12 weeks to patients with SCD.Methods: Healthy adult subjects over 18 years of age without significant current or prior health conditions received a single intravenous (IV) dose of 20 mg/kg inclacumab infused over approximately one hour (Cohort 1). Following a review of safety, a second cohort received a single IV dose of 40 mg/kg infused over approximately one hour (Cohort 2). The total study duration and sample collection period was 29 weeks. Final safety and preliminary pharmacokinetics (PK), anti-drug antibody (ADA), and ex vivo thrombin receptor-activating peptide (TRAP)-activated PLA formation data are reported.Results: Fifteen subjects received a single dose of inclacumab 20 mg/kg (n=6) or 40 mg/kg (n=9). Fourteen subjects completed the study. Median age was 42 years (range 22 - 52 years); median body weight was 73.6 kg (range 63.7 - 89.3 kg). Through the pre-specified 72-hour post-infusion safety assessment period in both cohorts, no treatment-emergent adverse events (AEs) > grade 1 (mild) nor dose-limiting toxicities were reported. During the duration of the study, there were no serious AEs, infusion-related reactions, or hypersensitivity reactions. Additionally, no clinically significant changes in vital signs, laboratory findings, or ECGs were observed. The most common AEs were headache, myalgia, and contact dermatitis. The only events assessed by the investigator as potentially related to inclacumab were headache and dizziness, which were experienced by one subject (20 mg/kg) and occurred 4 hours following the end of infusion. In healthy subjects, inclacumab demonstrated dose-proportional PK over the dose range tested; PK parameter estimates were consistent with those reported for monoclonal antibodies. Geometric mean C max following single doses of 20 and 40 mg/kg were 402 and 970 µg/mL, respectively. Mean TRAP-activated predose PLA formation was 33 - 39% across cohorts and decreased to 9-14% at 2 hours following end of infusion. PLA inhibition was sustained through at least 12 weeks for both the 20 and 40 mg/kg doses. Two subjects in the 40 mg/kg cohort were ADA-positive on Week 12 and thereafter; a preliminary analysis demonstrated no apparent impact on PK or safety in these subjects.Conclusions: Inclacumab displayed a well-tolerated safety profile for up to 29 weeks following a single dose of 20 or 40 mg/kg in healthy subjects. Durable inhibition of TRAP-activated PLA formation was observed through at least 12 weeks, consistent with prior observations. Overall, the results support a Phase 3 dose of 30 mg/kg every 12 weeks in patients with SCD-related VOCs.Funding: This study was supported by Global Blood Therapeutics. DisclosuresMayer: Global Blood Therapeutics: Consultancy. Redfern: Linear Clinical Research: Current Employment; Novartis: Other: Advisory Board; Pfizer: Other: Advisory Board; Roche: Other: Advisory Board; Eisai: Other: Advisory Board; Astra Zeneca: Other: Advisory Board. Geng: Global Blood Therapeutics: Current Employment. Shi: Global Blood Therapeutics: Current Employment. van Zutphen-van Geffen: Global Blood Therapeutics: Consultancy. Kuan: Global Blood Therapeutics: Consultancy. Koeck: Global Blood Therapeutics: Consultancy. Kastrissios: Global Blood Therapeutics: Consultancy. Patel: Global Blood Therapeutics: Consultancy. Davis: Global Blood Therapeutics: Current Employment. Yue: Global Blood Therapeutics: Current Employment.

The role of neutrophils in the pathogenesis of ischemic stroke

Background. Immune responses and inflammation play an important role in the pathogenesis of ischemic stroke.Aim. To analyze the involvement of neutrophils in the pathogenesis of ischemic stroke.Results. Data on the contribution of neutrophil granulocytes to the development of local sterile inflammation and secondary brain injury in acute ischemic stroke were summarized. Mechanisms of neutrophil influence on thrombosis, neutrophil extracellular trap formation (NETosis), protease release, and direct interaction with platelets with subsequent formation of platelet-leukocyte aggregates were discussed. Available information on the effectiveness of reperfusion therapy and an association of changes in neutrophil activity with development of infectious complications of stroke were presented. In addition, research data were presented on the contribution of neutrophils to atherogenesis, which is one of the most important etiological factors in ischemic stroke. The review showed that the contribution of neutrophils to the pathogenesis of ischemic stroke is associated with implementation of their secretory, regulatory, and phagocytic functions, as well as with NETosis.Conclusion. It was shown that neutrophils are involved in the pathogenesis of ischemic stroke and modulate a response to treatment.

