scholarly journals Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia

2020 ◽  
Vol 21 (24) ◽  
pp. 9515
Author(s):  
Alessandro Aliotta ◽  
Manuel Krüsi ◽  
Debora Bertaggia Calderara ◽  
Maxime G. Zermatten ◽  
Francisco J. Gomez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cytosolic Calcium ◽  
Procoagulant Activity ◽  
Ion Fluxes ◽  
Glanzmann Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Healthy Donors ◽  
Thrombin Activation

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

scholarly journals Platelet function and bleeding at different phases of childhood immune thrombocytopenia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anastasia A. Ignatova ◽  
Elena V. Suntsova ◽  
Alexey V. Pshonkin ◽  
Alexey A. Martyanov ◽  
Evgeniya A. Ponomarenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Platelet Function ◽  
Whole Blood ◽  
Immune Thrombocytopenia ◽  
Disease Duration ◽  
Cytosolic Calcium ◽  
Healthy Children ◽  
Chronic Itp ◽  
Healthy Donors

AbstractImmune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.

scholarly journals Tumor microenvironment and clonal monocytes from chronic myelomonocytic leukemia induce a procoagulant climate

2019 ◽  
Vol 3 (12) ◽  
pp. 1868-1880
Author(s):  
Johanna Zannoni ◽  
Natacha Mauz ◽  
Landry Seyve ◽  
Mathieu Meunier ◽  
Karin Pernet-Gallay ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Myeloproliferative Neoplasms ◽  
Tumor Development ◽  
Chronic Myelomonocytic Leukemia ◽  
Procoagulant Activity ◽  
Hematopoietic Stem ◽  
Thrombin Generation Assay ◽  
Healthy Donors ◽  
Myelomonocytic Leukemia

Abstract Chronic myelomonocytic leukemia (CMML) is a myeloid hematological malignancy with overlapping features of myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (MPNs). The knowledge of the role of the tumor microenvironment (TME), particularly mesenchymal stromal cells (MSCs), in MDS pathogenesis is increasing. Generally, cancer is associated with a procoagulant state participating in tumor development. Monocytes release procoagulant, tissue factor (TF)–bearing microparticles. We hypothesized that MSCs and clonal monocytes release procoagulant extracellular vesicles (EVs) within the CMML TME, inducing a procoagulant state that could modify hematopoietic stem cell (HSC) homeostasis. We isolated and cultured MSCs and monocytes from CMML patients and MSCs from healthy donors (HDs). Their medium EVs and small EVs (sEVs) were collected after iterative ultracentrifugations and characterized by nanoparticle tracking analysis. Their impact on hemostasis was studied with a thrombin generation assay and fibrinography. CMML or HD HSCs were exposed to sEVs from either CMML or HD MSCs. CMML MSC sEVs increased HD HSC procoagulant activity, suggesting a transfer of TF from the CMML TME to HD HSCs. The presence of TF on sEVs was shown by electron microscopy and western blot. Moreover, CMML monocyte EVs conferred a procoagulant activity to HD MSCs, which was reversed by an anti-TF antibody, suggesting the presence of TF on the EVs. Our findings revealed a procoagulant “climate” within the CMML environment related to TF-bearing sEVs secreted by CMML MSCs and monocytes.

scholarly journals BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy: an ephemeral existence?

2019 ◽  
Author(s):  
Rodrigo Jiménez-Saiz ◽  
Yosef Ellenbogen ◽  
Kelly Bruton ◽  
Paul Spill ◽  
Doron D. Sommer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Allergic Disease ◽  
Peripheral Circulation ◽  
Memory B Cells ◽  
Healthy Donors ◽  
Bona Fide ◽  
The Subject ◽  
Flow Cytometry Protocol

