Total TGF-β1 is released as LLCs within 5 minutes after platelet stimulation. Platelets (109 per mL) were stimulated with thrombin (0.125 U/mL) at 37°C for the indicated time periods and centrifuged at 14 000g for 20 minutes at 4°C. Pellets were lysed in buffer containing Triton-X-100 (1%). (A) Total TGF-β1 in platelet lysates and releasates before and after thrombin stimulation for the indicated times was measured by ELISA after acidification (top panel) and immunoblotting with anti–TGF-β1 (bottom panel); results shown are from 1 of more than 10 similar experiments. (B) Both lysates and releasates were immunoblotted with antibodies to LTBP1, LAP, TGF-β1, and actin. The immunoblots demonstrate release of TGF-β1, LAP, and LTBP-1, the 3 components of LLCs. LAP and the upper band of LTBP-1 migrated at Mr approximately 270 kD, consistent with their forming a 1:1 complex. The lower LTBP-1 band of Mr approximately 160-kD identified in the immunoblot has the same Mr reported for free LTBP-1.31 The lower bands identified in the LAP immunoblot have Mr's consistent with precursor molecules that are not covalently coupled to LTBP-1.
