Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that is involved in a variety of physiological processes such as tumor cell adhesion. invasion. and metastasis, It has been hypothesized that TSP-1 provides an adhesive matrix for osteosarcoma cells. Here we present data showing that TSP-1 can promote cell substrate adhesion to U2OS and SAOS cells through the alpha4beta1 integrin. The dose-dependent adhesion to TSP-1 was inhibited by anti-integrin antibodies directed against the alpha4 or beta1 subunit, but not by control antibodies against other integrins. To localize the potential alpha4beta1-binding site within the TSP-1 molecule, the protein was subjected to limited proteolysis with chymotrypsin in the absence of calcium, The stable 70-kDa core fragment produced under these conditions promoted alpha4beta1-dependent osteosarcoma cell adhesion in a manner similar to that of the intact protein, Moreover adhesion experiments with neutralizing antibodies revealed that the adhesion was totally dependent on the alpha4beta1 interaction. Further blocking experiments with potential inhibitory peptides revealed that the alpha4beta1-mediated adhesion was not influenced by peptides containing the RGD sequence. Attachment to the 70-kDa fragment was strongly inhibited by the CS-1 peptide, which represents the most active recognition domain for alpha4beta1 integrin in fibronectin. The present data provide evidence that TSP-1 contains an alpha4beta1 integrin-binding site within the 70-kDa core region. (C) 2002 Elsevier Science (USA). All rights reserved.