scholarly journals P2Y12 antagonist ticagrelor inhibits the release of procoagulant extracellular vesicles from activated platelets

2020 ◽  
Vol 26 (6) ◽  
pp. 782-789 ◽  
Author(s):  
Aleksandra Gasecka ◽  
Rienk Nieuwland ◽  
Edwin Van der Pol ◽  
Najat Hajji ◽  
Agata Ćwiek ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Activated Platelets ◽  
P2y12 Antagonist

Abstract 40: Platelet Fragmentation During the Hemostatic Response and its Prevention by a P2Y12 Antagonist

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Maurizio Tomaiuolo ◽  
Chelsea N Matzko ◽  
Izmarie Poventud-Fuentes ◽  
Leonard Nettey ◽  
Brad A Herbig ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Surface Expression ◽  
Large Mass ◽  
Major Step ◽  
Jugular Veins ◽  
Injury Site ◽  
Activated Platelets ◽  
Platelet Signaling ◽  
P2y12 Antagonist

Previous studies using intravital microscopy have shown that hemostatic plugs formed in the mouse microvasculature have a characteristic architecture in which the extent of platelet activation reflects gradients in the distribution of platelet agonists radiating outwards from the injury site. In that setting, we found minimal overlap of thrombin and ADP signaling, with thrombin primarily responsible for robust platelet activation close to the injury site and P2Y 12 -mediated ADP signaling resulting in accumulation of minimally activated platelets. Here we have taken these studies a major step forward by integrating fluorescence with scanning electron microscopy. Hemostatic plugs produced by needle injury in mouse jugular veins were imaged in situ 1 to 20 min after injury. The results show with unprecedented detail what could only be inferred previously, showing that platelet size, morphology and packing density vary remarkably depending on spatial localization within the hemostatic plug. The intraluminal and extravascular portions of the hemostatic mass presented distinct architectures. A large mass comprised almost exclusively of platelets was observed on the interior surface of the vein. Platelets closest to the injury edge had a highly activated morphology, including P-selectin surface expression, dense packing and platelet fragmentation, while those farther from the injury edge often remained discoid. In contrast, the extravascular portion of the hemostatic mass was rich in densely-packed, platelet-derived fragments intertwined with fibrin. Hemostatic plugs from mice treated with a P2Y 12 inhibitor were significantly smaller. The platelet activation gradient described above was less apparent and, notably, fragmentation of the platelets close to the injury edge was not observed with the inhibitor present. In conclusion, our findings indicate that 1) the development of a platelet activation gradient is a conserved feature of the hemostatic response across different vessels, 2) fragmentation of platelets closest to the injury site occurs very rapidly following injury, and 3) clinically relevant platelet signaling pathways play a role in regulating its formation.

scholarly journals Crizanlizumab Therapy Is Associated with Lower Levels of Circulating Extracellular Vesicles in Sickle Cell Disease Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 955-955
Author(s):  
Cristiane Maria de Souza ◽  
Carolina Lanaro ◽  
Irene Pereira dos Santos ◽  
Oladele Olatunya ◽  
Sara T Olalla Saad ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Board Of Directors ◽  
Extracellular Vesicles ◽  
Sickle Cell ◽  
Treated Group ◽  
Clinical Severity ◽  
Cell Disease ◽  
Advisory Committees ◽  
Activated Platelets

Abstract Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity. Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD. This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab. We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019). We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively). Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation. Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug. Disclosures Benites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Extracellular vesicles from activated platelets: a semiquantitative cryo-electron microscopy and immuno-gold labeling study

Platelets ◽  
2017 ◽  
Vol 28 (3) ◽  
pp. 263-271 ◽  
Author(s):  
Alain R. Brisson ◽  
Sisareuth Tan ◽  
Romain Linares ◽  
Céline Gounou ◽  
Nicolas Arraud
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Cryo Electron Microscopy ◽  
Activated Platelets

scholarly journals Epigallocatechin gallate inhibits release of extracellular vesicles from platelets without inhibiting phosphatidylserine exposure

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarah L. Millington-Burgess ◽  
Matthew T. Harper
Keyword(s):  
Myocardial Infarction ◽  
Cause Of Death ◽  
Extracellular Vesicles ◽  
Therapeutic Target ◽  
Arterial Thrombosis ◽  
Epigallocatechin Gallate ◽  
Annexin V ◽  
Phospholipid Scramblase ◽  
Activated Platelets ◽  
Phosphatidylserine Exposure

AbstractArterial thrombosis triggers myocardial infarction and is a leading cause of death worldwide. Procoagulant platelets, a subpopulation of activated platelets that expose phosphatidylserine (PS), promote coagulation and occlusive thrombosis. Procoagulant platelets may therefore be a therapeutic target. PS exposure in procoagulant platelets requires TMEM16F, a phospholipid scramblase. Epigallocatechin gallate (EGCG) has been reported to inhibit TMEM16F but this has been challenged. We investigated whether EGCG inhibits PS exposure in procoagulant platelets. PS exposure is often measured using fluorophore-conjugated annexin V. EGCG quenched annexin V-FITC fluorescence, which gives the appearance of inhibition of PS exposure. However, EGCG did not quench annexin V-APC fluorescence. Using this fluorophore, we show that EGCG does not inhibit annexin V binding to procoagulant platelets. We confirmed this by using NBD-labelled PS to monitor PS scrambling. EGCG did not quench NBD fluorescence and did not inhibit PS scrambling. Procoagulant platelets also release PS-exposing extracellular vesicles (EVs) that further propagate coagulation. Surprisingly, EGCG inhibited EV release. This inhibition required the gallate group of EGCG. In conclusion, EGCG does not inhibit PS exposure in procoagulant platelets but does inhibit the EV release. Future investigation of this inhibition may help us further understand how EVs are released by procoagulant platelets.

