Identification of Binding Site for Heparin and Other Polysulfated Glycosaminoglycans on Human Thrombocytes

摘要

Heparin and other polysulf ated glycosaminoglycans (GAGPS) can evoke an immune response. when complexed to the surface of platelets in vivo. We have previously reported on the detection and characteristics of such antibodies leading to thrombocytopenia. Here we report data concerning the biochemical characterization of the heparin-/GAGPS-binding site on human thrombocytes. By means of heparin affinity chromatography, GAGPS chromatography and subsequent ‘Western blotting’ of specifically eluted proteins we were able to detect two proteins of apparent molecular mass of 207 and 170 kilodaltons, which could be reduced to 57 and 142 kilodaltons, respectively. The 170-kilodalton glycoprotein was stained by the carbohydrate specific PAS-reagent and by surface labeling with 3H. The second molecule binding to heparin (GAGPS)-Sepharose was detected by platelet surface labeling with 125I, protein-specific staining Coomassie brilliant blue and PAS staining. Binding studies using enzyme-labeling techniques with five monoclonal antibodies (MCA) against platelet surface proteins GPI, GPIIb/IIIa complex, GPIIIa and CRI (C3b receptor) revealed specific interaction of the 170-kilodalton heparin-binding glycoprotein with an MCA to GPI, suggesting identity or immunological cross-reactivity of these two moieties. No specific reactions were observed between the 207-kilodalton glycoprotein and any of the MCA tested. For further evaluation of the antigenicity of separated platelet proteins, an ELISA in microtiter plates was developed, and a cellular sandwich ELISA for serological detection of antibodies against whole native thrombocytes or GAGPS-derivatized proteins was set up. The identification of two thrombocytes surface glycoproteins as major binding sites for heparin (GAGPS) may provide the means for the elucidation of the molecular basis of the development of the immunologic mechanisms leading to thrombocytopenia in patients treated with these substances. Furthermore, a simple and sensitive ELISA can now be applied for the diagnosis of this severe complication of heparin (GAGPS) ther