Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (&) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (& = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81 % inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calciumdependent as shown by a 50 -80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A -Sepharose 4B. Elution of the antithrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.Thrombospondin is a platelet a-granule glycoprotein which is secreted in response to thrombin [l]. This glycoprotein has a molecular mass of 450 kDa and is composed of three equivalent disulphide-linked chains of 150 -160 kDa [2]. Each thrombospondin chain is made up of several protease-resistant domains, which bind specifically with heparin, fibrinogen, fibronectin, collagen, calcium, histidine-rich glycoprotein and plasminogen (for review see [3]).Thrombospondin is synthesized by a wide range of cultured cells [3 -71 including fibroblasts, monocytes, macrophages, endothelial and smooth muscle cells [3] and is incorporated into their extracellular matrices [8,9]. Source-specific differences in fragmentation patterns between thrombospondin molecules were reported following exposure to proteolytic enzymes [lo -121, suggesting thrombospondin polymorphism [ll].The exact physiological function(s) of thrombospondin is unknown; however, there is growing evidence that it takes part in the platelet aggregation process [13, 141 and promotes cell adhesion [6, 71. Recently human platelets have been shown to contain and secrete a 30-kDa phosphoprotein [15], namely osteonectin, which is a major protein of mineralized bone [16, 171. Platelet osteonectin is identical to bovine bone osteonectin in terms of molecular mass and immuno...