Cloning of Platelet aggregation factors from tumor cells

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One of the main causes of death of cancer patients is thrombosis. This is probably because cancer cells activate platelet aggregation and blood coagulation. Furthermore, it is fairly well accepted that above phenomena might be responsible for metastasis of cancer. In the initial year of our research program we did following experiments. We found using a neuroblastoma cell GOTO that platelet aggregation was induced upon addition of this cell to platelet-rich plasma. We also found that the addition of GOTO cells to normal plasma activated Factor X and it in turn activated prothrombin and produced thrombin. We then found that both activities, the activation of Factor X and prothrombin, disappeared upon differentiation of GOTO cells to Shwanian-like cells. Based on the results obtained in the initial year, we did subtraction cloning and production of monoclonal antibodies in the second year with the aim of elucidating the underlining mechanism of thrombin production by GOTO cells in molecular terms. In the final year we are looking into the specific binding of Factor X to the cells to carry out an expression cloning. As far as the production of monoclonal antibodies is concerned, we have been able to clone hybridomas directly on soft : agar plates and we have obtained several clones with the desired properties. We hope the time will soon come to start the actual cloning experiment with these antibodies. On the other hand we have made much improvement as to the purification of highly polar lipid, probably glycolipid, which is the antigen for the monoclonal antibody we established last year which specifically recognized the surface of neuroblastoma cells. We hope we could do NMR and mass spectrometric analyses to elucidate the chemical structure of this interesting lipid. We will chemically synthesize the lipid and elucidate the biological function of this lipid.