The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

Begin with a multi-well plate containing platelet-rich plasma. Platelets express surface mechanoreceptor complexes — responsive to mechanical stimuli.

The mechanoreceptor complex contains the extracellular ligand-binding domain and macro-glycopeptide region linked to the mechanosensory domain.

Introduce anti-mechanoreceptor antibodies, cross-linking with the ligand-binding domains of the receptors on opposing platelets.

Place a droplet of the antibody-treated plasma suspension between the assemblies of the viscometer.

Apply shear stress, simulating mechanical force, pulling the antibody-bound receptors away from the platelets, leading to macro-glycopeptide region dissociation from the mechanosensory domain.

This triggers a conformational change in the mechanosensory domain, activating platelets and resulting in the expression of surface markers.

Transfer the sheared platelet suspension to a new well within the multi-well plate.

Introduce a cocktail of fluorophore-labeled marker-specific molecules interacting with corresponding platelet surface markers and fix the cells with a fixative solution.

In flow cytometry, distinct fluorescence signals correlate with the effects of shear stress on antibody-treated platelets.

For ligand treatment, slowly, add the antibody or ligand of interest to the platelet-rich plasma, and mix the platelets gently. During the incubation, turn on the cone-plate viscometer, and set the plate temperature to 22 degrees Celsius. After 5 to 10 minutes, transfer the treated platelet-rich plasma onto the temperature-controlled cone-plate viscometer directly at the center of the plate, taking care that all of the sample is deposited between the cone and plate at the point of contact.

To apply shear to the sample, calculate the shear according to the viscometer manual, and apply shear to the sample at the appropriate rate and duration. At the end of the application, lift the cone about 2 millimeters off the plate, so that the sample remains in contact with both the plate and the cone, and use a gel-loading pipette tip to collect 5 to 10 microliters from the center of the sample volume. Then, incubate the sheared sample with antibodies against the surface markers of interest for 20 minutes at room temperature, and fix the samples in 2% paraformaldehyde for 20 minutes at room temperature.

To analyze the samples by flow cytometry, collect at least 20,000 events for each condition, and quantify the signal strength of each fluorescent marker using the height value for the intensity of each fluorophore. Then, use a negative control with bovine serum albumin or vehicle to draw a gate excluding negative background fluorescent events, and quantify the percentage of events inside the gate for all experimental conditions.