Figure 3. Statin treatment reduces platelet recruitment to the endothelial surface.

(a) HUVEC monolayers treated with vehicle (DMSO), 0.5 or 2.5 μmol/L simvastatin for 24 h were perfused with washed platelets from healthy non-medicated volunteers in the presence of histamine (100 μmol/L) to stimulate vWF secretion. Left, video-microscopy frames taken during perfusion show platelet-decorated strings (highlighted by dotted boxes). Right, samples were fixed and immuno-stained to visualize vWF strings and platelets (CD41). Scale bar: 50 μm. (b) Analysis of the length of platelet-decorated vWF strings, pooled from two independent experiments, in duplicate for each treatment. Median and interquartile ranges are shown. Number of strings measured: DMSO, 271; simvastatin 0.5 μmol/L, 217; simvastatin 2.5 μmol/L, 278. ****P < 0.0001 (Mann-Whitney test). (c) Statin treatment primarily affected the longest (>100 μm) strings. Error bars represent the range of variation for the two experiments. (d) Integrin β3, which in endothelial cell is present in complex with integrin αν and functions as an anchor for vWF strings, was quantified from western blots by densitometry following incubation for 24 h with 0.5, 2.5 and 12.5 μmol/L statins (indicated by the triangle symbols). n = 3; mean ± SEM. *P < 0.05 or lower (not shown for simplicity) for all treatments compared to DMSO control (Student’s t-test). γ-Adaptin (Ad-γ) was used as a loading control. (e) Cell-free WPBs prepared from cells treated for 24 h as indicated were detergent-permeabilised and the resulting vWF filaments immuno-stained and imaged (insets show magnifications of the dashed boxes). Scale bar: 25 μm. (f) vWF multimerization in cell-free WPBs was analysed by agarose gel electrophoresis. Highly multimerized vWF is indicated (high-molecular weight, HMW). The multimeric patterns of plasma vWF (p-vWF) untreated or reduced (dithiothreitol, DTT) and alkylated (N-ethylmaleimide, NEM) to generate monomers are shown for comparison.