Complex formation of human thrombospondin with osteonectin.
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 (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 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 calcium-dependent 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 anti-thrombospondin 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.
Clezardin
P
P
Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Alexis-Carrel, Lyon, France.
Malaval
L
L
Ehrensperger
A S
AS
Delmas
P D
PD
Dechavanne
M
M
McGregor
J L
JL
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D001842:Bone and Bones / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D015676:Osteonectin; D011485:Protein Binding; D019699:Thrombospondins
D002352:Carrier Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D015676:Osteonectin; D019699:Thrombospondins