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 risk6,7. 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 fibrinolysis14,15,16,17. 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 changes20,21. 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 activity22,23. 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.
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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
- 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.
- 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.
- 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
- 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. - 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
- 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). - 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.
- 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.
- 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.
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Representative 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 test platform and identify any physical disturbances to the probe. Performance quality control involves testing with standard viscosity oil to ensure that the resistance value detected by the probe falls within the set range.
After obtaining EV-rich plasma, EVs were measured using flow cytometry for sample quality control. Unqualified samples exhibit a distinct cluster with slightly larger particle size near the "gate" of EV (Figure 2A). In contrast, qualified EV-rich samples show that most signals fall within the "gate" of EV as a single group (Figure 2B).
To evaluate EV concentration in EV-rich plasma samples from healthy volunteers, superspeed centrifugation (1,00,000 x g, 70 min) was performed. The results revealed that an increase in EV concentration shortened EV-ACT, while a decrease in EV concentration prolonged EV-ACT (Figure 3A). EV-ACT time may exhibit a negative correlation with EV levels. Several samples from clinical patients were examined, and the findings demonstrated that EV-rich plasma samples from patients with preeclampsia, hip fractures, and lung cancer (from left to right) had significantly shorter EV-ACT times compared to healthy volunteers (Figure 3B).
In conclusion, a rapid and cost-effective experimental method for detecting EV procoagulant activity was preliminarily established, holding promise for clinical applications in bedside examinations.
Figure 1: The schematic diagram and physical image of the clot analyzer. (A) and (B) represent the main components of the analyzer, and the operation settings, respectively. Please click here to view a larger version of this figure.
Figure 2: Representative flow cytometry of EV in EV-rich plasma. (A) Unqualified EV-rich plasma samples. (B) Qualified EV-rich plasma samples. Please click here to view a larger version of this figure.
Figure 3: Representative results of the EV-ACT of the sample. (A) EV-ACT results after EV removal and EV concentration in EV-rich samples from healthy volunteers (from left to right, suspension of sediment after supercentrifugation, EV-rich plasma, and supernatant after ultracentrifugation). (B) EV-ACT results of EV-rich samples from healthy volunteers and patients (From left to right, preeclampsia,hip fracture, lung cancer, and healthy volunteer). Please click here to view a larger version of this figure.
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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, exhibited prolonged EV-ACT times. These findings suggest a close relationship between EV-ACT results and EV levels. In Figure 3B, the EV-ACT of plasma samples from patients with preeclampsia, hip fractures, and lung cancer were compared to healthy volunteers, revealing significantly shorter EV-ACT times in the disease groups. Previous reports have indicated elevated levels of coagulation-promoting EVs in plasma for these diseases27,28,29. However, it should be noted that other factors present in plasma may influence EV-ACT outcomes, and further exploration of these influencing factors is warranted. This assay proposes to recognize the crucial role of EVs in the hypercoagulable state of certain diseases and addresses the lack of a low-cost and rapid detection method for EV procoagulant activity.
Within the isolated blood sample system, coagulation-related substances can be divided into blood cells, platelets, cell metabolites (mainly EVs), and other "invisible" components (such as coagulation factors and anticoagulant factors)30,31,32. Given the complexity of the coagulation mechanism, the detection system was simplified to EV-rich plasma to minimize interference from other factors. Key steps in this process include preventing the generation of artificial EVs during sample processing and minimizing platelet contamination. Therefore, it is necessary to initiate the differential centrifugation procedure within 0.5 h and complete the detection within 2 h after sample collection. Previous studies have demonstrated that using sufficiently high centrifugal force and retaining the upper one-half of the supernatant can effectively reduce platelet contamination33. Flow cytometry was used to detect residual platelets in EV-rich plasma, confirming their very low presence. Unqualified samples primarily result from platelet contamination and cell fragmentation caused by the handling process. Platelet contamination is characterized by the presence of particles outside the "gate" of EVs, while cell fragmentation appears as a distinct cluster within the "gate" of EVs.
Recent studies have confirmed significantly elevated levels of plasma EVs in certain diseases34,35. These EVs primarily promote coagulation through the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. Therefore, the ACT time of EV-rich plasma is largely dependent on the level of coagulant EVs. One limitation of this test is the need to ensure that levels of clotting factors do not change significantly to the extent that they impact clotting time during EV-ACT analysis. However, studies on changes in clotting factor levels after disease onset are relatively scarce, and their effect on EV-ACT needs to be evaluated in further research. Moreover, excessive use of anticoagulant drugs can significantly affect EV-ACT results, so this method should not be employed during such treatment.
Several methods exist for determining procoagulant EVs, each with its advantages and disadvantages. Piwkham et al. evaluated EV procoagulant activity by mixing EV suspension with normal plasma and observing the time of blood clot formation in a water bath at 37 °C36. Although this method is simple, the subjective observation of clot formation time can lead to result variations among different observers. Patil et al. employed an in-house standardized clotting assay for procoagulant microparticle testing using Russell viper venom on a semi-automated coagulometer37. This detection method utilizes semi-automatic equipment, offering high efficiency and simplicity. However, the addition of Russell viper venom to activate factor X overlooks the presence of other anticoagulants in plasma, such as lactadherin protein. This method assesses outcomes based on the balance between procoagulant EVs and anticoagulant components in the plasma, providing a more accurate reflection of the clotting function of the plasma sample.
Existing coagulation function tests primarily focus on thrombin activation time in whole blood or platelet-rich blood, or detect deficiencies in coagulation factors24. Developing a test method for EV procoagulant activity will help elucidate the role of EVs in disease and future treatments. The simple sample preparation process, involving the addition of only CaCl2, makes the assay convenient for clinicians to quickly determine the coagulation function status of patients. The EV-ACT test is easy to perform, with results available within 10-15 minutes, making it well-suited for development as a bedside test. Previous studies have tentatively identified applications of EV-ACT in preeclampsia, tumors, and fractures. Future research will continue to explore the clinical application prospects of EV-ACT.
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Disclosures
All authors declared that there are no potential conflicts of interest.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China, grant no. 81930031, 81901525. In addition, we thank Tianjin Century Yikang Medical Technology Development Co., Ltd. for providing us with machines and technical guidance.
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. |
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