Temporal contribution of the platelet body and balloon to thrombin generation

Agbani, Ejaife O.; Hers, Ingeborg; Poole, Alastair W.

doi:10.3324/haematol.2017.166819

Cited by 27 publications

(26 citation statements)

References 11 publications

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“…4J), and in particular sodium or potassium kinetics show later peaks or nadirs (Figs. 5 and 6), we estimated that the initial "blind window" should not be a major obstacle for the application of this method to the study of platelet heterogeneity-in particular procoagulant platelets, which are visualized minutes after strong stimuli (35,52,53). Nevertheless, as we do not have a complete continuous kinetic measurement, this initial "blind window" after platelet activation is a limitation for the measurement of rapid calcium fluctuations induced by single agonists, which could be overcome as already published (29,30).…”

Section: Discussionmentioning

confidence: 99%

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

2020

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The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow‐cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real‐time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo‐3 (Ca2+), ION NaTRIUM Green‐2 (Na+), and ION Potassium Green‐2 (K+). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR‐1 agonist), AYPGKF (PAR‐4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry

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Section: Discussionmentioning

confidence: 99%

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

2020

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“…10 Since then, several studies have investigated the procoagulant attributes of this platelet phenotype and its contribution to arterial thrombus formation. 8,10,17,20,21,33,34 It is clear that ballooned platelets not only bind annexin-V as indication of PS exposure but also provide an extended surface area for the assembly of the prothrombinase complex and contribute to the acceleration of coagulation at the wound site. 8,20,21,33,34 Ballooned phenotypes have been reported as either ballooned platelets (BPs), 8,10,34 sustained calcium-induced platelets (SCIP), 17 fibrinogen capped platelets (FIB-CAP), 20,21 or ballooned and procoagulantspread (BAPS) platelets 8 (Figure 1) and show attributes similar to other candidates of the "procoagulant platelet," such as the MPTP and COATED platelets.…”

Section: Balloon-shaped or Ballooning Phenotypesmentioning

confidence: 99%

Procoagulant platelets: generation, function, and therapeutic targeting in thrombosis

Agbani

Poole

2017

Blood

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147

169

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Current understanding of how platelets localize coagulation to wound sites has come mainly from studies of a subpopulation of activated platelets. In this review, we summarize data from the last 4 decades that have described these platelets with a range of descriptive titles and attributes. We identify striking overlaps in the reported characteristics of these platelets, which imply a single subpopulation of versatile platelets and thus suggest that their commonality requires unification of their description. We therefore propose the term procoagulant platelet as the unifying terminology. We discuss the agonist requirements and molecular drivers for the dramatic morphological transformation platelets undergo when becoming procoagulant. Finally, we provide perspectives on the biomarker potential of procoagulant platelets for thrombotic events as well as on the possible clinical benefits of inhibitors of carbonic anhydrase enzymes and the water channel Aquaporin-1 for targeting this subpopulation of platelets as antiprocoagulant antithrombotics.

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“…However, recently, it has been shown that the procoagulant activity initially located on the “cap” on the remnant platelet body moves to the balloon platelets 46. Using imaging flow cytometry, we observed that more than 90% of procoagulant PS‐exposing platelets possessed what we termed here, a platelet‐associated EV.…”

Section: Discussionmentioning

confidence: 75%

“…22 However, recently, it has been shown that the procoagulant activity initially located on the "cap" on the remnant platelet body moves to the balloon platelets. 46 Using imaging flow cytometry, we observed that more than 90% of procoagulant PS-exposing platelets possessed what we termed here, a platelet-associated EV. Forty to fifty percent of procoagulant platelets possessed only one platelet-associated EV, while the remaining platelets possessed multiple associated EVs.…”

Section: Discussionmentioning

confidence: 82%

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Reddy

Wang

Christensen

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.ObjectiveTo evaluate procoagulant PS‐exposing platelets by imaging flow cytometry.MethodsPlatelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure.ResultsA subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS‐exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non‐PS‐exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS‐exposing platelets possessing platelet‐associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.ConclusionsInnovative imaging flow cytometry allowed detailed fluorescence‐based, quantitative morphometric analysis of PS‐exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical “balloons,” almost devoid of their normal internal architecture. Almost all PS‐exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet‐associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

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Temporal contribution of the platelet body and balloon to thrombin generation

Cited by 27 publications

References 11 publications

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

Procoagulant platelets: generation, function, and therapeutic targeting in thrombosis

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

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