Early platelet dysfunction in patients receiving extracorporeal membrane oxygenation is associated with mortality

Extracorporeal membrane oxygenation (ECMO) is used for patients with cardiopulmonary failure and is associated with severe bleeding and poor outcome. Platelet dysfunction may be a contributing factor. The aim of this prospective observational study was to characterize platelet dysfunction and its relation to outcome in ECMO patients. Blood was sampled from thirty ECMO patients at three timepoints. Expression of CD62P, CD63, activated GPIIb/IIIa, GPVI, GPIbα and formation platelet-leukocyte aggregates (PLA) were analyzed at rest and in response to stimulation. Delta granule storage-pool deficiency and secretion defects were also investigated. Fifteen healthy volunteers and ten patients with coronary artery disease served as controls. Results were also compared between survivors and non-survivors. Compared to controls, expression of platelet surface markers, delta granule secretion and formation of PLA was reduced, particularly in response to stimulation. Baseline CD63 expression was higher and activated GPIIb/IIIa expression in response to stimulation was lower in non-survivors on day 1 of ECMO. Logistic regression analysis revealed that these markers were associated with mortality. In conclusion, platelets from ECMO patients are severely dysfunctional predisposing patients to bleeding complications and poor outcome. Platelet dysfunction on day 1 of ECMO detected by the platelet surface markers CD63 and activated GPIIb/IIIa is associated with mortality. CD63 and activated GPIIb/IIIa may therefore serve as novel prognostic biomarkers, but future studies are required to determine their true potential.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

Pharmacokinetics, Pharmacodynamics and Safety of Ticagrelor in Children with Sickle Cell Disease: Results of a Two-Part Phase 2 Study

▪ INTRODUCTIONActivated platelets promote the adhesion of sickle cells to endothelial cells and the formation of platelet-leukocyte aggregates, contributing to vaso-occlusion (VOC). It has been suggested that inhibition of platelet reactivity may reduce the rate of VOCs in sickle cell disease (SCD). A study with ticlopidine reported a reduced rate of VOCs versus placebo. A recent, placebo-controlled, pediatric study with prasugrel showed a non-significant reduction in VOCs events, which was likely due to the low level of platelet inhibition. Ticagrelor is a reversible P2Y12 receptor antagonist shown to inhibit ADP-induced platelet aggregation and is approved for use in adults with coronary artery disease (CAD). A phase 2 study was conducted to characterize the pharmacokinetic (PK), pharmacodynamic (PD) and safety profiles of ticagrelor in children aged 2-17 years with SCD (HbSS or HbS/β0; NCT02214121). METHODSThis study was conducted in 2 parts: Part A was an open-label, randomized, dose-ranging study in which patients received two single doses of ticagrelor 1 week apart, followed by twice daily (bid) ticagrelor for 1 week. Initially, single doses of 0.125-0.563 mg/kg were given, followed by repeated dosing with 0.125 mg/kg bid. Individual platelet response was used to inform further dosing. After an interim analysis including the first 14 patients in Part A, single doses were increased to a maximum of 2.25 mg/kg and repeated dosing up to 0.75 mg/kg bid. Part B (optional) immediately followed the repeated dosing in Part A: patients were randomized 2:1 to continue ticagrelor with the same dose used during Part A or matching placebo as a double-blind treatment for 4 weeks. Safety was assessed during both parts, pain and analgesic use were recorded daily in an eDiary, and painful crises were reported at each visit. Exposure to ticagrelor and the active metabolite (AR-C124910XX), as well as the subsequent platelet inhibition, were determined after single and repeated doses in Parts A and B. RESULTSA total of 45 children with SCD, mean age 11.2 years (range: 3-17) received study drug in Part A and 23 (15 ticagrelor, 8 placebo) children, mean age 10.0 years (range: 3-17) in Part B. Most patients were of African descent (35/45 in Part A and 22/23 in Part B). Ticagrelor exposure increased approximately dose-linearly, with maximum plasma concentration values ranging from 15.2 to 566.6 ng/mL (with 0.125 mg/kg to 2.25 mg/kg) for ticagrelor and 3.1-156.0 ng/mL for AR-C124910XX. The parent:metabolite ratio in children with SCD was similar to that previously observed in adults. Mean P2Y12 reaction units (PRU) after single doses of ticagrelor decreased from pre-dose to 2 hours post-dose (change from baseline: -23.4 to -207.8 PRU for 0.125 mg/kg to 2.25 mg/kg), and returned towards baseline up to the last timepoint (6 or 8 hours' post-dose). In Part B, no difference was seen between the placebo and ticagrelor groups in pain ratings and analgesic use, although the study was not statistically powered to detect differences in these outcomes. Ticagrelor was well tolerated, with no bleeding during treatment and no patient discontinued treatment due to an adverse event. CONCLUSIONSThe PK/PD profile of ticagrelor in children with SCD was suitably characterized and the exposure-response relationship appeared similar to that previously observed in adults with CAD. Ticagrelor was well tolerated and no safety concerns were raised. The potential to impact SCD-related pain crises needs further evaluation, as the number of patients, low doses for some patients and short study duration limit the efficacy conclusions from this study. DisclosuresHsu:AstraZeneca: Other: steering committee for HESTIA trial; Emmi: Other: consultant for Emmi. Sarnaik:AstraZeneca: Other: Advisory Board. Amilon:AstraZeneca: Employment. Rensfeldt:AstraZeneca: Employment. Berggren:AstraZeneca: Employment.

