Stirring enhances latent TGF-β1 activation. (A) Platelet releasate or (B) serum (200 μL) was placed in a glass aggregometer cuvette containing a metal stir bar and incubated at 37°C with and without stirring (1200 rpm) for the indicated time periods. Active TGF-β1 was measured directly in the ELISA, whereas total TGF-β1 (latent + active) was measured after activating latent TGF-β1 by treatment with acid. Stirring dramatically increased the amount of active TGF-β1 in both platelet releasates (3.5 ± 0.5 ng/mL, P < .001; n = 3) and sera (2.5 ± 0.6 ng/mL, P < .001; n = 4). Increasing the stirring speed enhanced latent TGF-β1 activation in both platelet releasates (C) and sera (D) when measured after 2 hours. Error bars represent SD. (E) MLECs were stimulated for 2 hours in serum-free medium with stirred or unstirred platelet releasates in the presence or absence of anti–TGF-β1 neutralizing antibody (α–TGF-β1; 2 μg/mL). Smad2 phosphorylation was detected by immunoblotting with a mAb specific for phosphorylated Smad2 (P-Smad2), and total Smad2 was detected with a polyclonal antibody that reacts with both phosphorylated and unphosphorylated Smad2. Exposure to platelet releasates stirred at 1200 rpm increased the amount of phosphorylated Smad2 but not total Smad2.