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

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.