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Complex formation of human thrombospondin with osteonectin.
P Clezardin1, L Malaval, A S Ehrensperger
1Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Alexis-Carrel, Lyon, France.
European Journal of Biochemistry
|August 1, 1988
Summary
Human thrombospondin and osteonectin, both found in platelets, specifically bind to each other. This calcium-dependent interaction is confirmed through various assays, indicating a novel protein complex formation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Thrombospondin (TSP) is a 450-kDa glycoprotein found in platelets and endothelial cells.
- Osteonectin (ON) is a 30-kDa protein isolated from bone and platelets.
- The interaction between TSP and ON has not been previously characterized.
Purpose of the Study:
- To investigate the specific interaction between human thrombospondin and osteonectin.
- To characterize the binding affinity and conditions required for complex formation.
- To determine if osteonectin forms a complex with thrombospondin upon platelet activation.
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA) to measure binding affinity (Kd).
- Affinity chromatography using anti-thrombospondin monoclonal antibodies.
- SDS-PAGE and autoradiography for complex identification.
- Western blotting to confirm complex formation.
Main Results:
- Purified osteonectin binds specifically to thrombospondin with high affinity (Kd = 0.7-0.86 nM).
- Complex formation is calcium-dependent and significantly inhibited by excess fluid-phase osteonectin.
- Known thrombospondin-interacting proteins did not inhibit TSP-ON binding.
- Osteonectin was found complexed with thrombospondin released from activated platelets.
Conclusions:
- Human thrombospondin and osteonectin form a specific, calcium-dependent complex.
- This interaction occurs independently of other known thrombospondin binding partners.
- Osteonectin is associated with thrombospondin in the platelet-release reaction, suggesting a functional role in platelet biology.