TGF-β1 from mice expressing an LAP (C33S) mutation is present in small latent complexes rather than large latent complexes and cannot be activated by stirring. (A) Platelet releasates from wild-type (WT) or mutant (C33S) mice were either unstirred or stirred at 1200 rpm for 2 hours at 37°C, and active and total TGF-β1 were measured by ELISA (WT increased from 0.2% to 13%; mutant was 0.2% both before and after stirring; P < .005; n = 3). Platelet releasates were also immunoblotted with anti-LAP antibody. LAP migrated at an Mr compatible with large latent complex (Mr 270 kD) in WT mice but at an Mr compatible with small latent complex (Mr 80 kD) in C33S mutant mice (inset). (B) Sera from WT or C33S contained similar amounts of total TGF-β1 (WT mice: 63 ± 6 ng/mL; C33S mice: 67 ± 16 ng/mL). Mouse sera were either unstirred or stirred for 120 minutes at 37°C and TGF-β1 activity was measured by ELISA. TGF-β1 activity increased from 0.25% to 4.2% in WT mice but increased only from 0.23% to 0.3% in mutant mice (P < .001; n = 6). Sera were also immunoblotted with anti-LAP antibody and yielded results similar to those obtained with platelet releasate, but with several additional unidentified bands (inset). Error bars represent SD.