Platelets in dengue infection: more than a numbers game

Dengue virus (DENV) infection is responsible for the development of dengue illness, which can be either asymptomatic, present mild manifestations or evolve to severe dengue. Thrombocytopenia is an important characteristic during DENV infection, being observed both in mild and severe dengue, although the lowest platelet counts are encountered during severe cases. This review gathers information regarding several mechanisms that have been related to alterations in platelet number and function, leading to thrombocytopenia but also platelet-mediated immune and inflammatory response. On this regard, we highlight that the decrease in platelet counts may be due to bone marrow suppression or consumption of platelets at the periphery. We discuss the infection of hematopoietic progenitors and stromal cells as mechanisms involved in bone marrow suppression. Concerning peripheral consumption of platelets, we addressed the direct infection of platelets by DENV, adhesion of platelets to leukocytes and vascular endothelium and platelet clearance mediated by anti-platelet antibodies. We also focused on platelet involvement on the dengue immunity and pathogenesis through translation and secretion of viral and host factors and through platelet-leukocyte aggregates formation. Hence, the present review highlights important findings related to platelet activation and thrombocytopenia during dengue infection, and also exhibits different mechanisms associated with decreased platelet counts. Graphical abstract: Schematic mechanistic representation of platelet-mediated immune responses and thrombocytopenia during dengue infection. (A) DENV-infected platelets secrete cytokines and chemokines and also adhere to activated vascular endothelium. Platelets aggregate with leukocytes, inducing the secretion of NETs and inflammatory mediators by neutrophils and monocytes, respectively. (B) DENV directly infects stromal cells and hematopoietic precursors, including megakaryocytes, which compromises megakaryopoiesis. Both central and peripheric mechanisms contribute to DENV-associated thrombocytopenia.

Platelet and Endothelial Activation as Potential Mechanisms Behind the Thrombotic Complications of COVID-19 Patients

The authors hypothesized that the cytokine storm described in COVID-19 patients may lead to consistent cell-based tissue factor (TF)-mediated activation of coagulation, procoagulant microvesicles (MVs) release, and massive platelet activation. COVID-19 patients have higher levels of TF+ platelets, TF+ granulocytes, and TF+ MVs than healthy subjects and coronary artery disease patients. Plasma MV-associated thrombin generation is present in prophylactic anticoagulated patients. A sustained platelet activation in terms of P-selectin expression and platelet-leukocyte aggregate formation, and altered nitric oxide/prostacyclin synthesis are also observed. COVID-19 plasma, added to the blood of healthy subjects, induces platelet activation similar to that observed invivo. This effect was blunted by pre-incubation with tocilizumab, aspirin, or a P2Y12 inhibitor.

Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates.

Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This article focuses on the underlying cause of the more common phenotype 1, investigating the effects of ASO on platelet production and platelet function. Five phosphorothioate ASO were studied: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phenotype 1), and 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord bloodderived megakaryocytes were treated with these ASO to study their effects on proplatelet production. Platelet activation (determined by surface P-selectin) and platelet-leukocyte aggregates were analyzed in ASO-treated blood from healthy human volunteers. None of the ASO inhibited proplatelet production by human megakaryocytes. All the ASO were shown to bind to the platelet receptor glycoprotein VI (KD ~0.2-1.5 μM). CpG ASO had the highest affinity to glycoprotein VI, the most potent platelet-activating effects and led to the greatest formation of platelet-leukocyte aggregates. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYKdependent formation of platelet-leukocyte aggregates. Donors with higher platelet glycoprotein VI levels had greater ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and platelet-leukocyte aggregates may explain phenotype 1 (moderate drops in platelet count). Platelet glycoprotein VI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.

Procoagulant State of Sleep Apnea Depends on Systemic Inflammation and Endothelial Damage

IntroductionGrowing evidence shows a hypercoagulable state in obstructive sleep apnea (OSA) that could be a risk factor for thromboembolic disease. ObjectivesWe aimed to elucidate mechanisms involved in the procoagulant profile observed in patients with OSA and to investigate the potential utility of global tests in its characterization. MethodsThirty-eight patients with severe OSA without previous history of thrombosis and nineteen healthy age- and sex-matched controls were included.Kinetic of clot formation was determined using rotational thromboelastometry.Haemostatic capacity of plasma and microparticles was determined by Calibrated Automated Thrombinography.Platelet surface receptors, activation markers and formation of platelet/leukocytes aggregates were analyzed by flow cytometry. ResultsThromboelastometry showed a procoagulant state in patients with OSA that did not seem to be related to a basal activation of platelets but by the increased existence of platelet/leukocyte aggregates.Patients with OSA presented many signs of endothelial damage such as increased plasma levels of E-selectin and cfDNA and enhanced thrombin generation due to the presence of microparticles rich in tissue-factor, which is related to OSA severity. ConclusionsOSA induces an enhancement in the dynamics of clot formation which appears to be caused by at least two pathological mechanisms. First, a greater formation of platelet-leukocyte aggregates; secondly, endothelial damage which provokes a greater procoagulant potential due to the increase in tissue factor-rich microparticles. Moreover, this study has identified thromboelastometry and thrombin generation assay as useful tools to evaluate the prothrombotic state in these patients.

Антикоагулянтное ведение беременных с коронавирусной инфекцией

Введение. Частота распространения новой коронавирусной инфекции COVID-19 (НКИ COVID-19) среди беременных сравнима с общепопуляционными показателями, что требует особого внимания в связи с повышенным риском материнской смертности в результате осложнений инфекционного процесса. Учитывая наличие гиперкоагуляционного статуса во время физиологически протекающей беременности, инфицирование вирусом COVID-19 может способствовать еще большему усилению прокоагулянтных свой ств крови. Патогенетически обоснованным является применение антикоагулянтной терапии во время беременности при условии верифицированной коронавирусной инфекции с целью снижения риска развития тромботических осложнений. Материалы и методы. Представлены 2 клинических случая успешного применения антикоагулянтов (низкомолекулярного гепарина) у беременных с подтвержденным инфицированием COVID-19. Результаты. Ретроспективный анализ лабораторных и клинических данных продемонстрировал эффективность парнапарина натрия в качестве средства профилактики тромботических осложнений у беременных с НКИ COVID-19. Заключение. Применение антикоагулянтной терапии у беременных с НКИ COVID-19 позволяет снизить риск развития тромботических эпизодов и улучшить репродуктивные исходы беременности. Background. The COVID-19 frequency among pregnant women is comparable to general population but it requires specific approach due to increased risk of maternal mortality as a result of infectious complications. The hypercoagulation is during physiological pregnancy, and COVID-19 can enhance further a procoagulant conditions. In such cases the anticoagulant therapy is aimed to reduce the thrombotic risk. Patients / Methods. The article presents 2 clinical cases of successful use of anticoagulants (low molecular weight heparin) in pregnant women with confirmed COVID-19. Results. A retrospective analysis of laboratory and clinical data has demonstrated the effi cacy of parnaparin sodium as a means of preventing thrombotic complications in pregnant women with COVID-19. Conclusions. Anticoagulant therapy in pregnant women with COVID-19 allows to reduce the thrombotic risk and improve the reproductive outcomes of pregnancy.

