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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV21008	1/2	Homo sapiens	Blood plasma	DG (d)(U)C	Annalisa Radeghieri	2022	89%
Study summary Full title Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles All authors Annalisa Radeghieri, Silvia Alacqua, Andrea Zendrini, Vanessa Previcini, Francesca Todaro, Giuliana Martini, Doris Ricotta, Paolo Bergese Journal Journal of Extracellular Biology Abstract Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clott (show more...)Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV-protein corona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms. (hide) EV-METRIC 89% (99th 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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle 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 DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Adam 10/ Alix/ Antithrombin 3 non-EV: Argonaute2/ Apo A1/ GM130 Proteomics no EV density (g/ml) 1.11-1.22 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 8% Highest density fraction 75% Total gradient volume, incl. sample (mL) 4,8 Sample volume (mL) 1 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0,4 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ Adam 10/ TSG101/ Alix/ CD81 Not detected contaminants Apo A1/ GM130 Detected EV-associated proteins CD63/ TSG101 Detected contaminants Argonaute2 Detected EV-associated proteins Antithrombin 3 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force-EM Image type Close-up, Wide-field Report size (nm) 50

	EV21008	2/2	Homo sapiens	Blood plasma	DG (d)(U)C	Annalisa Radeghieri	2022	50%
Study summary Full title Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles All authors Annalisa Radeghieri, Silvia Alacqua, Andrea Zendrini, Vanessa Previcini, Francesca Todaro, Giuliana Martini, Doris Ricotta, Paolo Bergese Journal Journal of Extracellular Biology Abstract Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clott (show more...)Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV-protein corona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms. (hide) EV-METRIC 50% (82nd 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 Blood plasma Sample origin Antithrombin 3 deficiency Focus vesicles extracellular vesicle 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 DG (d)(U)C Protein markers EV: Antithrombin 3 non-EV: None Proteomics no EV density (g/ml) 1.11-1.22 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 8% Highest density fraction 75% Total gradient volume, incl. sample (mL) 4,8 Sample volume (mL) 1 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0,4 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins Antithrombin 3 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV21008
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	Antithrombin 3 deficiency
separation protocol	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2
EV-METRIC %	89	50