Thrombogenicity of vascular smooth muscle cells under flow conditions. (A) Representative images of platelet thrombus formation (brightfield), VSMC nuclei (Hoechst33342, blue) and AF647-fibrin formation (red) on calcified synthetic VSMC under blood flow. (B) Quantification of fluorescent fibrin formation indicates an increase in the presence of calcification, and a reduction in the presence of either CTI or ASIS. (C) Platelet deposition on calcified VSMC was reduced in the presence of ASIS. Scale bar = 20 µm. (D) Quantification of fluorescent fibrin indicates an increase of fibrin generation in VSMCs treated with warfarin and calcium (P = 0.0337). (B) Quantification of platelet deposition of synthetic VSMCs under flow. Cells treated with warfarin and calcium show an increase in platelet deposition (P = 0.0059). All experiments were done with at least three biological replicates, with duplicate technical replicates. Data were analysed using Mann–Whitney U test and Dunnett’s multiple comparison test.