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 (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. Copyright © 1988, Wiley Blackwell. All rights reserved