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Journal of Thrombosis and Haemostasis :... Aug 2023Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of...
BACKGROUND
Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation.
OBJECTIVES
To determine the importance of GPVI clusters for thrombus formation in whole blood under shear.
METHODS
We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials.
RESULTS
Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation.
CONCLUSION
Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.
Topics: Humans; Blood Platelets; Phosphatidylserines; Platelet Membrane Glycoproteins; Platelet Activation; Collagen; Thrombosis; Platelet Aggregation
PubMed: 37150294
DOI: 10.1016/j.jtha.2023.04.028 -
Cytometry. Part a : the Journal of the... Apr 2022Platelets are small anucleate blood cells that contribute to hemostasis, immunity, and inflammation. Circulating platelets are heterogeneous in size, age, receptor...
Platelets are small anucleate blood cells that contribute to hemostasis, immunity, and inflammation. Circulating platelets are heterogeneous in size, age, receptor expression, and reactivity. They inherit many features from megakaryocytes and are further modified on exposure to bioactive substances in the bloodstream. Among these substances, prothrombotic agonists, vasodilators, and bloodborne pathogens modulate platelet phenotypes via distinct signaling cascades. The ability of platelets to respond to (patho)physiologic signals is incompletely understood but likely depends on their repertoire of surface receptors, which may partition them into discrete subsets with specialized functions and divergent abilities. The single-cell resolution of flow and mass cytometry is ideal for immunophenotyping and allows the identification of platelet subsets in remarkable detail. In this report, we describe the surface markers and gating strategies needed for the comprehensive characterization of platelets.
Topics: Biomarkers; Blood Platelets; Flow Cytometry; Hemostasis; Humans; Immunophenotyping; Megakaryocytes; Platelet Activation
PubMed: 34997669
DOI: 10.1002/cyto.a.24531 -
Frontiers in Bioscience (Landmark... Jun 2015Diverse Streptococcus species including Streptococcus Pneumoniae, Sanguis, Gordonii, Mitis and Mutans cause life-threatening conditions including pneumonia, bacteremia... (Review)
Review
Diverse Streptococcus species including Streptococcus Pneumoniae, Sanguis, Gordonii, Mitis and Mutans cause life-threatening conditions including pneumonia, bacteremia and meningitis. These diseases bear a high morbidity and mortality and for this reason, understanding the key events in the pathogenesis of these infections have a great significance in their prevention and/or treatment. Here, we describe as how the activation of the platelets and their affinity to bind to bacterial proteins act as early key events in the pathogenesis of Streptococcal infections.
Topics: Bacterial Proteins; Blood Platelets; Humans; Platelet Activation; Platelet Aggregation; Streptococcal Infections; Streptococcus
PubMed: 25961531
DOI: 10.2741/4345 -
Current Protocols Feb 2023Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these...
Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.
Topics: Platelet Aggregation; Platelet Function Tests; Calcium; Blood Platelets; Platelet Activation
PubMed: 36786557
DOI: 10.1002/cpz1.668 -
BioMed Research International 2017The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant...
The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.
Topics: Blood Platelets; Enzyme Inhibitors; Female; Fibrinolysis; Flow Cytometry; Humans; Male; Marine Toxins; Oxazoles; Platelet Activation
PubMed: 28680886
DOI: 10.1155/2017/9795271 -
British Journal of Haematology Oct 2021
Topics: Adolescent; Adult; COVID-19; Child; Family Characteristics; Humans; Platelet Activation; SARS-CoV-2; Young Adult
PubMed: 34101171
DOI: 10.1111/bjh.17629 -
American Journal of Physiology. Cell... Dec 2013
Tubulin acetylation a valuable accessory of the platelet cytoskeleton. Focus on "Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation".
Topics: Blood Platelets; Cytoskeleton; Histone Deacetylase 6; Histone Deacetylases; Humans; Platelet Activation; Signal Transduction; Tubulin
PubMed: 24108865
DOI: 10.1152/ajpcell.00309.2013 -
The Journal of Thoracic and... Aug 2004Platelet function plays a major role in the understanding of thromboembolic events in prolonged mechanical support. We studied the platelet activation, platelet...
BACKGROUND
Platelet function plays a major role in the understanding of thromboembolic events in prolonged mechanical support. We studied the platelet activation, platelet aggregation profile, and efficacy of aspirin in patients in whom an external ventricular assist device had been implanted.
PATIENTS AND METHODS
Fifteen patients were studied prospectively up to 6 weeks after implantation of the same type of ventricular assist device. Platelet function was studied weekly before daily aspirin administration. Aspirin efficacy was tested ex vivo by measuring platelet aggregation triggered by arachidonic acid. Flow cytometry was used to quantify the spontaneous and induced (adenosine diphosphate stimulation) expression of glycoproteins alphaIIbbeta3, Ibalpha, and CD62P on platelet membranes. The plasma levels of von Willebrand factor (von Willebrand factor activity and von Willebrand factor antigen) and fibrinogen were also determined.
RESULTS
Six of the 15 patients (26%) maintained an arachidonic acid-induced platelet aggregation despite daily aspirin treatment (250 mg). CD62P values remained increased during a 5-week postoperative period. Spontaneous levels of glycoproteins alphaIIbbeta3 and Ibalpha on platelet membranes remained within a normal range with a preserved reactivity. The plasma levels of fibrinogen and von Willebrand factor remained increased during the entire study period.
CONCLUSION
In patients with an implanted external ventricular assist device, the platelet activation profile displays a persistent activation with a preserved reactivity associated with a persistent high inflammatory state and endothelial activation.
Topics: Adolescent; Aspirin; Female; Heart-Assist Devices; Humans; Male; Middle Aged; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Prospective Studies; Time Factors
PubMed: 15282455
DOI: 10.1016/j.jtcvs.2003.11.059 -
Journal of Thrombosis and Haemostasis :... Jun 2010Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in...
BACKGROUND AND OBJECTIVE
Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms.
METHODS AND RESULTS
SAA dose-dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP-enhanced platelet P-selectin expression and fibrinogen binding, which consequently hampered ADP-induced platelet-leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside-in signaling. Under an arterial shear rate of 1000 s(-1), SAA decreased platelet adhesion on collagen surfaces by approximately 40%. Western blot analysis showed that SAA, like the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and TGX-221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild-type mice (35 + or - 2 min without SAA and 56 + or - 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr(tm1Her) mutant mice (21 + or - 2 min without SAA and 45 + or - 4 min with SAA; P < 0.01).
CONCLUSIONS
SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.
Topics: Adenosine Diphosphate; Adult; Animals; Arteries; Caffeic Acids; Collagen; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Lactates; Male; Mice; Phosphoinositide-3 Kinase Inhibitors; Platelet Activation; Platelet Aggregation; Signal Transduction; Thrombin; Thrombosis; Thromboxanes
PubMed: 20345719
DOI: 10.1111/j.1538-7836.2010.03859.x -
International Journal of Molecular... Jan 2022Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet... (Review)
Review
Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.
Topics: Blood Platelets; Extracellular Traps; Humans; Neoplasms; Neutrophils; P-Selectin; Platelet Activation; Thromboplastin; Thrombosis; Venous Thrombosis
PubMed: 35163180
DOI: 10.3390/ijms23031257