Intracellular origin and ultrastructure of platelet‐derived microparticles

Пономарева, А. А.; Невзорова, Т. А.; Mordakhanova, Elmira R.; Andrianova, Izabella A.; Rauova, Lubica; Litvinov, Rustem I.; Weisel, John W.

doi:10.1111/jth.13745

Cited by 79 publications

(77 citation statements)

References 43 publications

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“…Recent studies have challenged this view, demonstrating diversity among platelet-derived EVs. 41,47 In keeping with these reports, we observed heterogeneity within the released EV…”

Section: Discussionsupporting

confidence: 88%

“…Since EVs are formed from procoagulant platelets following activation, it is anticipated that EVs should retain the properties of the activated platelet membrane. Recent studies have challenged this view, demonstrating diversity among platelet‐derived EVs 41, 47. In keeping with these reports, we observed heterogeneity within the released EV population.…”

Section: Discussionsupporting

confidence: 88%

“…Gerrard et al 39 observed similarly altered ultrastructure of A23187-stimulated platelets in suspension by electron microscopy, and reported that platelets exposed to high A23187 concentrations lost their alpha and dense granules, and often had the cytoplasmic gel concentrated in a dense mass, with swollen membranes that were pulled away from the cell contents. We observed extracellular vesicles (EVs) in T+C-or A23187-stimulated conditions; in some cases, these EVs appeared to be associated with or blebbing from the platelet surface, similar to the EVs budding from platelets incubated in the presence of fibroblastlike synoviocytes observed by Boilard et al 40 and from platelets activated with thrombin observed by Ponomareva et al, 41 suggesting that the free EVs had been released from the balloon platelets.…”

Section: Discussionsupporting

confidence: 73%

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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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“…Recent studies have challenged this view, demonstrating diversity among platelet-derived EVs. 41,47 In keeping with these reports, we observed heterogeneity within the released EV…”

Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 73%

See 1 more Smart Citation

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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“…The third potential explanation involves platelet disintegration via microvesiculation [6,7], but whether this mechanism is responsible for the low platelet count is unclear. Our recent studies suggest that platelets activated by thrombin undergo fatal dysfunction and cytoplasmic fragmentation clearly distinct from what happens when platelets are activated by adenosine diphosphate (ADP) or collagen [8,9]. Based on these and other findings, we hypothesized that platelets activated in HIT through FcγRIIA undergo activation followed by death, leading to thrombocytopenia with the removal of dead platelets perhaps by neutrophils and macrophages.In this work, we studied the direct effects of PF4-containing HIT-pathogenic immune complexes on isolated human platelets.…”

mentioning

confidence: 94%

Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

Mordakhanova

Невзорова

Synbulatova

et al. 2020

IJMS

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Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-X L , and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.Despite a lot of information being available on triggering factors and pathogenic mechanisms involved in HIT, the causes of thrombocytopenia in HIT are not well delineated. One possibility is that a low platelet count is triggered by the clearance of Ab-coated platelets in the spleen and potentially the liver. The second possibility is that platelets are consumed within thrombi; however, it is unclear if sufficient platelets are incorporated into thrombi to cause thrombocytopenia. The third potential explanation involves platelet disintegration via microvesiculation [6,7], but whether this mechanism is responsible for the low platelet count is unclear. Our recent studies suggest that platelets activated by thrombin undergo fatal dysfunction and cytoplasmic fragmentation clearly distinct from what happens when platelets are activated by adenosine diphosphate (ADP) or collagen [8,9]. Based on these and other findings, we hypothesized that platelets activated in HIT through FcγRIIA undergo activation followed by death, leading to thrombocytopenia with the removal of dead platelets perhaps by neutrophils and macrophages.In this work, we studied the direct effects of PF4-containing HIT-pathogenic immune complexes on isolated human platelets. As a tool, we used two isotype-matched murine anti-human PF4/heparin monoclonal Abs that mimic their human counterparts in vitro [10], and in vivo the pathogenic monoclonal anti-PF4/heparin antibody (KKO) causes HIT in an animal model, while the non-pathogenic monoclonal anti-PF4/heparin antibody (RTO) does not [11]. Importantly, ELISA-positive plasma samples from patients suspected of having HIT contain Abs that show hep...

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“…Within this chapter, we will review the methods and applications of multiple modalities of platelet imaging including light transmission, super-resolution, electron, and intravital microscopy. Advances in our ability to image platelets have enabled a more complete understanding of platelet biology in health and disease, and these techniques remain powerful clinical and research tools [15][16][17][18][19]. We will also detail the challenges of these imaging methods specific to platelets, and future barriers that should to be addressed.…”

Section: Introductionmentioning

confidence: 99%

Platelet Imaging

Matthay¹,

Kornblith²

2020

Platelets

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The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlativefluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation in vivo, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for analysis by electron microscopy.

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Intracellular origin and ultrastructure of platelet‐derived microparticles

Cited by 79 publications

References 43 publications

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

Platelet Imaging

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