Chronolog 700 aggregometer

Platelet function testing was measured using LTA (Light Transmission Aggregometry) [24 (link)]. In brief, blood was collected on citric acid and then platelet aggregation was measured photometrically using a Chronolog 700 Aggregometer (Chronolog Corp., Havertown, PA, USA). Aggregation was induced by 1 μmol/L adenosine diphosphate (ADP) (Sigma-Aldrich, Vienna, Austria). The duration of platelet aggregation run ranged from 5 to 10 min. The intensity (A), lag (L), time (T), and the rate (V) of aggregation were determined from the aggregation plot.

Following sample collection (4 mLs/time point), samples were divided, one aliquot was subjected to centrifugation (10 min at 2500× g), and the supernatant collected for use as the plasma poor platelet (PPP) sample. Light transmission aggregometry was performed using a two-channel Chronolog 700 Aggregometer (Chronolog Corporation, Havertown, PA, USA). Following preliminary analyses using 250,000 platelets/μL, an unstable optical baseline was observed. To correct for this platelet concentration of PRP, samples were adjusted using PPP to a final concentration of 500,000 cells/μL with 500 μL of adjusted PRP used for analysis. Platelet aggregation was induced using 5μL adenosine diphosphate (ADP, final concentration 10 μM, CHRONO-PAR^® ADP, Chronolog Corporation) or 5 μL arachidonic acid (AA, 0.5 mM final concentration, CHRONO-PAR^® Arachidonic Acid; Chronolog Corporation). An amount of 500 μL of PPP was used as a control representing 100% light transmission or 100% aggregation, while PRP represented 0% light transmission or 0% aggregation prior to stimulation. The aggregation tracing was run for a minimum of 6 min or when tracings reached full amplitude after agonist addition (ADP or AA). Results are represented as % ADP or AA aggregation representing maximum amplitude after stimulation.

Simultaneous monitoring of platelet aggregation and ATP secretion was performed at 37 °C with constant stirring (1000 rpm) in a Chronolog 700 aggregometer (Chronolog, Havertown, PA, USA). For all experiments involving pharmacological inhibitors, platelets (2 × 10⁸ mL^− 1) were pre-treated with vehicle control or inhibitors for 10 min. Also, for experiments involving functional blockade of platelet CXCR7, platelets were pre-incubated for 10 min with 10 μg/mL^− 1 IV.3 prior to a 10 min incubation with 10 μg/mL^− 1 anti-CXCR7 (11G8) or IgG₁ control to prevent non-specific platelet activation via the FcγRIIA receptor. Before agonist stimulation, 5 μL of luciferin-luciferase (Chronolog) was added. Post aggregation, 1 nM ATP standard was added as a reference value for quantification of secreted ATP. Data analysis was performed with Aggro/Link5 software (Chronolog).