Abciximab reopro

Purified anti-human TLR2, TLR4 blocking antibodies and their matching isotype controls were obtained from BioLegend, Inc., USA. Bay 61–3606 and Phorbol 12-myristate 13-acetate (PMA), ticagrelor and Cell Detection ELISA PLUS kit were from Sigma-Aldrich, Australia. ML-171, aspirin, RGDS, cathepsin G inhibitor I, neutrophil elastase inhibitor (1-(3-methylbenzoyl)-1H-indazole-3-carbonitrile), myeloperoxidase inhibitor 1 (4-Aminobenzoic acid hydrazide), losartan were obtained from Cayman Chemical, USA. Abciximab (ReoPro) and low molecular weight heparin (Clexane enoxaparin sodium) were from Eli Lilly and Sanofi Aventis Australia Pty Ltd., respectively. DNAse I solution was purchased from STEMCELL Technologies Australia Pty Ltd. Collagen and thrombin were from Chrono-log Corporation, USA. Human PMN Elastase ELISA kit was obtained from Abcam Biotechnology, Cambridge, UK.

To visualize distinctly fibrin and platelets in a fluorescent confocal microscope, Alexa-Fluor 594-labeled human fibrinogen (Molecular Probes, Grand Island, NY, 40 µg/ml final concentration) and calcein green, AM (Molecular Probes, 0.2 µg/ml final) were added to PRP samples and incubated for 10 min at 37 °C before initiation of clotting. Clotting was induced by adding CaCl₂ (40 mM final concentration) and thrombin (0.75 or 1 U/ml final concentration) to PRP containing the fluorescent probes. Thirty microliters of samples was quickly transferred onto 35 × 10 mm PELCO clear wall glass bottom cell culture dishes in the environmental chamber of the confocal microscope for the time course z-stack imaging. In some experiments, the glass was pre-coated with 4% (v/v) Triton X-100 in PBS to prevent attachment of fibrin and allow for unconstrained clot contraction. In inhibitory experiments abciximab (ReoPro; Eli Lilly, Indianapolis, IN, USA) was added to PRP at 100 µg/ml or blebbistatin (Sigma-Aldrich, St. Louis, MO, USA) was used at 300 µM (both final concentrations) prior to the fluorescent probes and incubated for 15 min at 37 °C.