Thrombospondin (TBSP) is a 450 kDa glycoprotein secreted by a wide range of cells
including platelets, endothelial cells and fibroblasts. Using non-denaturating conditions,
we recently reported that platelet TBSP was structurally different from endothelial
and fibroblast TBSP (P. Clezardin et al., Eur. J. Biochem., 1986, 159, 569-579). The
aim of this study was to compare the structure of TBSP purified from platelets, endothelial
cells and fibroblasts using denaturating conditions. Moreover, the interaction of
fibrinogen with these three forms of TBSP was also investigated. TBSPs were first
purified by heparin-Sepharose and immunoaffinity chromatography followed by Mono O
anion-exchange chromatography on a FPLC system. Thermolysin digests of purified TBSPs
were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions
and the subsequent electrophoresed proteolytic fragments identified by Coomassie and
silver staining. The interaction of undigested and digested TBSPs with solid-phase
adsorbed fibrinogen was investigated by enzyme-linked immunosorbent assay using an
anti-TBSP monoclonal antibody (P10). when using Coomassie staining, a 70 kDa proteolytic
fragment of thermolysin-treated platelet TBSP was absent from the endothelial and
fibroblast TBSP digests. Moreover, a 18 kDa fragment from thermolysin-treated endothelial
and fibroblast TBSP was undetectable in the platelet TBSP digest when using silver
staining on SDS-polyacrylamide gels. The binding of undigested TBSPs to solid-phase
adsorbed fibrinogen was different from that obtained with digested TBSPs. These results
indicate that the observed structural differences might induce functional differences
between platelet and the two other forms of TBSP.
