An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

Platelets are crucial in preventing blood loss after vessel injury, fighting infections, and maintaining vascular integrity during inflammation. While hemostatic plugs require the collective activation and aggregation of platelets, their role in protecting the inflamed blood vessels is performed at the single-cell level. In this context, recent studies have found that platelets can migrate autonomously, a process dependent on mechanosensing of their adhesive microenvironment.

This approach allows precise control of the substrate adhesive properties and serves as a simple in vitro assay to study the mechanisms underlying platelet migration. The results indicate that migrating platelets binding to integrin ligands can exert force capable of disrupting the avidin-biotin bond. This assay provides a simple and reliable method to visualize platelet migration.

Combined with live cell imaging, it will help us better understand the interplay and the regulation of different cytoskeletal components in this important platelet function. To begin, sonicate glass cover slips in 20%nitric acid for one minute, followed by sonication in isopropanol, ethanol, and water for one minute each. Treat the pre-cleaned cover slips with oxygen plasma in a plasma cleaner for two minutes, then assemble them with sticky slides.

Now fill the channel with 2.5 microliters of PLL-PEG-biotin diluted in 97.5 microliters of PLL-PEG and incubate for 30 minutes at room temperature, then wash three times with PBS. Next, add 100 microliters of neutravidin-FITC and incubate for 30 minutes in the dark at room temperature, then wash three times with PBS. Finally, add 100 microliters of cyclo-RGD biotin, incubate for 30 minutes at room temperature, and wash three times with PBS.

After anesthetizing the mouse and removing the thoracic skin, insert the needle between the second and third ribs on the left side of the sternum to draw blood from the heart. Mix the blood with one milliliter of Tyrode's buffer and centrifuge at 70 g for 20 minutes at room temperature with the brake switched off, then take the upper part containing platelet-rich plasma. Mix with three milliliters of Tyrode's buffer and add 100 nanograms per milliliter prostacyclin to prevent platelet activation.

Afterward, centrifuge at 1200 g for five minutes at room temperature. Discard the supernatant and resuspend the pellet in 500 microliters of Tyrode's buffer. Then, using a hemocytometer, measure platelet counts.

To begin, take mouse platelets in biotin-neutravidin-biotin cRGD coating channels and record live platelet migration using an inverted microscope equipped with a stage incubator. To count the platelet adhesion or migration numbers, right click on the dropdown menu of the point tool in the toolbar and select the multi-point tool. Extract the distance of migration from fixed samples by measuring the length of the migration path imprinted in the neutravidin-FITC coating.

Right click on the dropdown menu of the straight line tool in the toolbar and select the free handline tool. To quantify the platelets'shape select Image, Adjust, and Threshold in the toolbar to generate binary masks by segmenting fluorescent platelets. Select shape descriptors in Analyze and Set Measurements, then select Display Results using Analyze and Analyze Particles.

Platelets exhibited optimal migration at 2.5%PLL-PEG-biotin concentration. Migration was reduced at both lower and higher ligand densities. Platelets spread inadequately at 1%ligand density, indicated by low area and perimeter, suggesting insufficient integrin activation.

At 2.5%ligand density, platelets spread effectively, disrupting cRGD ligands and migrating. High ligand density caused platelets to spread without polarization, reducing migration due to failure to break cRGD ligands.