Prothrombin promotes the invasiveness of melanoma cells by a different mechanism from thrombin

The migration of tumor cells through the extravascular matrix is a crucial step in the process of tumor invasion and metastasis. It has been recognized that activation of blood coagulation may contribute to the growth and the invasive behavior of tumor cells. We have previously found that prothrombin and its derivative, fragment 1, bind to and stimulate the motility of melanoma cells by a different mechanism from thrombin, The present study showed that prothrombin and fragment 1 also significantly increase the invasive ability of melanoma cells in an in vitro matrigel system by a different mechanism from thrombin. The optimal concentration of each factor for stimulating invasiveness of tumor M2 cells was between 0.5 similar to 1 mu M. The M2 cells with highly metastatic potential showed strong invasive ability compared to the CL10 cells with low metastatic potential in the presence of the same concentrations of each factor. Hirudin inhibited the stimulatory activity of thrombin but not that of prothrombin or fragment 1 on the invasive ability of M2 cells. The activity of plasmin and urokinase-type plasminogen activator (uPA) was significantly increased in conditioned media from M2 cells stimulated with thrombin, prothrombin or fragment 1, hirudin inhibited the stimulative activity of thrombin but not that of prothrombin or fragment 1. Western blot analysis showed that thrombin increases the generation of the active forms of gelatinase A on the surface of M2 cells; hirudin inhibited this stimulative activity of thrombin. Prothrombin also increases the active forms of gelatinase A, but hirudin did not inhibit this effect of prothrombin. The results of this study suggest that prothrombin enhances the invasiveness of melanoma cells in vitro by a mechanism different from thrombin. (C) 199 Harcourt Publishers Ltd.