Inflammation Drives Coagulopathies in Sars-Cov-2 Patients

Background:A hypercoagulable state has been consistently reported in patients with severe Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), characterized by elevated D-dimer, prolonged PT, and mild thrombocytopenia, though the mechanism is unclear. We have previously shown that human immunodeficiency virus (HIV) infection causes depletion of the anticoagulant protein S and virus-mediated platelet activation. Based on early reports, we hypothesized that a similar process contributed to COVID-19-associated thrombosis.Aim:To probe platelet activation and coagulation factor activity in SARS-CoV-2-infected patients.Methods:Blood was collected from consenting patients with differing COVID-19 severity: outpatients (15), hospitalized inpatients (15), and healthy controls (8). Platelet-leukocyte aggregate (PLA) formation and monocyte profiling were measured by flow cytometry. Coagulation factors were assessed by enzymatic assays. PS, von Willebrand Factor (vWF), PC, cytokines, and anti-S-Protein (viral spike protein) IgG were measured by ELISAs.Results:Ninety percent of SARS-CoV-2+ out-patients and in-patients had circulating anti-S-Protein IgG, but plasma IL-6 and TNFα were only elevated in three in-patients, consistent with reports that systemic inflammation is relatively rare in this population. Immune response did not correlate with disease severity. Unlike in HIV1+/AIDS patients, total PS was not reduced in SARS-CoV-2+ patients. However, the anticoagulant pool of PS (“free PS”) was reduced in plasma samples from in-patients compared to controls (47.2%±23.3% vs. 100.8±42.6%, p=0057), while out-patients had an intermediate concentration (73.1%±28.9%). Specific loss of free PS is likely mediated by an increase in C4-binding protein (C4bp), which binds PS. In-patients also had a trend toward elevated plasma tissue factor (TF) compared to controls (79.5±121.4 fM vs. 37.8±39.7 fM, p = 0.32). Endothelial cells and monocytes can express TF under inflammatory conditions. We evaluated endothelial damage and dysfunction by measuring E-Selectin, which was unchanged in either in-patients or out-patients, and von Willebrand Factor (vWF), which was elevated in in-patients compared to controls (143±29.8 ng/mL vs. 56.2±41.9 ng/mL, p=0.0023). Plasma from in-patients also had elevated myeloperoxidase (524±187 ng/mL vs. 127±35 ng/mL, p=0.0026) and had a trend toward increased platelet-leukocyte aggregates (14.6±11.7% vs. 5.2±3.7%, p=0.24), indicating platelet and leukocyte stimulation. Unlike in the HIV1+/AIDS patients, no virus was detectable in any of the SARS-CoV-2+ patient plasmas. Consistent with a lack of direct platelet-virus interaction, plasma PF4 and platelet Akt phosphorylation were unchanged in the patient samples. We also observed a trend toward increased TF on TF+/CD64+/CD11b+ monocytes from in-patients compared to controls (MFI = 3244±2340 vs. 1741±382, p=0.18). Two inpatients were followed until they were SARS-CoV-2-negative. In both, PLAs, IL-6, vWF, and plasma TF remained elevated out to 28 days and PS remained reduced, suggesting that hemostatic dysregulation persists after SARS-CoV-2 is undetectable.Conclusions:We propose that localized inflammation in SARS-CoV-2+ patients results in a decrease in anticoagulant PS, through a shift of the free and C4bp-bound forms. At the same time, this inflammation causes stimulation of endothelial cells, which secrete procoagulant vWF, monocytes, which express TF and release it into plasma on microvesicles, and platelets, which form platelet-leukocyte aggregates. These changes may not return to baseline post-infection, suggesting that long-term monitoring of thrombotic risk may be necessary for SARS-CoV-2+ patients.DisclosuresNo relevant conflicts of interest to declare.

