Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Platelet procoagulant formation is crucial to maintain hemostasis. This process involves expression of phospholipids and microvesicle release at the platelet surface, which is essential for the coagulation steps. Assessments of this process have provide new data on how platelet procoagulant responses are in diverse human disease.

Flow cytometry has been a cornerstone and has gained widespread acceptance in the evaluation of phosphatidylserine expression on platelets and microvesicle release. The isolation and analysis of purified platelets from blood are time-consuming and necessitates specialized laboratory equipment, and are thus not currently available for routine clinical diagnosis. Analyzing phosphatidylserine exposure and microvesicle release is also challenging due to their small size and the low number of positive events for markers of interest.

To begin, collect peripheral venous blood in collection tubes containing the active anticoagulant trisodium citrate with citric acid and dextrose. Gently invert the tube to mix the blood with the anticoagulant and let the mixture rest at room temperature for 15 minutes. Count platelets using an automated cell counter to determine the correct speed for centrifugation.

To prepare the platelet-rich plasma, centrifuge the blood at 250 g for 10 minutes at room temperature without applying the break. Then add 1 milliliter of ACDA and 6.25 microliters of apyrase to 9 milliliters of the platelet-rich plasma. Centrifuge the mixture at 1, 100 g for 10 minutes at room temperature.

Using a Pasture pipette, remove the supernatant containing platelet-poor plasma and re-suspend the platelet pellet gently in 1 milliliter of washing buffer. Then add 3 to 4 milliliters of washing buffer to the tube and centrifuge it again. After removing the supernatant, re-suspend the pellet in 1 milliliter of washing buffer.

Transfer 150 microliters of the platelet suspension to a 1.5 milliliter tube for counting the cells in an automated cell counter. Add 3 to 4 milliliters of washing buffer to the remaining suspension to perform an additional wash as demonstrated earlier. Next, re-suspend the platelet pellet in the reaction buffer to achieve a final concentration of 5 x 10 to the power of 11 platelets per liter.

To begin, obtain washed platelets in the desired buffer for flow cytometry. Prepare all agonists and fluorochromes in the reaction buffer. For internal assays, add 100 microliters of washed platelets at a concentration of 50 x 10 to the power of 9 platelets per liter to different treatment tubes.

Gently shake each aliquot and incubate at 37 degrees Celsius for 5 minutes. Then add 5 microliters of non-diluted Annexin V-FITC to each microtube and incubate. Afterward, add 500 microliters of reaction buffer to each tube to stop the process.

Gate the platelet population using forward scatter or FSC and use density plots to identify each population independently. Set the negative cutoff at the far right of the resting state platelet population. After activation, classify events to the right of this cutoff as platelets exposing phosphatidylserine.

For differentiating microvesicles, set the cutoff at the lowest FSC value observed in the resting platelet population. After activation, classify phosphatidylserine positive events below this threshold as microvesicles. Baseline measurements indicated that less than 2.9%of non-activated platelets exhibited phosphatidylserine exposure.

Upon activation, 26.7%of platelets became phosphatidylserine positive with collagen, 9.1%with thrombin, and 36.2%with both collagen and thrombin. Ionophore treatment resulted in 49.6%phosphatidylserine positive platelets. The baseline proportion of microvesicles was 0.3%Significant increases were observed only with collagen and thrombin together at about 11.4%and more so with ionophores at 44%