AbstractImmunoglobulin (Ig) E is the critical effector molecule in allergic reactions. Consequently, research efforts to understand the biology of IgE-expressing cells is of paramount importance. In particular, the role of IgE+ memory B cells (MBCs) in the perpetuation of allergic reactivity has been the subject of intense research. Studies in mice have convincingly established that IgE+ B cells are rare and transient and, therefore, an unlikely candidate to maintain allergic disease. In contrast, IgE+ MBCs have been detected by flow cytometry in the sputum and peripheral blood of humans and have been proposed as a clinical marker of allergic disease. We established a method to genetically validate, at the single-cell level, the putative IgE+ MBCs identified by flow cytometry from humans. We, then used this information to develop an enhanced flow cytometry protocol that more accurately identifies bona fide IgE+ MBCs. We found that IgE+ MBCs were detected in some patients with atopic dermatitis, but at a frequency that was ~100 times lower than previously reported. We also found that IgE+ MBCs were undetectable in PBMCs from peanut allergic patients. These findings provide tools to identify bona fide IgE+ MBCs, demonstrate their extreme rarity in circulation and are consistent with the lack of a central role for IgE+ MBCs in the maintenance of allergic sensitivity.One Sentence SummaryThe frequency of IgE+ MBCs in the peripheral circulation of humans is orders of magnitude lower than previously reported and comparable between allergic and healthy donors, which cautions about the clinical utility of their assessment.

Characterization of angiotensin IV-degrading enzymes and receptors on rat mesangial cells

1998 ◽  
Vol 275 (4) ◽  
pp. F535-F542 ◽  
Author(s):  
Dominique Chansel ◽  
Stanislas Czekalski ◽  
Sophie Vandermeersch ◽  
Emmanuel Ruffet ◽  
Marie-Claude Fournié-Zaluski ◽  
...  
Keyword(s):  
Mesangial Cells ◽  
Biological Effects ◽  
Neutral Endopeptidase ◽  
Cytosolic Calcium ◽  
Ang Ii ◽  
Degrading Enzymes ◽  
Specific Receptors ◽  
Ang Iv

Because mesangial cells (MC) are a target and a degradation site for angiotensin II (ANG II), we characterized the degrading enzymes and receptors of ANG IV, a metabolite of ANG II, on these cells. ANG IV was metabolized into its NH2-terminal deleted peptides, ANG II-(4–8), ANG II-(5–8), and ANG II-(6–8) by rat MC. Total protection of ANG IV was obtained only when PC-18, a specific aminopeptidase N (APN) inhibitor, and JFH-27A, a mixed inhibitor of dipeptidylaminopeptidase (DAP) and neutral endopeptidase (NEP), were simultaneously added. In contrast, thiorphan, an NEP inhibitor, was inactive. These results demonstrate the exclusive role of APN and DAP in ANG IV degradation.125I-labeled ANG IV binding was studied in the presence of PC-18 and JFH-27A to suppress ligand degradation. Under these conditions, ANG IV-specific receptors could be demonstrated with a K D of 1.8 nM and a density of 55 fmol/mg. In contrast with MC, no evidence for ANG IV receptors could be obtained in freshly isolated glomeruli. ANG IV stimulated cytosolic calcium concentration in MC, whereas its NH2-terminal deleted metabolites were inactive. Therefore, ANG IV must be protected from degradation by APN and DAP in studies on the nonimmediate biological effects of this peptide.

scholarly journals Enhancement of Antigen-specific functional responses by neutrophils from allergic patients.

1996 ◽  
Vol 183 (6) ◽  
pp. 2571-2579 ◽  
Author(s):  
J Monteseirín ◽  
M J Camacho ◽  
R Montaño ◽  
E Llamas ◽  
M Conde ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Respiratory Burst ◽  
Functional Role ◽  
Specific Binding ◽  
Flow Cytometry Analysis ◽  
Functional Responses ◽  
E Proteins ◽  
Healthy Donors

