Increased circulating platelet-derived extracellular vesicles in severe COVID-19 disease

Figures & data

Figure 1. High sensitivity flow cytometry (hsFCM) and single-particle interferometric reflectance imaging sensor (SP-IRIS) were used to isolate and characterize medium and large EVs (200–4000 nm) and small EVs (50–200 nm), respectively. EV, extracellular vesicle; SEC, size-exclusion chromatography; RC, residual cell.

Table I. Summary of the laboratory test results of the 50 patients with COVID-19 infection. Results between high and low D-dimer groups are presented in Supplemental Table S2 and statistically significant differences in supplemental figure S3.

	Median	Range	Interquartile range	Reference range	NAs
PT (%)	99	23–133	90–106	70–130	1
APTT (s)	35	27–138	32–39	28–37	4
Fibrinogen (g/L)	5.8	0.4–11.5	4.3–7.4	2.0–4.0	0
FVIII (IU/mL)	162	35–489	113–239	60–160	2
Thrombin time (s)	22	16–37	19–24	17–25	7
VWF:Act (IU/dL)	256	10–563	129–305	50–190	0
VWF:Ag (IU/dL)	386	135–874	254–486	50–190	0
VWF:CB (IU/dL)	186	4–530	64–248	55–180	8
Antithrombin (IU/dL)	86	7–137	72–105	85–125	3
D-dimer (mg/L)	0.7	0.2–11.3	0.4–1.8	<0.5	0
Plt (×10^9/L)	294	93–694	215–434	150–360	1
Anti-Xa (IU/mL)	0.12	0–0.59	0.04–0.22	-	6
Hgb (g/L)	118	73–147	102–129	^1	2
Leuk (×10^9/L)	6.5	2.0–20.6	4.7–9.8	3.4–8.2	1
CRP (mg/L)	61	4–398	21–115	<4	2
ADAMTS13%)	72	19–140	42–92	60–130	4
CAT Peak (nM)	64.5	1.6–324.6	27–141	142–178^2	7
CAT ETP (nM*min)	1069.3	109–2173	573–1450	1140–1391^2	15
CAT LT (min)	5.6	2.7–41.5	3.9–7.2	3.00–3.67^2	7

¹Hgb ref interval 134–167 g/L for men, 117–155 g/L for women. ²range of CAT TG from normal plasma.

PT, prothrombin time; APTT, activated partial thromboplastin time; Fibr, fibrinogen; VWF:Act, von Willebrand factor GPIb activity; VWF:Ag, VWF antigen; Plt, platelet count; Hgb, hemoglobin; Leuk, leukocytes; CRP, C-reactive protein; VWF:CB, von Willebrand factor, collagen binding assay; CAT ETP, thrombin generation with CAT®, endogenous thrombin potential; CAT Peak, peak thrombin generation, CAT LT, Lag Time of thrombin generation; SD, standard deviation; NAs, samples with no results

Figure 2. Residual cells/large extracellular vesicles (1000–4000 nm) and medium-sized extracellular vesicles (200–1000 nm) measured by high sensitivity flow cytometry. Markers for red blood cells (CD235a), endothelial cells (CD31), leukocytes (CD45), platelets (CD61), and phosphatidyl serine exposure (lactadherin) were measured. Significant differences between low D-dimer and control groups compared with the high D-dimer >1.5 mg/L are marked with asterisks: *p < .05, **p < .01, ***p < .001. One outlier sample, with particle counts exceeding 6 × 10⁹/L is not shown. Pairwise comparisons were determined using Mann–Whitney U test, with Bonferroni correction. Box plots show median, 25th and 75th percentiles and range.

Figure 3. Particle counts and diameters of small EVs 50–200 nm by SP-IRIS (exoview R100). CD41a+, CD63+, CD81+, and CD9+ EV counts were all significantly higher in the high D-dimer group >1.5 mg/L compared to low D-dimer (≤1.5 mg/L) or controls. In terms of particle diameter, CD9+ particles were significantly larger in high D-dimer patients in comparison to low D-dimer patients. Significant differences between low D-dimer and control groups compared with the high D-dimer >1.5 mg/L are marked with asterisks: *p < .05, **p < .01, ***p < .001. Three outliers, with particle count exceeding 3 × 10⁵ are not shown. Pairwise comparisons were done using Mann–Whitney U test, with Bonferroni correction. Box plots, show median, 25th and 75th percentiles and range.

Figure 4. SP-IRIS tetraspanin colocalization analysis with platelet marker CD41a used as the capture antibody, as well as colocalizations with CD63, CD81, and CD9. Results are shown in stacked boxplots as proportions of bound antibodies. Significant differences between controls and low ≤1.5 mg/L and high >1.5 mg/L D-dimer groups are marked with white asterisks: *p < .05, **p < .01, ***p < .001. Significant differences between the high and low D-dimer groups are marked with red asterisks: *p < .05, **p < .01 three outliers, with particle count exceeding 3 × 10⁵ are not shown.

Ayers L, Pink R, Carter DRF, Nieuwland R. Clinical requirements for extracellular vesicle assays. J Extracell Vesicles. 2019;8(1). doi:10.1080/20013078.2019.1593755.

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Puhm F, Flamand L, Boilard E. Platelet extracellular vesicles in COVID-19: potential markers and makers. J Leukoc Biol. 2022;111(1):63–74. doi:10.1002/JLB.3MIR0221-100R.

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