Platelets Amplify Inflammation in Arthritis via Collagen-Dependent Microparticle Production

Boilard, Éric; Nigrović, Peter A.; Larabee, Katherine; Watts, Gerald F.; Coblyn, Jonathan S.; Weinblatt, Michael E.; Massarotti, Elena; Remold‐O′Donnell, Eileen; Farndale, Richard W.; Ware, Jerry; Lee, David M.

doi:10.1126/science.1181928

Cited by 964 publications

(1,029 citation statements)

References 28 publications

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“…Gerrard et al39 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 fibroblast‐like synoviocytes observed by Boilard et al40 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: 74%

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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Section: Discussionsupporting

confidence: 74%

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 flow cytometry instrument settings and MP gating were adopted from previous works [2,12,16]. We used Megamix beads (BioCytex, Marseille, France) to determine the gate for MPs, as described previously [2,18].…”

Section: Flow Cytometric Analysis Of Pfp Samplesmentioning

confidence: 99%

“…However, there are remaining debated questions including the use of anticoagulants for MP analysis. Although most studies used sodium-citrate tubes, some groups applied ACD (acid-citrate-dextrose) tubes in their studies [14][15][16]. A recent report by Jayachandran et al suggested that neither citrate nor ACD tubes were suitable for MP measurement as these anticoagulants may eliminate MPs from plasma [17].…”

Section: Introductionmentioning

confidence: 99%

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube

György

Pálóczi

Kovacs

et al. 2014

Thrombosis Research

108

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Introduction: Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples. Materials and Methods: We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes. Results: Agitation and storage of blood samples at 37°C for 1 hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes. Conclusions: We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses.© 2013 Elsevier Ltd. All rights reserved. IntroductionExtracellular vesicles (EVs) are membrane surrounded structures of various sizes (30-5000 nm) that have received significant attention recently [1]. EVs may be classified on the basis of their biogenesis, diameter and membrane markers. The two best characterized types of EVs include exosomes of endosomal origin and plasma membrane-derived microparticles (MPs) (recently often referred to also as microvesicles or ectosomes in the literature) [1]. EVs are present in all biological fluids including blood plasma, synovial fluid, cerebrospinal fluid, urine, tears and breast milk [1]. MPs are in between 100 and 1000 nm in diameter, and they are also detectable by flow cytometry [2,3]. Thus, MP profiles are easily analyzed in the routine clinical laboratory practice, and represent novel biomarkers of various diseases. In circulation, most MPs are derived from platelets, red blood cells, endothelial cells and leukocytes. Because of their abundance, platelet-derived MPs (PMPs) received the highest attention during the past few years. Elevated PMP counts are characteristic for nearly all autoimmune disorders [4,5], and also for several cardiovascular and metabolic diseases [1]. EVs not only contain proteins but also RNA molecules [1]. Extracellular RNA (exRNA) in blood plasma is encapsulated in EVs, or bound to either proteins or HDL molecules [6]. exRNAs, particularly miRNAs, are specific and sensitive biomarkers...

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“…For example, elevated levels of CD3+ and CD8+ T cell-derived and platelet-derived EVs are detected in RA SF [2,3]. Furthermore, SF EVs are described as having potent pro-inflammatory properties that might contribute to the perpetuation of joint inflammation in RA [1,3–5]. Understanding the composition and function of SF EVs is therefore of great interest and may provide insight into pathogenesis of joint diseases.…”

Section: Introductionmentioning

confidence: 99%

Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

Foers

Chatfield

Dagley

et al. 2018

J of Extracellular Vesicle

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As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease.

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Platelets Amplify Inflammation in Arthritis via Collagen-Dependent Microparticle Production

Cited by 964 publications

References 28 publications

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube

Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

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