It has been demonstrated that neutrophils from healthy donors or from patients with inflammatory disorders can bind immunoglobulin (Ig) E proteins through binding to Mac-2/epsilon bp. Functional responses to allergens were assessed by measuring the respiratory burst and intracellular Ca2+ levels, and binding of allergens to neutrophils was assessed by flow cytometry analysis and fluorescence microscopy. In this article, we demonstrate that neutrophils sensitized to specific allergens (from allergic patients), but not from healthy donors, are sensitive to allergens of the same type as those that produce clinical allergic symptoms. The activation of neutrophils was analyzed by the induction of a respiratory burst that was detected with luminol-dependent chemiluminescence. Intracellular Ca2+ levels increased parallel to those of the inducing allergens. In addition, the specific binding of allergens on the cell surface was revealed by flow cytometry and allergen-FITC-labeled staining analyses. The present data suggest a restricted recognition of allergen by sensitive neutrophils, probably associated with the specific binding of the allergen to its corresponding IgE molecule, which is bound to the Mac-2/epsilon bp structure. These findings demonstrate a functional role of allergen-associated neutrophils during the allergic state.

scholarly journals PRODUCTION AND CHARACTERIZATION OF ANTIBODIES TO TISSUE PLASMINOGEN ACTIVATOR: APPLICATION FOR THE PLATELET FLOW CYTOMETRY ASSAY

2020 ◽  
Vol 13 (5) ◽  
pp. 62-72
Author(s):  
E. I. Yusova ◽  
Keyword(s):  
Flow Cytometry ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Immune Serum ◽  
Flow Cytometry Assay ◽  
Tissue Plasminogen

Tissue plasminogen activator (tPA) is one of the key protein of plasminogen/plasmin system that converts plasminogen in the active proteinase plasmin. Platelets are able to bind both tPA and plasminogen on their surface, thus providing stimulatory effects on activation of zymogen. The present study was aimed to produce polyclonal antibodies against tPA and characterize their immunochemical capacities for further application in flow cytometry assay to study interaction between tPA and platelets. The experimental methods involved immunization of rabbit with tPA, collection of immune serum, synthesis of tPA-containing immunoaffine sorbent, ELISA, and flow cytometry. Polyclonal monospecific antibodies against tPA with high affinity to the antigen (Кd = 4.05・10–9 М) were obtained. Flow cytometry assay based on the use of the produced antibodies showed the presence of binding sites for tPA on the plasma membrane of inactive platelets. Moreover, agonist-stimulated platelets were revealed to expose more binding sites than their resting counterparts. Certain subpopulations of platelets that differ in the ability to bind tPA on their surface were also identified. Obtained data are of significant importance for further investigation of mechanisms underlying the role of platelets to regulate fibrinolytic rates.

High-dose epinephrine enhances platelet aggregation at the expense of procoagulant activity

2021 ◽  
Author(s):  
Alessandro Aliotta ◽  
Debora Bertaggia Calderara ◽  
Maxime G Zermatten ◽  
Lorenzo Alberio
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Light Transmission ◽  
Shape Change ◽  
Annexin V ◽  
Cytosolic Calcium ◽  
Calcium Mobilization ◽  
Procoagulant Activity ◽  
High Dose

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein [GP] IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of epinephrine in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PARs) agonists, epinephrine (EPI), and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-thrombin (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V positivity and increased binding of PAC-1 with the triple activation (CVX+THR+EPI) com-pared with CVX+THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1000 µM) compared with CVX+THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity and en-hancing platelet aggregation.

Sodium–Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation

2020 ◽  
Author(s):  
Alessandro Aliotta ◽  
Debora Bertaggia Calderara ◽  
Maxime G. Zermatten ◽  
Lorenzo Alberio
Keyword(s):  
Platelet Activation ◽  
Intracellular Signaling ◽  
Critical Role ◽  
Ion Fluxes ◽  
Sodium Calcium Exchanger ◽  
Potassium Efflux ◽  
Sodium Calcium ◽  
Activated Platelets ◽  
Thrombin Activation

AbstractProcoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium–calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 583-586 ◽  
Author(s):  
Edith van de Vijver ◽  
Iris M. De Cuyper ◽  
Anja J. Gerrits ◽  
Arthur J. Verhoeven ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Diagnostic Methods ◽  
Severe Bleeding ◽  
Bleeding Tendency ◽  
Aggregation Assay ◽  
Glanzmann Thrombasthenia ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Receptor

Abstract Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbβ3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2β1) instead of integrin αIIbβ3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

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