Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

Summary

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

Abstract

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Introduction

Thrombosis, which is caused by hypercoagulability, plays a significant role in various diseases, including brain trauma¹, pre-eclampsia², tumors³, and fracture patients⁴. The mechanism underlying hypercoagulability is complex, and recent emphasis has been placed on the role of extracellular vesicles (EV) in coagulation disorders. EVs are vesicle-like bodies with a bilayer structure that detach from the cell membrane, ranging in diameter from 10 nm to 1000 nm. They are associated with a variety of disease processes, particularly coagulation disorders⁵. Several studies have identified EVs as a promising predictor of thrombosis risk^6,7. The procoagulant activity of EVs depends on the expression of coagulation factors, primarily tissue factor (TF) and phosphatidylserine (PS). EVs with robust procoagulant activity significantly enhance the catalytic efficiency of tenase and prothrombin complex, thereby promoting thrombin-mediated fibrinogen and local thrombosis⁸. Elevated levels of EVs and their causal relationship with hypercoagulability have been observed in numerous diseases⁹. Consequently, standardizing the detection of EVs and reporting their procoagulant activity is an important area of investigation¹⁰.

To date, only a few commercial kits are available for detecting the procoagulant activity of EVs. The MP-Activity assay and the MP-TF assay, produced by a commercial company, are functional assays used to measure EV's procoagulant activity in plasma¹¹. These assays employ a principle similar to that of enzyme-linked immunosorbent assays to detect PS and TF on EVs. However, these kits are expensive and limited to a few high-level research institutions. The process is complex and time-consuming, making it challenging to implement them in clinical settings. Additionally, a commercially developed procoagulant phospholipid (PPL) assay mixes PS-free plasma with test plasma, measuring clotting time to quantitatively detect levels of PS-positive EVs¹². However, these assays primarily focus on PS and TF on EVs, overlooking other clotting pathways that circulating EVs may be involved in¹².

The plasma coagulation system is intricate and comprises both "invisible" and "visible" components, including coagulants, anticoagulants, fibrinolytic systems, and EVs suspended in the plasma. Physiologically, these components maintain a dynamic balance. In pathological conditions, significantly increased EVs in circulation contribute to hypercoagulability, particularly in patients with brain trauma, preeclampsia, fractures, and various types of cancer¹³. Currently, the evaluation of coagulation status in clinical laboratories primarily involves assessing the coagulation system, anticoagulation system, and fibrinolysis^14,15,16,17. Prothrombin time, activated partial thromboplastin time, thrombin time, and international normalized ratio are commonly used to evaluate coagulation factor levels in the coagulation system¹⁸. However, recent studies have revealed that these tests do not fully reflect the hypercoagulability of certain diseases¹⁹. Other assay methods, such as thromboelastometry (TEG), rotational TEG, and Sonoclot analysis, measure whole-blood viscoelastic changes^20,21. Since whole blood samples contain numerous blood cells and platelets, these tests are more likely to indicate the clotting status of the sample as a whole. Some researchers have reported on the role of blood cells and platelets in procoagulant activity^22,23. A recent study also discovered that previous coagulation function tests face difficulties in detecting changes in microparticles' procoagulant activity²⁴. Therefore, a hypothesis has been proposed that the procoagulant function of EVs can be evaluated by viscoelastic measurements of the activated clotting time (ACT) in EV-rich plasma.

Protocol

The collection of human samples was approved by the Medical Ethics Committee of Tianjin Medical University General Hospital. The collection of human venous blood strictly followed the guideline issued by the National Health Commission of China, namely WS/T 661-2020 Guideline for Collection of Venous Blood Specimens. Briefly, blood was collected from healthy individuals with informed consent from the anterior brachial area vein, and the samples were mixed using 3.2% sodium citrate anticoagulant in a ratio of 1:9. When only sodium citrate anticoagulant specimens were collected, the first collection vessel was discarded. The processing flow was initiated within 0.5 h of sample collection. Adult healthy subjects were recruited for sample collection after obtaining informed consent. Patient exclusion criteria were: (1) a recent intravascular thrombosis, (2) liver and kidney function impairment, (3) hypertension, hyperlipidemia, diabetes, and other chronic diseases, (4) aspirin or anticoagulant treatment, (5) menstruation and pregnancy.

