Active TGF-β1 can be extracted from platelet-rich thrombi formed in vivo. (A) Mouse platelet releasate was stirred at 1200 rpm for 2 hours at 37°C and active and total TGF-β1 were measured by ELISA (P < 005; n = 5). (B) Mouse serum was stirred or unstirred for 120 minutes at 37°C and TGF-β1 activity was measured by ELISA (P < .001; n = 19). (C) Thrombosis was induced in the carotid arteries of C57Bl/6 mice by exposure to ferric chloride (8%) for 3 minutes. After thrombi formed (∼ 5 minutes), arterial segments (∼ 4 mm) were excised either 5 or 120 minutes thereafter. Thrombi were removed from the segments and dispersed in buffer (200 μL) on ice for 1 hour, after which the supernatants were collected by centrifugation. Total TGF-β1 was measured by ELISA and both active and total TGF-β1 were measured by the MLEC assay in the absence and presence of a TGF-β1 neutralizing antibody. Active TGF-β1 was detected in both samples, but the percentage of active TGF-β1 in the 120-minute thrombi was greater than in the 5-minute sample (P < .005; n = 6). Error bars represent SD. (D) Immunoblotting with an anti–TGF-β1 antibody confirmed the presence of TGF-β1 in the supernatants of 5- and 120-minute thrombi. The intensities corresponded to the amounts of total TGF-β1 found by ELISA (∼ 5 ng/mL at 5 minutes and ∼ 4 ng/mL at 120 minutes). (E) Immunoblotting of carotid lysate with anti-αIIb and actin antibodies also confirmed the presence of equal numbers of platelets in the thrombi.