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

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.

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 vesicle activity clotting time. Our teams put attention on the research of brain injury and in the brain injury, there are 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 vesicles we call 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 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 use in other disease such as Suplasia preeclampsia, tumor and hematology disease.

In the future we have planned to investigate the zoo of EV ACT in scanning for clinical hypercoagulability, a condition that increase the risk of blood clot. To begin centrifuge the human blood at 120 G for 20 minutes at room temperature to remove the blood cells, then transfer the upper half of the supernatant to a new tube. Next centrifuge the platelet rich plasma to remove the platelets and transfer the upper half of the supernatant to a new tube.

Then remove the cell debris by centrifuging the platelet poor plasma at 13, 000 G for two minutes. Transfer the upper half of the supernatant to a new tube, turn on the clot analyzer to detect extracellular vesicle or EV activated clotting time and preheat the instrument to 37 degrees Celsius. Install the disposable probe and test cup, then start the quality control procedure of the machine.

After the quality control run, enter sample information into the system. Then add 200 microliters of EV rich plasma to the test cup, followed by 20 millimolar, 170 microliters of calcium chloride. Click the start button, close the cover and allow the probe to detect the change in sample resistance.

The unqualified EV samples exhibit a distinct cluster with a slightly larger particle size near the gate of EV.In contrast, qualified EV rich samples showed most signals within the gate of EV as a single group. An increase in EV concentration, shortened EV activated clotting time, while a decrease in EV concentration, prolonged EV activated clotting time. EV rich plasma samples from patients with preeclampsia, hip fractures, and lung cancer had significantly shorter EV activated clotting time compared to healthy volunteers.