1. Isolation of EV-rich plasma

1. Centrifuge the samples at 120 x g for 20 min at room temperature to remove blood cells. The supernatant is platelet-rich plasma. Then, transfer the upper 1/2 of the supernatant to a new centrifuge tube with a pipette.
2. Centrifuge the platelet-rich plasma at 1500 x g for 20 min at room temperature to remove the platelets. The supernatant is platelet-poor plasma. Then transfer the upper 1/2 of the supernatant to a new centrifuge tube.
3. Centrifuge the platelet-poor plasma at 13000 x g for 2 min at room temperature to remove cell debris. The supernatant is EV-rich plasma. Then, transfer the upper 1/2 of the supernatant to a new centrifuge tube for subsequent testing.

2. Detection of EV-ACT of the sample by the analyzer

1. Turn on the clot analyzer (see Table of Materials) and preheat the instrument to 37 °C. Install the disposable probe and test cup, then start the quality control procedure of the machine.
   NOTE: When the viscous resistance signal value on the screen is displayed as a straight line, it means that the detection is not interfered with and the quality control of analyzer status is qualified. The testing procedure can be started after the self-test is qualified.
2. Enter sample information into the system. Then, transfer 200 µL of EV-rich plasma to the test cup, followed by 20 mM 170 µL of calcium chloride. Click the start button. The magnetic stirring rod in the test cup will fully mix the sample and calcium chloride. Close the cover, and the probe will begin to detect the change in sample resistance.
   ​NOTE: After the test is finished, the analyzer will automatically prompt "test completed". The time of EV-ACT is displayed and recorded by the system. The result is expressed in the unit of time "second". Discard the probe and test the cup.

3. Flow cytometry-based quality control of the EV-rich plasma sample

1. EVs were first identified by their size (0.1-1 µm). Adjust the parameters of the flow cytometer in advance and set the "gate" of EV using commercially available polystyrene beads (0.5, 0.9, and 3.0 µm) (see Table of Materials).
   NOTE: The bead mixture consists of fluorescent spheres of different diameters covering the size range of microvesicles (0.5 and 0.9 µm) and platelets (0.9 µm and 3 µm).
2. Adjust the voltage of the Forward Scatter and the Side Scatter, and ensure that the 0.9 µm microspheres fall in the appropriate region. The EV region can be drawn according to the diameter of the microsphere.
3. Transfer 50 µL of EV-rich plasma into the flow tube, followed by 450 µL of filtered phosphate buffer saline.Thoroughly mix the liquid in the flow tube.Finally, detect the samples using flow cytometry²⁵.
4. Use Ultra Rainbow Fluorescent Particles (see Table of Materials) to quantify the number of EVs.In brief, add 10 µL of the particles to the sample before the flow detection, and calculate the EV concentration using the following formula after the sample detection is complete: 10,120 * EV (#) / (microbeads * volume) * dilution factor²⁶.

Results

The thrombin activation time of EV-rich plasma was measured using a viscoelastic method analyzer for plasma coagulation time measurement. The machine consists of four main components: an electronic signal converter, a probe, a detection tank, and a heating element (Figure 1A,B). The probe utilizes high-frequency and low-amplitude oscillations to detect changes in plasma viscosity. Daily quality control primarily involves air quality control to assess the stability of the tes...

Discussion

In this study, the preparation of EV-rich plasma was described, and the method's rationality was verified using flow cytometry. Subsequently, the recalcified plasma samples were analyzed for ACT time using a clot analyzer based on viscoelasticity principles²⁴. As shown in Figure 3A, the concentration of EVs obtained through ultracentrifugation was found to shorten the EV-ACT time, while the supernatant after ultracentrifugation, which had reduced EV levels, exhibi...

Materials

Name	Company	Catalog Number	Comments
AccuCount Ultra Rainbow Fluorescent Particles	3.8 microm; Spherotech, Lake Forest, IL, USA		For quantitative detection of MP
Calcium chloride	Werfen (china)	0020006800	20 mM
Century Clot analyzer	Tianjin Century Yikang Medical Technology Development Co., Ltd		The principle is to measure plasma viscosity by viscoelastic method
Disposable probe and test cup	Tianjin Century Yikang Medical Technology Development Co., Ltd
LSR Fortessa flow cytometer	BD, USA		Used to detect MP
Megamix polystyrene beads	Biocytex, Marseille, France	7801	The Megamix consists of a mixture of microbeads of selected diameters: 0.5 µm, 0.9 µm and 3 µm.