Thrombin down-regulates the serine-phosphorylated endoglin through PAR1 in ECs. (A) HAECs were serum-started for 2 hours and then stimulated with thrombin (3 U/mL) for the indicated time periods. (B) HAECs were left untreated (–) or preincubated with hirudin (6 U/mL), a monoclonal PAR1 antibody (ATAP2, 10 μg/mL), or an antibody against TGF-β1/2/3 (10 μg/mL) for 30 minutes, then stimulated with thrombin (3 U/mL) for 30 minutes. (C) HAECs were stimulated with thrombin (3 U/mL), thrombin receptor-activating peptide (TRAP, 40 μM), peptide agonists (100 μM) for human PAR1 (PAR1-AP), human PAR2 (PAR2-AP), or PAR3 (PAR3-AP). (D) HAECs were stimulated for 30 minutes with thrombin (3 U/mL), tumor necrosis factor (TNF; 10 ng/mL), interleukin 8 (IL-8; 2 nM), angiotensin II (Ang II; 100 nM), or lysophosphatidic acid (LPA; 10 μM). Lysates were subjected to immunoblotting with the antibody against the serine-phosphorylated endoglin as described in Figures 1 and 2. The serine phosphorylation of endoglin was shown as percentage relative to untreated control cells by densitometric analysis. The same blot was stripped and reprobed with an endoglin antibody (SN6h) to show the equal loading. Results shown are representative immunoblots of 3 independent experiments.