Platelet-T cell aggregates in lung cancer patients: Implications for thrombosis.

Platelet-leukocyte aggregates (PLAs) are associated with increased thrombosis risk. The influence of PLA formation is especially important for cancer patients, since thrombosis accounts for approximately 10% of cancer-associated deaths. Our objective was to characterize and quantify PLAs in whole blood samples from lung cancer patients compared to healthy volunteers with the intent to analyze PLA formation in the context of lung cancer-associated thrombosis. Consenting lung cancer patients (57) and healthy volunteers (56) were enrolled at the Dana Cancer Center at the University of Toledo Health Science Campus. Peripheral blood samples were analyzed by flow cytometry. Patient medical history was reviewed through electronic medical records. Most importantly, we found lung cancer patients to have higher percentages of platelet-T cell aggregates (PTCAs) than healthy volunteers among both CD4+ T lymphocyte and CD8+ T lymphocyte populations. Our findings demonstrate that characterization of PTCAs may have clinical utility in differentiating lung cancer patients from healthy volunteers and stratifying lung cancer patients by history of thrombosis.

Haem oxygenase protects against thrombocytopaenia and malaria-associated lung injury

BackgroundMalaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host’s response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65.MethodsC57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer.ResultsPlasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia.ConclusionData here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.

The Immune Response To Cardiorespiratory Exercise Testing In Heart Failure Patients With Reduced Ejection Fraction

PURPOSE: Long-term exercise training reduces systemic inflammation in heart failure patients. However, due to the impaired immune system in these patients, an acute exercise challenge may trigger pro-inflammatory responses. We compared the acute response to a standardized cardiopulmonary exercise test (CPET) in patients with heart failure with reduced ejection fraction (HFrEF) to age and gender matched controls. METHODS: Patients with HFrEF (n=13; left ventricular ejection fraction [LVEF] < 40%) and controls (n=14, LVEF > 50%) participated in a CPET. Blood samples were taken before, immediately after and 2 hours after CPET. Flow cytometry was used to assess quantitative and morphological alterations in leukocyte subpopulations as well as the formation of leukocyte-platelet aggregates. Only significant findings are reported (p < 0.05) and are given as median and inter-quartile range (IQR). RESULTS: HFrEF (mean LVEF: 36%) and controls (mean LVEF: 57%) were 59 (range: 41 to 80) and 57 (range: 50 to 65) years old, respectively. CPET increased the leukocyte (control: 1.37-fold [IQR: 1.16 to 1.49]; HFrEF: 1.24-fold [IQR: 1.20 to 1.32]), natural killer (NK) cell (controls: 2.11-fold [IQR: 1.30 to 3.13]; HFrEF: 1.67-fold [IQR: 1.56 to 1.71]) and NK-T cell (control: 1.69-fold [IQR: 1.52 to 3.60]; HFrEF: 1.62-fold [IQR: 1.60 to 2.53]) concentration in the HFrEF and control group. CD4+ cells (control: 1.15-fold [IQR: 0.84 to 1.54]; HFrEF: 1.45-fold [IQR: 1.10 to 1.98]) and CD8+ T-cells (control: 1.33-fold [IQR: 1.01 to 1.68]; HFrEF: 1.70-fold [IQR: 1.25 to 2.15]) only increased in HFrEF patients. Aggregation of thrombocytes with monocytes (control: 0.86-fold [IQR: 0.78 to 1.49]; HFrEF: 1.59-fold [IQR: 1.05 to 7.30-fold]), T-lymphocytes (control: 1.27-fold [IQR: 1.05 to 1.68]; HFrEF: 1.49-fold [IQR: 1.03 to 2.64]) and neutrophils (control: 1.08-fold [IQR: 0.87 to 1.25]; HFrEF: 2.13-fold [IQR: 1.62 to 2.19]) increased 2 hour post CPET in patients with HFrEF, but not in controls. CONCLUSIONS: CPET differentially induced specific immune responses in patients with HFrEF compared to age and gender matched controls. The prolonged immune response in these patients suggests differences in immune resolving mechanisms which deserve further research.

Platelets Endocytose Viral Particles and Are Activated via TLR (Toll-Like Receptor) Signaling.

Thrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients. Collectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.