Angiopoietin 1

ChIP assay was performed using EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, upstate) according to the manufacturer’s instructions. Briefly, cells cultured under the indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 ml of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from soluble chromatin preparations were approximately 400–800 bp in length. Immunoprecipitation (IP) was carried out overnight with Angiopoietin 1 (Abcam, ab102015, USA), SFTPC (Abclonal, A11764, USA), FGF10 (Abclonal, A1201, USA), or normal mouse IgG as a negative control. Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed with 2 μl DNA samples with the following primers: SPC: forward, 5′-AAGAGATCCCTCTCCCAGCA-3′; reverse, 5′-TGGGGTTTGCCGCCATC-3′; Ang-1: forward, 5′-AACAATTTCTCCTTTGATAGGTGGT-3′; reverse, 5′-GCCTTTCCGGATATCATGACC-3′;
FGF-10: forward, 5′-TCGCCATAAAGTGCGTTTGC-3′; reverse, 5′-GCCCTTCACTGAATCATGCG-3′.

At the end of the incubation of platelets with or without the agonists and after taking platelet samples for analysis in flow cytometry and microscopy, supernatants of the remaining platelets were collected after two centrifugation steps: 12 min, 1200 g at RT and 3 min, 13000 g at RT. Aliquots of supernatants were kept frozen at −80°C until analysis. The following ELISAs were used in accordance with the manufacturer’s instructions: serotonin (GenWay, San Diego, CA, USA), platelet factor 4 (PF4) (RayBiotech, Norcross, GA, USA), TGF-β1 (Enzo Life Science, Villeurbanne, France) and angiopoietin-1 (Abcam, Paris, France).