Extracellular vesicles and coagulation in blood from healthy humans revisited

Abstract

Background: In 2001, we studied the presence and coagulant properties of "microparticles" in the blood of healthy humans. Since then, multiple improvements in detection, isolation and functional characterization of the now called "extracellular vesicles" (EVs) have been made, and shortcomings were identified. Aim: To revisit the presence and function of EVs in blood from healthy humans. Methods: Blood was collected from 20 healthy donors. EV-containing plasma was prepared according to new guidelines, and plasma was diluted to prevent swarm detection. Single EVs were measured by flow cytometry with known sensitivity of fluorescence and light scatter. The haemostatic properties of EVs were measured by thrombin-, fibrin-, and plasmin generation. Plasma concentrations of thrombin-antithrombin complexes and prothrombin fragment 1 + 2 were measured to assess the coagulation status in vivo. Results: Compared to 2001, the total concentrations of detected EVs increased from 190- to 264-fold. In contrast to 2001, however, EVs are non-coagulant which we show can be attributed to improvements in blood collection and plasma preparation. No relation is present between the plasma concentrations of EVs and either TAT or F1 + 2. Finally, we show that EVs support plasmin generation. Discussion: Improvements in blood collection, plasma preparation and detection of EVs reveal that results from earlier studies have to be interpreted with care. Compared to 2001, higher concentrations of EVs are detected in blood of healthy humans which promote fibrinolysis rather than coagulation.

Keywords: Coagulation; exosomes; extracellular vesicles; fibrinolysis; flow cytometry; microparticles.

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.

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.