Complex formation of human thrombospondin with osteonectin

Abstract

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...

Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor β

“…SPARC is found at high levels in bone [22,48] and periodontal tissues [26], and has been shown to be synthesized by fibroblasts from various tissues [27,491. SPARC has also been found in human platelets and is released as a complex with thrombospondin [50] by activation of the platelets with throm-A B (1)], Interleukin-I (IL-1) and secreted phosphoprotein I/osteopontin (SppI) cDNA, and the plasmid vector pT7T3, which were immobilized on a Bio-Trans membrane. In a separate experiment (B), nuclei from 3 x lo7 human fibroblasts were isolated 7 h after addition of TGF-P (T) or vehicle (C) and 2 x lo7 dpm radiolabeled RNA hybridized to SPARC and fibronectin (FN) cDNA immobilized on Bio-Trans bin [32].…”

Common deleted genes in the 5q− syndrome: thrombocytopenia and reduced erythroid colony formation in SPARC null mice

“…32 Peripheral blood values from SPARC-null versus wild-type mice showed significantly lower levels of platelets. A possible explanation relates to the known loss of the anti-aggregation effect of SPARC on platelets; [15][16][17] this could result in their clumping and rapid clearance from the circulation. However, no increased incidence of thrombosis has been reported in mice-lacking SPARC.…”

“…12 Interestingly, bone marrow-derived SPARC can inhibit the binding of thrombospondin to the surface of platelets, and, thereby, inhibit platelet aggregation. 15,16 Recently, SPARC was shown to inhibit breast cancer cell invasion, platelet aggregation and metastasis. 17 Increased methylation of SPARC in lung and pancreatic tumors is consistent with its role as a potential tumor suppressor gene that is silenced in selected cancers.…”

Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells

“…In addition to the production of thrombospondin, human osteoblasts and osteosarcoma cells also synthesize and secrete osteonectin, a 30-kDa phosphoprotein [24,251. We have recently demonstrated that osteonectin isolated from platelets and bone interacts with thrombospondin in a specific manner [15]. Studies are currently in progress to determine the physiological significance of this complex in human cells such as osteoblasts and osteosarcoma cells.…”

“…Each thrombospondin chain is made up of several protease-resistant domains, which bind specifically to heparin, fibrinogen, fibronectin, collagen, histidine-rich glycoprotein and plasminogen (for review see [14]). We have recently demonstrated that thrombospondin also forms a specific complex with osteonectin [15], a bone-related protein which is present in human platelets [16]. These findings, taken together with the fact that thrombospondin is secreted by several cultured cell lines, prompted us to examine whether or not it is also present in bone.…”