LPS and Stx1-treated ASTs release factors that increase platelet activation and adhesion to HUVECd.

<p>Platelets or HUVECd were stimulated with CM-Control, CM−LPS, CM−Stx1 or CM−LPS+Stx1. After 1.5 h density expression of P-selectin and the % of activated platelets was determined by FACS (<b>A</b> and <b>B</b>). To determine platelets adhesion to CM-stimulated HUVECd (<b>C</b>), platelets were seeded on stimulated HUVECd (CM-Control/Pt, CM−LPS/Pt, CM−Stx1/Pt, CM−LPS+Stx1/Pt). Free platelets were washed away after 1.5 h and the platelet acid phosphatase (AcP) percentage of enzymatic activity (% EA) was determined by spectrophotometry. As control of maximal platelet adhesion, total platelets on untreated HUVECd were left unwashed (Total Pt). <b>A</b>: P-selectin relative median fluorescence (RF) (Mean ± SEM, n = 6). *p<0.05 (CM−LPS+Stx1 vs. CM-Control); ^#p<0.05 (CM−LPS+Stx1 vs. CM−Stx1 or CM−LPS) (Upper graph). % of activated platelets was defined by high P-selectin expressing subpopulation. *p<0.05 (CM−LPS+Stx1 vs. CM-Control) (Lower graph). <b>B</b>: Representative FACS analysis of an independent experiment showing histogram and scatter plots. <b>C</b>: AcP (% EA) respect to % EA of Total Pt (Mean ± SEM, n = 6). *p<0.05 (CM−LPS+Stx1/Pt vs. CM-Control/Pt); ^#p<0.05 (CM−LPS+Stx1/Pt vs. CM−LPS/Pt, n = 6).