E selectin

Physical exams and routine laboratory tests were performed on the suspected patients. Serum ADAMTS13 activity was measured by the Fluorescence Resonance Energy Transfer (FRET) assay (United States Patent No.7270976) [17] (link). Serum anti-ADAMTS13 IgG antibodies (Sekisui Diagnostics, USA), vWF (von Willebrand factor) (Sunbiote, Shanghai, China), thrombomodulin (Diaclone Research, Besancon, France), E-selectin (R&D Systems, Minneapolis, Minnesota, USA) and soluble vascular cellular adhesion molecule-1 (sVCAM-1, VCAM) (R&D Systems, Minneapolis, Minnesota, USA) were measured using enzyme linked immunosorbent assays. The concentrations of endothelial cells in the circulation were sorted using a magnetic microbead sorting system (MACS, Miltenyi Biotec, Germany), according to the manufacturer’s instructions [18] (link).

Recombinant human L-selectin (10 ng/ml), P-selectin (10 ng/ml) or E-selectin (30 ng/ml) Fc chimeras (R&D Systems) were mixed with increasing concentrations of heparin or heparin derivatives in PBS before addition to 96-well ELISA plates precoated with BSA-Sialyl Lewis^x (110 ng/well, R&D Systems) for 45 minutes at room temperature. The plates were washed three times with 200 μl of Quantikine wash buffer (R&D Systems) and the bound selectins were detected using an HRP-anti Fc antibody and Substrate Reagent Pack (R&D Systems) according to the manufacturer's instructions.

TC, HDL-C, TG, and Fasting plasma glucose concentration (FPG) were measured by the autoanalyser (HITACHI, Tokyo, Japan) with commercially available kits (Randox Laboratories, Crumlin, UK). Serum sPLA2-IIa protein was determined using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI, USA). Serum sPLA2 activity was measured by a selective fluorometric assay (Cayman Chemicals) by using diheptanoyl thio-phosphocholine as fluorescent substrate. We used a solution of 0.1U bee venom PLA2 as a positive control to hydrolyze fluorescent substrate completely. The hydrolysis of substrate in the absence of serum sample was used as negative control. The intra-assay coefficient of variation was 4.2%. ELISAs were used to determine VCAM-1, ICAM-1, E-selectin, and P-selectin levels (R & D Systems, Minneapolis, MNUSA). All measurements were performed according to the manufacturers’ instructions. Each sample was tested in duplicate, and the means of the duplicates were used for the statistical analysis.

Peripheral blood was collected via cardiac puncture 30 min after APE challenge using a 1-ml heparinized syringe. A total of 100 μl aliquots were immediately sampled and analyzed using a veterinary Hematrue hematology bench top analyzer (Heska Corporation Loveland, Colorado, U.S.A.). The number of platelets was measured. Commercial ELISA kits were used to determine CTS (CUSABIO, Cedarlane, Burlington, Canada), P-selectin, E-selectin, MCP-1, (R&D Systems, Minneapolis, MN, U.S.A.), MPO and VWF levels (Abcam, Shanghai, China).

For adhesion of neutrophils to immobilized E-selectin in combine with ICAM-1 under flow, a laminar flow chamber slide (μ-Slide I^0.8 Luer, ibidi, Martinsried, Germany) was filled with protein G (25 μg/mL; Invitrogen, USA) overnight. After washing with phosphate-buffered saline, the slide was filled with E-selectin (5 μg/mL; R&D system, USA) and ICAM-1 (5 μg/mL; R&D system, USA) for 2 hours and blocked with 1% bovine serum albumin (Sigma-Aldrich, USA) for 1 hour. Freshly isolated human neutrophils (5x10⁵ cells) were preincubated without or with IND02 at RT for 1 hour, followed by perfusion at a constant flow rate of 1 dynes/cm². The number of adherent cells (stationary over 5 seconds) was determined after 3 minutes of perfusion. The shear flow was generated by using the perfusion loop system and an air pressure pump (ibidi, Martinsried, Germany), and the images were captured by the microscope. The number of adherent cells was calculated in 10–15 random fields in a single experiment and the statistical test was calculated from 3 experiments.

To analyze selectin-mediated neutrophil rolling of patient samples, we used a previously described whole-blood-perfused human microfluidic chamber system [14 (link)]. In this model, it has been shown that above 90% of all rolling cells are polymorphonuclear neutrophils (PMNs) [15 (link)]. Briefly, the chambers were coated with E-selectin (2 μg/mL) (R&D Systems, Minneapolis, MN, USA), P-selectin (15 μg/mL) (R&D Systems, Minneapolis, MN, USA), E-selectin/ICAM-1 (2/6 μg/mL) (R&D Systems, Minneapolis, MN, USA) or P-selectin/ICAM-1 (15/6 μg/mL) (R&D Systems, Minneapolis, MN, USA) for 2 h. The chambers were then blocked with 1% casein (Fisher Scientific, Waltham, MA, USA) for 1 h and subsequently perfused with heparinized whole blood at a constant shear stress of 5–6 dynes/cm². For analysis of chemokine-induced arrest of rolling PMNs, flow chambers were coated with P-selectin (15 μg/mL), ICAM-1 (6 μg/mL) and Interleukin-8 (IL-8) (50 μg/mL, Peprotech, Rocky Hill, NJ, USA) or P-selectin and ICAM-1 as control. After two minutes of perfusion with heparinized whole blood at a shear stress of 5–6 dynes/cm², rolling and adhering cells were counted per field of view.

The VWF (Abcam), P-selectin and E-selectin (R&D Systems) in endothelial cell supernatant were determined by ELISA, following the manufacturer’s instructions strictly.