Ca 510

Blood plasma obtained from 1.5 ml blood (3,820 r/min, 20°C, 15 min) was put into a Sysmex CA-510 fully automatic blood coagulation analyzer to determine activated partial thromboplastin time (APTT), prothrombin time (PT), and plasma viscosity (PV). Blood (0.9 ml) was put directly into a fully automatic self-cleaning hemorheology analyzer (LBY-N6B) to measure the erythrocyte aggregation index (EAI) and the red corpuscle electrophoresis index (RCEI), as well as whole-blood viscosity (WBV) at 5/s, 30/s, 50/s, 150/s, and 200/s shear rates. Blood (0.9 ml) was centrifuged (3,820 r/min, 20°C, 15 min) for hematocrit detection by an automatic dynamic blood sedimentation tester (LBY-XC40). Platelet-rich plasma (PRP) was separated from 3.0 ml blood sample (500 r/min, 20°C, 10 min), then the remaining without PRP was used to obtain platelet-poor plasma (PPP) (3,000 r/min, 20°C, another 10 min). Platelet counts were kept approximate 200×10⁹/L in each PRP sample. Platelet aggregation was induced by 5 µl adenosine diphosphate (ADP). PRP (300 µl) and PPP (300 µl) were used to detect maximum platelet aggregation rate (MPAR) on a platelet aggregation analyzer (LBY-NJ4). All the tests were completed in the drug non-clinical evaluation research center of Guangzhou General Pharmaceutical Research Institute.

Blood samples were obtained on days −1, 0, 1, 3 and 5 from the femoral vein, and the data for blood count (XN‐2000, Sysmex Inc.), blood chemistry (7180 Clinical Analyzer, Hitachi High‐Tech Technologies) and coagulation tests (CA‐510, Sysmex Inc.) were acquired. Serum samples were stored at −80°C before cytokine analysis and examined for interleukin (IL)‐1β, IL‐2, IL‐6, interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α using Bio‐Plex 200 (Bio‐Rad Laboratories Inc.) and a Non‐Human Primate Cytokine Magnetic Beads panel (EMD Millipore).

The rats were anesthetized with pentobarbital (30 mg/kg, i.p.). Blood samples were collected in 3.2% sodium citrate tubes (Terumo Corporation, Tokyo, Japan) by cardiac puncture at the following zeitgeber times (ZTs): 0, 4, 8, 12, 16, and 20 hours, with ZT0 defined as lights on and ZT12 as lights off (8:45 a.m. and 8:45 p.m., respectively). The plasma samples were centrifuged (1,500 g) at 4°C for 10 minutes and then stored at −80°C until assayed. The plasma activities of FII, FV, and FX were determined by measuring the clotting time of a sample using the appropriate reagents (factor-deficient plasma and the HemosIL RecombiPlasTin kit; IL Japan, Tokyo, Japan) and an automated coagulation analyzer (ACL TOP; IL Japan). The activity levels were expressed as percentages (relative to normal human plasma). PT and APTT were measured using a coagulation analyzer (CA-510; SYSMEX Corporation, Kobe, Japan) according to the manufacturer's instructions.