Low molecular weight heparin

Tau protein was diluted to 40 µM in 100 mM sodium acetate pH 7 with 2 mM DTT and heated for 10 min at 55 °C. Low molecular weight heparin (Sigma) was added to 40 µM. The mixture was distributed to a 96-well plate and shaken in a tabletop Thermomixer (Eppendorf) at 37 °C and 1,000 rpm for 7 days. Insoluble Tau was pelleted by ultracentrifugation at 100,000 g for 30 min at 4 °C. The pellet was washed twice, with resuspension by vigorous pipetting in 100 mM sodium acetate pH 7. Aggregate formation was monitored by thioflavin assay and electron microscopy.

After a 24 h serum deprivation, 20 × 10³EPC per well were seeded on a fibronectin (100 μg/mL, BD Biosciences Pharmingen, Le Pont de Claix, France) pre-coated 8-well Labtek and incubated for 2 h at 37 °C with 3 nM RANTES to assess their spreading. In a parallel experiment, EPC were pre-incubated for 2 h at 37 °C with anti-CCR5, anti-CD44, or anti- SDC-4 antibodies or their isotypes (5 µg/mL). Alternatively, cells were preincubated with beta-D-xyloside (1 mM) for 72 h, or RANTES was pre-incubated with low molecular weight heparin (10 µg/mL, Sigma-Aldrich) for 2 h, as previously described^{12 (link)}. Cells were permeabilized in 0.05% Triton X-100 (Sigma-Aldrich), fixed with PFA (1%) then stained with AlexaFluor 568 phalloidin (Molecular Probes, Invitrogen) and filamentous actin was observed with a fluorescence microscope (Zeiss, AXIOPHOT, N°/MicMac, France S.A). Ten fields of stained cells were photographed for each treatment. Cell areas expressed in square inches were evaluated on 30 cells by treatment with the Scion Imager software (Scion Image Software and National Institutes of Health, Release Beta 3b Software).

Bioactivity of the Fcp5, peptide p5, and Fc2a control was assessed using a EuLISA. The wells of a 96-well polystyrene microplate (Corning, Corning, NY, USA) were coated either with poly-l-lysine (Sigma-Aldrich) followed by low molecular weight heparin (Sigma-Aldrich), amyloid-like fibrils (0.83 µM), or monomeric forms of rVλ6Wil, Aβ(1–40) or human IAPP(Ile26Pro). Wells coated with fibrils or monomeric proteins were incubated overnight at 37 or 4°C, respectively. The wells were then treated with 200 µL of blocking buffer (PBS containing 1% bovine serum albumin; BSA) for 1 h at room temperature before washing with PBS and addition of the appropriate concentration of Fcp5, biotinylated-p5, or Fc2a in PBS with 1% (w/v) BSA and 0.05% (v/v) tween 20. Following a wash step, the bound Fcp5 and Fc2a were detected by addition of biotinylated goat anti-mouse IgG (Sigma-Aldrich). After washing with PBS/tween, wells were incubated with 100 µL of europium–streptavidin (Perkin Elmer, Waltham, MA, USA) and washed, followed by addition of 100 µL enhancement solution (Perkin Elmer) before measurement of time-resolved fluorescence emission using a Wallac Victor 3 plate reader (Perkin Elmer).

Chemicals. Reagents of TT, PT and APTT, CaCl₂ and coagulation control plasma were produced in TECO (Germany). Tris–HCl was purchased from Amresco (USA). Reference anticoagulant drug (heparin, HEP; low molecular weight heparin, LMWH) and DMSO were produced in Sigma-Aldrich (USA).
The measurements were taken using the MC-4000 Optic coagulometer (Germany). Prior to the detection, coagulation control plasma were pre-incubated with the examined compounds (15 min, 37 °C) at the final concentrations of 200 μM.