In the previous study, we generated a
monoclonal antibody, 8F11, against NL-17, a high metastatic clone derived from a metastatic variant of murine
colon adenocarcinoma 26. 8F11 inhibited platelet aggregation induced by NL-17 and recognized a Mr 44,000
membrane protein as
antigen. In the present study, the reactivity of 8F11 to murine
B16 melanoma and its metastatic variants was examined, and the
antigen recognized by 8F11 on the cell surface was characterized. 8F11 was found to strongly react with 3 metastatic variants of
B16 melanoma. In contrast, only slight reactivity was observed with parent
B16 melanoma. The reactivity of the antibody to these cells was in the order B16F10 greater than B16BL-6 greater than B16F1 much greater than B16. Western blot analysis showed a Mr 41,000
protein as the
antigen recognized by 8F11 on the cell surface of B16F10 cells. The Mr 41,000
antigen appeared to be a
glycoprotein that bound to
wheat germ agglutinin as has been observed for the Mr 44,000
antigen of NL-17. To elucidate the functional role of the Mr 41,000
antigen in
B16 melanoma, platelet aggregation induced by B16 and B16F10 was compared. B16 was reported to stimulate platelet aggregation by the generation of
thrombin, whereas B16F10 was found to activate platelet by at least 2 mechanisms: one dependent on
thrombin and the other independent on
thrombin. The activity of B16 and its metastatic variants to induce platelet aggregation in the presence of
MD805, a synthetic antagonist of
thrombin, well correlated with the reactivity of 8F11 to these cells. 8F11 blocked platelet activation by B16F10 under conditions preventing
thrombin activity such as enzymatic formation of
lysolecithin through the treatment of the cell surface with
phospholipase A2 or in the presence of
MD805. These data indicate that Mr 41,000
glycoprotein recognized by 8F11 on metastatic variants of
B16 melanoma is involved in the
thrombin-independent platelet aggregation. A positive correlation was observed between the levels of Mr 41,000
glycoprotein expression of B16 and its metastatic variants and their pulmonary
metastasis after i.v. injection, suggesting Mr 41,000
glycoprotein, as well as other factors reported previously, may play an important role in the hematogenous spread of
B16 melanoma.