Lumi aggregometer model 560 ca

Platelet aggregation was measured in a two channel aggregometer (Chronolog Lumi-Aggregometer model 560-Ca, Havertown, PA, USA) at 37°C with stirring (1 000 rpm). Platelet aggregation assays were carried out using ADP (10 μM) and in some experiments the platelets were pre-incubated for 3 min with inhibitors of SFKs (PP2, 10 μM), PI3K (wortmannin, 100 nM; and LY294002, 10 μM), AKT (API-1, 20 μM), sGC (ODQ, 25 μM), PKG (Rp-8-Br-PET-cGMPS, 25 μM) or PKC (GF109203X, 10 μM). The concentrations of these inhibitors were used accordingly to previously published studies [20 (link)–24 (link)]. The same volume of DMSO (1%) was used as vehicle control for all the inhibitors, except for Rp-8-Br-PET-cGMPS that was dissolved in saline. Platelet aggregation data were obtained at an end-point of 5 min after ADP addition.

For the method used for blood sample collection and platelet aggregation refer to the work in [42 (link)]. The blood samples were collected up to the mark in sky blue capped vacutainer containing trisodium citrate, thus assuring 1:9 ratio of blood is to anticoagulant. The platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared using Tyroid buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO₃, 5.5 mM glucose, 1 mM MgCl₂, 0.3 mM NaHPO₄, pH 7.4) and centrifuged (2000 rpm, 7 min). Platelet aggregation was measured in a two channel aggregometer (Chrono-log Lumi-Aggregometer model 560-Ca, Havertown, PA, USA) at 37 °C with stirring (1000 rpm). Washed platelets were pre-incubated with various concentrations of either rice extract and cq-9 for 1 min in the presence of 1 mM calcium chloride (CaCl₂), followed by stimulation with various agonists (Collagen, ADP, or thrombin) for 6 min with continuous stirring at 37 °C.