Mepacrine

Five microliters whole blood was diluted 1:10 (v:v) in HEPES buffered saline (10 mM HEPES, 150 mM NaCl, 1 mM MgSO₄, 5 mM KCl, pH 7.4), which contained 100 µM mepacrine (Sigma Aldrich) and 15 µg/mL in‐house developed PE‐conjugated anti‐GP1b nanobodies (clone 17), with or without 25 µM protease activating receptor (PAR)‐1 activating peptide SFLLRN (PAR1‐AP; Bachem). Whole blood was incubated for 10 minutes at 37°C, after which samples were fixed with 0.148% formaldehyde, 137 mM NaCl, 2.7 mM KCl, 1.12 mM NaH₂PO₄, 10.2 mM Na₂HPO₄, 1.15 mM KH₂PO₄, 4 mM EDTA, pH 6.8 for 20 minutes at room temperature and analyzed on a BD FACSCanto II (BD Biosciences). The flow cytometer was calibrated every week to maintain stable fluorescent intensity. Platelets were identified based on forward and sideward scatter, as well as GPIbα‐expression. mepacrine fluorescence was normalized on the median fluorescence of the healthy control population and was expressed as normalized median fluorescent intensity. The coefficient of variation for mepacrine uptake was 2.3%. Flow cytometric analysis was reproducible within 6 hours after blood collection (data not shown).

Mepacrine (Sigma-Aldrich, USA) is an Acridine
derivative whose emission wavelength is within the range
of FITC. PLTs labeling, 20 μl of 20 mg/mL Mepacrine
was added to the 5×10⁷ PLTs to 30 μl PBS solution
and incubated for 30 minutes at ambient temperature.
Afterward, the PLTs were washed three times with PBS
by centrifugation at 1200 g for 15 minutes. Ultimately,
PLTs were prepared for adding to cultured HepG2 cells.

AV-shunt assay was performed in rats to investigate the blood compatibility of grafts. Rats were anesthetized with isoflurane gas throughout the procedure. Heparin (100 Units/kg) was administered as an anticoagulant agent. The grafts (PCL, PCL-epECM/H, PCL-epECM/H-IL-4)) were cut into a 1.5 cm and sterilized (n = 3). Then the grafts were connected to the abdominal aorta at one end and to the abdominal vein at the other end (As shown in Fig. 2A), using 24-G indwelling needles to establish extracorporeal circulation, and three grafts were connected in one circulation. After circulation for 1.5 h, the circuit was perfused with physiological saline. Samples were equally divided into two parts. One was fixed in 2.5% glutaraldehyde overnight and dehydrated by a gradient of ethanol for SEM assay, and the other was stained with mepacrine (Sigma, St Louis, USA) for 30 min at 37 °C and then observed under LSCM (Leica TCS SP8, Germany).

Washed platelets (10⁸/ml) were diluted 1:40 in Hanks' balanced salt solution. Ceefourin or vehicle was added to platelets to a final concentration of 10 μmol/l (30 min, 37°C). Platelets were then stimulated with thrombin (0, 0.05 or 0.25 U/ml, 5 min, 37°C). Mepacrine (10 μmol/l, Sigma-Aldrich) was added (30 min, 37°C) to stain dense granules. Samples, diluted 1:2 in Hanks' balanced salt solution were run on the flow cytometer. In these experiments, 10,000 platelets were collected off of forward and side scatter properties.