Measurements of platelet activation and adhesion

For the evaluation of platelet activity via LTA, citrate-anticoagulated blood was centrifuged within 15 minutes of collection at 200g for 10 minutes to obtain PRP. LTA was performed 30 minutes later on the AggRAM light transmission aggregometer. Platelet aggregation was assessed in response to submaximal ADP and epinephrine concentrations and recorded at maximum aggregation over a 10-minute evaluation.

For the evaluation of platelet activity via extent of MPA and NPA, platelet surface PAC-1 expression, and platelet surface P-selectin expression, citrate-anticoagulated blood was processed within 15 minutes of collection. Whole blood was incubated with CD61-FITC or CD42b-APC (platelets), CD14-APC (monocytes), CD45-APC (leukocytes), PAC-1-FITC, and CD62-FITC (P-selectin) antibodies and evaluated on an Accuri C6 flow cytometer. MPA was assessed using CD14 to identify the monocyte population, and NPA was assessed using CD45 and forward/side light scattering characteristics to identify the neutrophil population. Monocytes and neutrophils with adherent platelets were identified by CD61/CD42b positivity.

For the evaluation of platelet adhesion, platelets were isolated from citrate-anticoagulated blood and stained with 1 μM DiO. Following a second isolation process, stained platelets were resuspended in Tyrodes buffer at a concentration of 1 × 10⁵ platelets/μL. Coverslips (18 mm) were coated with 100 μg/mL collagen at 37°C for 1 hour, followed by addition of 0.1 U thrombin prior to addition of 1 mL isolated stained platelets. Platelets were incubated at 37°C for 30 minutes, and the wells were then washed twice and analyzed using an Evos FL fluorescence microscope (Thermo Fisher Scientific Inc., Waltham, MA). Platelet adhesion was quantified using Image J software (U. S. National Institutes of Health, Bethesda, Maryland).