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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140207	1/1	Homo sapiens	NAY	(d)(U)C Filtration IAF	Pook M	2014	33%
Study summary Full title Platelets store laminins 411/421 and 511/521 in compartments distinct from alpha- or dense granules and secrete these proteins via microvesicles All authors Pook M, Tamming L, Padari K, Tiido T, Maimets T, Patarroyo M, Juronen E, Jaks V, Ingerpuu S Journal J Thromb Haemost Abstract BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive (show more...)BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. OBJECTIVES: Our aim was to compare the subcellular localization of laminins 411/421 and 511/521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. METHODS: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. RESULTS AND CONCLUSIONS: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express ?-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF Adj. k-factor 255.8 (pelleting) Protein markers EV: CD63/ CD41/ CD62P/ CD61 non-EV: Proteomics no Show all info Study aim Other/Laminin secretion Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Filtration steps 0.2µm > x > 0.1µm Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD41/ CD61/ CD62P ELISA Antibody details provided? No Detected EV-associated proteins CD41/ CD61/ CD62P

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EV-TRACK ID	EV140207
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration IAF
Exp. nr.	1
EV-METRIC %	33