Platelet-osteosarcoma cell interaction is mediated through a specific fibrinogen-binding sequence located within the N-terminal domain of thrombospondin 1

Approximately 20% of patients with osteosarcoma have metastatic disease in lungs or bones at diagnosis. The requirement of platelets in hematogenous d issemination of metastatic cells is now well established. Tumor cells inter act with platelets and induce platelet aggregation. In this respect, metast atic potential of tumor cells correlates with their capacity to aggregate p latelets in vitro. We have previously shown that thrombospondin 1 (TSP-1) i s synthesized and expressed on the surface of MG-63 osteosarcoma cells and mediates plateletosteosarcoma cell interaction. However, active sites mimic king the function of TSP-1 during plateletosteosarcoma cell interaction are not known. In this study, a panel of antibodies directed against the N-ter minal and C-terminal domains and type 1, type 2, and type 3 repeats of TSP- 1 were first used to delineate the structural requirement for the binding o f osteosarcoma cell surface-associated TSP-I to platelets. A drastic inhibi tion of the platelet-aggregating activity of MG-63 cells was obtained in th e presence of a monoclonal antibody directed against the N-terminal domain of TSP-1. Among a series of 16 synthetic peptides spanning the whole N-term inal domain of TSP-1, only synthetic peptide N12/I encompassing amino acid residues 151-164 of the N-terminal domain of TSP-1 inhibited the platelet-a ggregating activity of MG-63 cells. Electron microscopy studies showed that peptide N12/I strongly inhibited platelet-osteosarcoma cell interaction. A polyclonal antibody directed against peptide N12/I specifically bound to t he surface of MG-63 cells, recognized TSP-1 and drastically inhibited the p latelet-aggregating activity of MG-63 cells. Zn addition, peptide N12/I spe cifically bound to fibrinogen and inhibited TSP-1/fibrinogen interaction. O verall, our results provide evidence that a fibrinogen-binding sequence loc ated within the N-terminal domain of TSP-1 mediates the binding of osteosar coma cell surface-associated TSP-1 to platelet-bound fibrinogen.