Specific inhibition of endothelial cell proliferation by thrombospondin

Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation.

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

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.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14194.x

Complex formation of human thrombospondin with osteonectin

Circulating activated platelets in myeloproliferative disorders.

Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4 degrees C with 10mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1986.tb09363.x

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin.

Depression of reverse transcriptase activity by hybridoma supernatants: a potential problem in screening for retroviral contamination.

The purpose of these studies was to investigate whether the cell-differentiating effect of anthracyclines can trigger an over-production and secretion of molecules that may interfere with the tumor-host relationship. We exposed mouse hybridoma B-cells, which are devoted to immunoglobulin production, to doxorubicin (10–40 ng/ml). We found that most doxorubicin-treated cells secreted 3- to 5-fold higher amounts of immunoglobulin than untreated cells, along with an accumulation of 50% of them in the G2+M phase of the cell cycle. The antigenic specificity of the immunoglobulin and its size pattern as determined by polyacrylamide gel electrophoresis were similar whether or not cells were treated with doxorubicin. The enhancement of immunoglobulin secretion by doxorubicin was associated with an increase of the intracellular pool of heavy and light chains of the immunoglobulin. Furthermore, an elevated synthesis of immunoglobulin was observed. The synthesis of other proteins also appeared to be modified in these circumstances. These data suggest that doxorubicin can potentiate the biological functions of target cells when used at low concentrations, elevating the production and secretion of effector molecules that interfere with the tumor-host relationship.

/pdf/effect-of-doxorubicin-on-mouse-hybridoma-b-cells-stimulation-yzyi8sx0un.pdf

Joseph Huppert

Papers

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin.

Depression of reverse transcriptase activity by hybridoma supernatants: a potential problem in screening for retroviral contamination.

Effect of Doxorubicin on Mouse Hybridoma B Cells: Stimulation of Immunoglobulin Synthesis and Secretion

Cytogenetic modifications of friend leukemia cells resistant to adriamycin