Cultured Human Fibroblasts Synthesize and Secrete Thrombospondin and Incorporate It into Extracellular Matrix

Thrombospondin, a major glycoprotein released from α granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 μ g per 106 cells per 24 hr, respectively); secretion was blocked by cycloheximide. [3H]Thrombospondin was isolated from [3H]leucine-labeled fibroblast postculture medium and from cell layers with rabbit polyclonal or mouse monoclonal anti-thrombospondin coupled to staphylococcal protein A-Sepharose. The immunologically isolated [3H]thrombospondin migrated in NaDodSO4/polyacrylamide gels with purified marker platelet thrombospondin both with and without reduction. Immunofluorescence microscopy using rabbit polyclonal and mouse monoclonal anti-thrombospondin antibodies localized thrombospondin to the fibrillar extracellular matrix surrounding the cells. Thus, cultured human fibroblasts secrete thrombospondin and incorporate it into the extracellular matrix.