Platelet aggregometer

Blood collection for complete blood count (CBC), D-Dimer, and anti-PF4/polyanionic antibodies were drawn from participants prior to the vaccination to day − 3, day 10 ± 3, and day 28 ± 3. Complete blood count was determined using Sysmex. D-dimer levels were determined by Innovance D-Dimer microparticle-enhanced immunoassay (Dade Behring). Results for D-dimer levels were reported in ng/mL FEU. The normal level was less than 500 ng/mL FEU. Anti-PF4/polyanionic antibodies were screened by IgG-specific ELISA (Hyphen Biomed Zymutest HIA IgG, Quadratech Diagnostics, UK) according to the manufacturer’s instructions. Results were interpreted as positive if the optical density (OD) was above 0.3. Positive samples in ELISA were tested by platelet aggregation on the CHRONO-LOG® platelet aggregometer (Chrono-log Corporation, PA, USA). Normal blood group O donor platelets were incubated with PF4/polyanion-positive sera in the presence of low-dose heparin (unfractionated heparin 1.0 IU/mL), high-dose heparin (unfractionated heparin 100 IU/mL), or saline buffer. A previously confirmed HIT serum was used as a positive control and normal pooled plasma as a negative control.

Rats were anesthetized with chloral hydrate (300 mg/kg). Blood was drawn from the abdominal aortas to determine. The blood was anticoagulated with heparin (20 U/mL). All platelet aggregation studies were performed using a Chrono-log platelet aggregometer (Chrono-log Co., USA). Single-use cuvettes containing a Teflon-coated stirrer (800 rpm) were filled with pre-warmed 500 μL physiologic saline and 500 μL whole blood. After 10 min of incubation, tests were initiated by adding ADP (10 μM) and AA (0.5 mM). Aggregation was recorded for 6 min.

Platelet aggregation assay was performed as previously described (Kim et al., 2017 (link)). Washed platelets in modified HEPES-Tyrode buffer were pre-incubated with 0.01% DMSO or various concentrations of HRT (30, 50, and 100 μg/ml) for 10 min for at 37°C and then stimulated with numerous agonists. Platelet aggregation was monitored in a platelet aggregometer (Chronolog, Corp., Havertown, PA, United States) at 37°C with stirring (1,000 rpm).

Murine blood was collected in tubes containing 0.2 μg/ml enoxaparin. Platelet-rich plasma (PRP) was obtained by two centrifugation cycles of 300 × g for 7 min at RT. Aggregation following ADP stimulation was analyzed in PRP. All other measurements were carried out with washed platelets. For this, PRP was pelleted at 700 × g in the presence of prostacyclin (PGI₂) (0.1 μg/ml) and apyrase (0.02 U/ml). The platelet pellet was washed twice in modified Tyrode-HEPES buffer (134 mM NaCl, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 5 mM HEPES, 1 mM MgCl₂, 5 mM glucose, 0.35% BSA, pH 7.4) containing PGI₂ and apyrase. Platelet suspensions (a total of 6 × 10⁷ platelets) in Tyrode-HEPES buffer containing 2 mM CaCl₂ were stimulated with the indicated agonists and light transmission was recorded on a Chronolog platelet aggregometer. The number of animals used for this study was as follows: collagen induced aggregation, WT n = 11, Sirt3-/- n = 9. ADP induced aggregation, WT n = 10, Sirt3-/- n = 7, thrombin induced aggregation, WT n = 7, Sirt3-/- n = 7.

Blood samples were collected from the retro-orbital plexus using a glass capillary tube containing 3.8% sodium citrate solution. Platelet rich plasma (PRP) was prepared from whole blood20 (link). Platelet counts were determined using a Hemavet 950 (Drew Scientific, Waterbury, CT) and adjusted to 300,000/μL with. Aggregation was initiated in PRP using collagen (3 μg/ml) and monitored by light transmittance using a platelet aggregometer (Chrono-log Corp., Havertown, PA)20 (link)22 (link).