AV leaflets were dissected from fresh porcine hearts acquired from commercial abattoirs and assigned into one of three age groups: YNG (6 weeks old), ADT (6 month old), or OLD (2 years old). Immunostains for numerous thrombotic and anti-thrombotic markers (Table 1) were performed on the AV leaflets of different ages, and the proportion of the tissue stained was quantified. The same hemostatic markers were also assessed using qRT-PCR and immunocytochemistry on porcine aortic valve endothelial cell (PAVEC) cultures of the three age groups, human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell (PPAEC) cultures. A sandwich ELISA was performed to quantify levels of VWF protein release and cleavage from histamine stimulated PAVECs, HUVECs, and PPAECs. To assess the effects of VEC-released VWF on calcific nodule formation in vitro, conditioned culture mediums consisting of 3% (v/v) PAVEC stimulation medium supernatant (via histamine stimulation) from the three age groups (YNG, ADT, OLD) were mixed with low serum VIC culture medium, and exposed to porcine aortic valve interstitial cells PAVICs) for 10 days. For study controls, PAVICs were also cultured in VIC low serum medium only, low serum VIC medium with 3% fresh PAVEC stimulation medium and no histamine, and low serum PAVIC medium with 3% fresh PAVEC stimulation medium with 8.4 nM histamine. PAVICs were stained with Alizarin Red S to dye calcified nodules red, and the number nodules and nodule sizes were measured. A full description of the materials and methods is available in the online-only Data Supplement.