scholarly journals Plasma Concentrations of Extracellular Vesicles Are Decreased in Patients with Post-Infarct Cardiac Remodelling

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 97
Author(s):  
Aleksandra Gąsecka ◽  
Kinga Pluta ◽  
Katarzyna Solarska ◽  
Bartłomiej Rydz ◽  
Ceren Eyileten ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Blood Cells ◽  
Plasma Concentrations ◽  
Left Ventricular ◽  
Ventricular Remodelling ◽  
Left Ventricular Remodelling ◽  
End Diastolic Volume ◽  
Activated Platelets ◽  
Baseline Concentrations

Background, the mechanisms underlying left ventricular remodelling (LVR) after acute myocardial infarction (AMI) remain obscure. In the course of AMI, blood cells and endothelial cells release extracellular vesicles (EVs). We hypothesized that changes in EV concentrations after AMI may underlie LVR. Methods, plasma concentrations of EVs from endothelial cells (CD146+), erythrocytes (CD235a+), leukocytes (CD45+), platelets (CD61+), activated platelets (P-selectin+), and EVs exposing phosphatidylserine after AMI were determined by flow cytometry in 55 patients with the first AMI. LVR was defined as an increase in left ventricular end-diastolic volume by 20% at 6 months after AMI, compared to baseline. Results, baseline concentrations of EVs from endothelial cells, erythrocytes and platelets were lower in patients who developed LVR (p ≤ 0.02 for all). Concentrations of EVs from endothelial cells and erythrocytes were independent LVR predictors (OR 8.2, CI 1.3–54.2 and OR 17.8, CI 2.3–138.6, respectively) in multivariate analysis. Combining the three EV subtypes allowed to predict LVR with 83% sensitivity and 87% specificity. Conclusions, decreased plasma concentrations of EVs from endothelial cells, erythrocytes and platelets predict LVR after AMI. Since EV release EVs contributes to cellular homeostasis by waste removal, decreased concentrations of EVs may indicate dysfunctional cardiac homeostasis after AMI, thus promoting LVR.

scholarly journals Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

2018 ◽  
Vol 16 (6) ◽  
pp. 1153-1163 ◽  
Author(s):  
Y. Yu ◽  
E. Gool ◽  
R.J. Berckmans ◽  
F.A.W. Coumans ◽  
A.D. Barendrecht ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Extracellular Vesicles ◽  
Human Saliva ◽  
Activated Platelets

257Ticagrelor decreases concentrations of prothrombotic extracellular vesicles compared to clopidogrel

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A Gasecka ◽  
R Nieuwland ◽  
M Budnik ◽  
F Dignat-George ◽  
C Eyileten ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Platelet Reactivity ◽  
Venous Blood ◽  
Plasma Concentrations ◽  
Underlying Mechanism ◽  
Distribution Fitting ◽  
Activated Platelets ◽  
Impedance Aggregometry

Abstract Introduction The platelet P2Y12 antagonist ticagrelor reduces all-cause and cardiovascular mortality in patients after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets release prothrombotic extracellular vesicles (EVs) exposing P-selectin, fibrinogen and phosphatidylserine (PS), we hypothesized that ticagrelor inhibits the release of EVs compared to clopidogrel. Purpose To compare the effects of ticagrelor and clopidogrel on the concentrations of platelet EVs in relation to platelet reactivity, and on the concentrations of EVs from other P2Y12-exposing cells (leukocytes, endothelial cells). Methods After the percutaneous coronary intervention, patients with first AMI (n=60, age 64.5±10.8 years, 68% male) were randomized in 1:1 ratio to antiplatelet therapy with ticagrelor or clopidogrel. Venous blood was collected from fasting patients at randomisation, 48 hours after randomisation, and 6 months following the index hospitalization with informed consent. Flow cytometry (Apogee A60 Micro) was used to determine plasma concentrations of EVs labelled with antibodies for activated platelets (CD61, CD62p; PEVs), fibrinogen, phosphatidylserine (PS), leukocytes (CD45; LEVs) and endothelial cells (CD146; EEVs). To maximize the reliability, the analysis of the 1,224 flow cytometry data files was performed with software enabling automatic flow rate stabilization, diameter and refractive index determination, size distribution fitting, fluorescent gate determination, and statistics reporting (MATLAB R2018a). To differentiate between EVs and small platelets, only particles <700 nm were included. Platelet reactivity was assessed by impedance aggregometry (Multiplate Analyzer) using the ASPI test (arachidonic acid, 0.5 mM) and the ADP test (adenosine diphosphate, 6.5 μM). Results Concentrations of PEVs exposing P-selectin, fibrinogen and PS were lower after 6 months of therapy with ticagrelor compared to clopidogrel, and decreased over time (p=0.034; p=0.021; p=0.018, respectively). Concentrations of LEVs were lower both after 72 hours and after 6 months of therapy with ticagrelor compared to clopidogrel (p=0.018; p<0.001, respectively). There was a trend towards higher concentrations of EEVs both after 72 hours and after 6 months of therapy with ticagrelor, compared to clopidogrel. There were no correlations between the concentrations of PEVs and platelet reactivity. Conclusions Ticagrelor decreases concentrations of prothrombotic EVs, compared to clopidogrel, suggesting that the clinical benefits of ticagrelor may be at least partly explained by inhibition of EV release from activated platelets. Further research is warranted to link this effect with clinical outcomes. Acknowledgement/Funding Medical University of Warsaw (1WR/PM2/18)

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