Thiol-disulfide exchange contributes to latent TGF-β1 activation. (A) Platelet releasates were stirred in the presence or absence of the indicated reagents for 120 minutes at 37°C and TGF-β1 activity was measured by ELISA. The concentrations used were as follows: N-ethylmaleimide (NEM) 1 mM, iodoacetamide (IODO) 1 mM, N-acetyl cysteine (NAC) 1 mM, MPB 100 μM, and glutathione (GSH) 200 μM. Stirring increased active TGF-β1 from approximately 0.2 to 5 ng/mL; total TGF-β1 was approximately 56 ng/mL. Data are expressed as percentage of the control value of active TGF-β1 in the absence of a thiol-reactive reagent (CON). Error bars represent SD. (B) MPB (100 μM) labeled a number of proteins in platelet releasates and the intensity of labeling increased when samples were stirred for 2 hours after adding the MPB. One of the labeled proteins was TGF-β1 itself (Mr, ∼ 25 kD) as judged by immunoprecipitation (middle panel) with a combination of anti-LAP and anti–TGF-β1 antibodies. (C) MPB labeling of the approximately 25-kD band corresponding to the migration of TGF-β1 (arrow), as well as some other proteins, was dramatically reduced when the MPB was added after 2 hours of stirring. Pretreatment with NEM (1 mM) prevented MPB labeling. (D) Densitometric quantification of biotin-MPB incorporation into the approximately 25-kD protein () in panels B and C (left). Error bars represent SD. (E) Platelet releasates were subjected to shear at 1800 s⁻¹ for the indicated time periods and then labeled with MPB (100 μM) for 2 hours at 37°C. MPB-labeled proteins in platelet releasates were detected with streptavidin-HRP. The sample sheared for 120 minutes from the same gel was also immunoblotted separately to identify TGF-β1. The vertical line separates the membranes reacted with streptavidin-HRP from the membrane reacted with the antibody to TGF-β1. (F) Thrombin-stimulated platelet releasates were incubated with the indicated thiol-reactive compounds followed by labeling with biotin-MPB (100 μM) for 2 hours. MPB-labeled proteins were detected with streptavidin-HRP (left panel) and immunoblotted to identify TGF-β1 (right panel). Vertical lines indicate deletion of intermediate lanes from the same gel.