Extracellular vesicles and coagulation in blood from healthy humans revisited

Figures & data

Table 1. Concentration and cellular origin of extracellular vesicles in human plasma.

		Concentration x 10^6/L (correction with IgG control) range			Concentration x 10^6/L (no correction with IgG control) range
Cellular origin	Biomarker	2001	2018	Fold difference	2001	2018	Fold difference
Erythrocyte	CD235a	74 (39–123) 15%	28,300 (14,000–120,000) 29%	382	80 (43–128) 15%	39,500 (25,600–186,000) 29%	494
Granulocytes	CD66e	46 (16–94)	ND		49 (22–103)	ND
	CD66b	ND	4400 (0–24,600)		ND	18,600 (4900–84,100)
Leukocyte	CD45	16 (7–31) 3%	13,800 (6300–82,200) 14%	862	22 (11–36) 4%	27,400 (9700–151,000) 20%	1245
Monocyte	CD14	0 (0–10)	1870 (0–10,800)		4 (2–14)	12,000 (2500–79,600)	3000
Endothelial	CD62E	88 (22–172)	1000 (0–75,600)		92 (27–179)	12,600 (1800–89,000)
Platelet	CD61	416 (148–839) 82%	54,100 (24,400–162,000) 56%	130	422 (152–843) 81%	71,400 (33,500–228,000) 52%	169
	PS (annexin V)	NA	ND		319 (146–623)	ND
	PS (lactadherin)	ND	NA		ND	257,000 (145,000–1,310,000)
Total platelet, erythrocyte, leucocyte EVs		506	96,200	190	524	138,300	264

Concentration and cellular origin of extracellular vesicles (EVs), with (left) or without (right) correction for isotype control, as reported in 2001 and 2018. Major differences between 2001 and 2018 occur not only from the improved sensitivity (lower limit of detection) of a new generation of flow cytometers, but also from differences in blood collection, plasma preparation, labelling of EVs, and the prevention of “swarm” detection. Data are presented as median and range. ND: Not determined; NA: Not applicable.

Figure 1. Comparison of the thrombin and fibrin generation tests in human plasma. Thrombin generation (TGT; thrombin generation test) 2001 (a) versus fibrin formation (FGT; fibrin generation test) 2018 (b) by extracellular vesicles, Innovin (c, d) or Synthasil (e, f). In both assays, calcium chloride is added at t = 0.

Table 2. Protocols to detect extracellular vesicles and their coagulant activity in 2001 and 2018.

	2001	2018	Ref
Blood collection
Collection tube	Glass	Plastic	[30]
Plasma preparation (1550g)	Once	Twice	[3,5]
Flow cytometry
Incubation time (labelling)	15 minutes	2 hours*
Centrifugation of EVs after labelling before measurement by flow cytometry	Yes	No	[3]
Prevention of “swarm”	No	Yes	[20]
Estimated scatter detector sensitivity to detect single EVs (lower limit of detection)	~500 nm	~170 nm	[17]
Coagulation
Concentration of EVs by centrifugation before measurement	Yes	No	[9]
EVs reconstituted in defibrinated pool plasma or coagulant activity measured directly in plasma	Reconstituted	Directly in plasma	[1]
Read-out	Thrombin activity	Clotting
Read-out	Chromogenic substrate	Fibrin formation	[9]
Coagulant activity	Likely contact activation	Not detectable

*The final concentration of antibodies now being used is 5- to 20-fold higher compared to 2001.

Figure 2. Effect of blood collection and handling on the coagulant properties of human plasma. Platelet-poor plasma (single centrifugation to remove platelets) collected in a siliconized glass tube (a) or plastic tube (b); (c) platelet-depleted plasma (double centrifugation) prepared from blood collected in a plastic tube. For the fibrin generation test (FGT, left), the prepared plasma samples (containing endogenous EVs) were recalcified at t = 0 as described in Methods. For the thrombin generation test (TGT, right), EVs were isolated from the prepared plasma samples and reconstituted in EV-depleted normal plasma as described in Methods, and thrombin generation was initiated by recalcification at t = 0. Please notice that the time scale (X-axis) differs for FGT and TGT. Representative data from one volunteer are shown. In total, six independent experiments were performed.

Figure 3. Presence of residual platelets in platelet-poor and platelet-depleted plasma. Platelet-poor plasma was prepared from blood collected in a glass tube (a) or plastic tube (b); (c) platelet-depleted plasma was prepared by double centrifugation from blood collected in a plastic tube.

Figure 4. Concentrations of plasma coagulation and fibrinolysis activation markers (a) Concentration of coagulation activation markers prothrombin fragment (F) 1 + 2 and thrombin-antithrombin complexes (TAT) in human plasma; (b) Plasmin generation by EVs in human plasma. Reference ranges are shown in grey.

Table 3. Publications presenting information on the coagulant properties of plasma EVs in healthy humans.

Assay	Plasma preparation	Detectable coagulant activity	References
Thrombin^1,2	1550g	Yes	[1]
	2800g → 10,000g	Yes	[31]
	2500g (2x)	Yes	[14]
	2000g (2x)	Yes	[32]
	1500g → 13,000g	Yes	[33]
	2500g (2x)	Yes	[34]
	3000g (2x)	Yes	[35]
	2500g (2x)	Yes	[36]
Thrombin^3	1500g → 12,000g	Yes	[33,37]
Fibrin^4	1550g	Yes	[38]
	1550g → 13,000g	Yes	[39]
	1550g (2x)	No	This manuscript
Factor Xa	1550g	Yes	[38]
	1550g → 13,000g	Yes	[39]
	2500g (2x)	Yes	[34]
PPL-dependent^5	1550g	Yes	[14,32,35,36,38,40,41]

¹TGT: thrombin generation test; ²TGA: thrombin generation assay (Calibrated Automated Thrombogram); ³Capture ELISA (enzyme-linked immunosorbent assay); ⁴FGT: fibrin generation test; ⁵PPL: phospholipid-dependent clotting assay.

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