Platelet TGF-β1 contributes to AS progression. (A) ELISA and immunoblot for total platelet TGF-β1 (left and middle panel) in TGF-β1^platelet-KO-LDLR mice (n = 5) and TGF-β1^flox-LDLR controls (n = 5). Immunoblot with anti-TGF-β1 antibody showing 25 kD TGF-β1 bands (lanes 1, 2, 3, and 4 represent platelet releasates from TGF-β1^flox-LDLR controls, and lanes 5, 6, 7, and 8 represent platelet releasates from TGF-β1^platelet-KO-LDLR mice). Equal number of platelets (5 × 10⁸) were used to prepare platelet releasates from each mouse. Total plasma TGF-β1 (right panel) in TGF-β1^platelet-KO-LDLR mice (n = 45) and TGF-β1^flox-LDLR controls (n = 14). (B) AS parameters: fractional valve opening and WSS in TGF-β1^platelet-KO- LDLR mice (n = 15) and TGF-β1^flox-LDLR controls (n = 7) at 6 months on HFD, as measured by echocardiography. (C) Representative images (left panel) of α-1 type I collagen stained aortic valves from TGF-β1^platelet-KO- LDLR mice and TGF-β1^flox-LDLR control mice after 6 months on HFD. Quantification of total valve area (middle panel) and collagen-positive area (right panel) in aortic valves of TGF-β1^platelet-KO- LDLR mice (n = 4) and TGF-β1^flox-LDLR controls (n = 4) at 6 months on HFD from immunohistochemical staining images. (D) Whole-mount confocal (maximum intensity projection) images showing activated platelets adjacent to valvular cells in a TGF-β1^platelet-KO-LDLR mouse and a TGF-β1^flox-LDLR control. Images also show intracellular p-Smad2 localization in the valvular cells. Whole-mount staining done for CD41 (green), CD62P (gray), p-Smad2 (red), and DAPI (blue). Quantification of nuclear or cytoplasmic p-Smad2 in aortic valves of TGF-β1^platelet-KO-LDLR and littermate TGF-β1^flox-LDLR control mice at 6 months on HFD (right panel). Two-tailed unpaired Student t test with Welch’s correction was used to compare 2 groups with different sample sizes. Data represented as mean ± SEM throughout.