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 ' ' ' I and incubated with monoclonal antibodies PI0 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 '251-labelled platelet releasates treated for 48 h at 4°C with 10 mM 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 251-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas '2SI-labelled PI2 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-fmed 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 calciumtreated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies PI0 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.Thrombospondin, a calcium-sensitive glycoprotein located in human blood platelet alpha-granules, is released from platelets upon stimulation [l]. Human platelet thrombospondin is composed of three equivalent disulphide-linked chains of 150 -160 kDa [2]. Isolated human platelet thrombospondin has been shown to interact with several proteins [2 -81. These thrombospondin-protein interactions suggest that thrombospondin may have several structural domains with independent functions. However, the functional domains of thrombospondin interacting with most of these proteins have still to be elucidated. The h...

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