Name: Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time
Uploaded: 2023-08-04T13:40:00
Description: Watch this Scientific Journal Video about Isolation of Extracellular Vesicle EV-Rich Plasma from Human Blood and Detection of Activated Clotting Time at JoVE.com

isolation of extracellular vesicle (ev)-rich plasma from human blood and detection of activated clotting time 

Evaluación de la hipercoagulación sanguínea del plasma rico en EV

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&#39;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 &#34;invisible&#34; and &#34;visible&#34; 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&#39; 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.

Autor destacado: Descifrando los trastornos de la coagulación en pacientes con lesiones cerebrales traumáticas 

Determinación de la actividad procoagulante de la vesícula extracelular (VE) mediante el tiempo de coagulación activado por EV (EV-ACT)

Our primary focus is on coagulation disorders in patients who have suffered brain trauma. In this study, we try to monitor changes in the clotting function with extracellular vesical activity clotting time. Our teams put attention on the research of brain injury.And in the brain injury, they are a severe condition we call the hyper coagulated state. It's very difficult to measure. And we find in the blood of patient there are some micro vesicle we called extracellular vesicles, including the apoptosis body, micro vesicle, and exosome.And they are different in the pro coagulated ability. So we must find a method to measure the ability. And this method, we can acute quickly to find the difference of the extracellular vesicles.That's very important. Depend on this measure, we can do many things. We have make sure it in the TPI mice, and the clinical patient, especially in the peripheral circulation, it will cost some embolization.That is very, very serious in the clinical. Our protocol offered a significant advantage compared to other techniques due to its simplicity, cost effectiveness, and speed. It allows for a blood clotting test to be conducted at the bedside, eliminating the need for extensive laboratory processing.Our research have established a new method that can measure the hyper coagulated states of the peripheral blood. It can be used not only in the TPI, we can also used in other disease such as sepsis, pre-eclampsia, tumor, and hematology disease. In the future, we have planned to investigated the of in scanning for clinical hyper a condition that increases the risk of blood clot.

Watch this Scientific Journal Video about Isolation of Extracellular Vesicle EV-Rich Plasma from Human Blood and Detection of Activated Clotting Time at JoVE.com

Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time   

Aislamiento de plasma rico en vesículas extracelulares (EV) de sangre humana y detección del tiempo de coagulación activado 

Para empezar, centrifuga la sangre humana a 120 g durante 20 minutos a temperatura ambiente para eliminar las células sanguíneas. A continuación, transfiera la mitad superior del sobrenadante a un nuevo tubo. A continuación, centrifuga el plasma rico en plaquetas para eliminar las plaquetas y transfiere la mitad superior del sobrenadante a un nuevo tubo.A continuación, eliminamos los restos celulares centrifugando la plaqueta a nuestro plasma a 13.000 G durante dos minutos. Transfiera la mitad superior del sobrenadante a un tubo nuevo. Encienda el analizador de coágulos para detectar vesículas extracelulares o tiempo de coagulación activado por EV y precaliente el instrumento a 37 grados Celsius.Instale la sonda desechable y el vaso de prueba. Luego inicie el procedimiento de control de calidad de la máquina. Después de la ejecución del control de calidad, introduzca la información de la muestra en el sistema.Luego agregue 200 microlitros de plasma rico en EV al vaso de prueba, seguido de 20 milimolares y 170 microlitros de cloruro de calcio. Haga clic en el botón de inicio, cierre la tapa y permita que la sonda detecte el cambio en la resistencia de la muestra. Las muestras de EV no calificadas exhiben un grupo distinto con un tamaño de partícula ligeramente mayor cerca de la puerta de EV.In contraste, las muestras calificadas ricas en EV mostraron la mayoría de las señales dentro de la puerta de EV como un solo grupo.Un aumento en la concentración de EV acortó el tiempo de coagulación activado por EV, mientras que una disminución en la concentración de EV prolongó el tiempo de coagulación activado por EV. Las muestras de plasma ricas en EV de pacientes con preeclampsia, fracturas de cadera y cáncer de pulmón tuvieron un tiempo de coagulación activado por EV significativamente más corto en comparación con las voluntarias sanas.

Research

JoVE Journal

Medicine

Isolation of Extracellular Vesicle (EV)-Rich Plasma from Human Blood and Detection of Activated Clotting Time 

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

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...