Animal blood counter

The blood samples were collected from the hearts each of 71 male BALB/c mice (21.1 ± 2.4 g), at five weeks old, after being anesthetized with a mixture of isoflurane and medical air (1.5–3% v/v), and were drawn into 3.13% trisodium citrate in a volume ratio of 9:1 at the end of the experiment. The blood cells were counted 3, 15, and 60 min following injection into the right femoral vein of the vehicle (phosphate buffered saline, 1 mL/kg), UFH (150 U/kg, 1 mL/kg), PS (1.5 mg/kg, 1 mL/kg), or both (Figure 8a), using the Animal Blood Counter (ABC Vet, Horiba ABX Sp. z o.o., Warsaw, Poland). At the same time, the points platelet aggregation was measured after the incubation of the whole blood (500 µL) and 0.9% NaCl solution (500 µL) for 20 min at 25 °C, and then for 15 min at 37 °C. The changes in impedance were registered for 6 min after the collagen addition (5 µg/mL) using a Chrono-log aggregometer (Chrono-log Corp., Havertown, PA, USA). The aggregation curve was described by the maximal extension, the slope of the platelet aggregation, lag phase, and the area under the curve.

Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fg) levels (Clauss method) were determined according to the kit manufacturer’s instructions by using the Coag-Chrom 3003 apparatus (Bio-ksel, Poland). For fibrin generation, we used a method described previously (He 2001 (link)) and modified by us (Buczko 2003 (link)). Briefly, fibrin generation curves were created by recalcinating rat plasma samples directly in microplate wells by using CaCl₂ (36 mM) dissolved in Tris buffer (66 mM Tris, 130 mM NaCl, pH 7.4) at 37 °C. Increases in optical density in the wells (as a result of fibrin generation) were measured with the microplate reader (Biotec EL808, USA) at 1-min intervals for 9 min and expressed as the area under the curve (AUC). Time-point analysis results were calculated as percentages of basal optical density.
Blood cell count was assessed with an Animal Blood Counter (ABC Vet, Horiba, Germany) in blood collected from the left ventricles of 2K1C hypertensive rats after thrombus removal (ex vivo) or untreated 2K1C rats (in vitro experiments).