BINDING AND ACTIVATION OF PLASMINOGEN ON THE SURFACE OF OSTEOSARCOMA CELLS

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorp tion, metastasis, and the activation of growth factors. The various co mponents of the Pm system are known to bind and function on the cell s urface of various cell types, but no pertinent data are available desc ribing membrane-bound Pm or its zymogen, plasminogen (Pg), in either n ormal or neoplastic bone cells. We report here that Pg binds to the su rface of the human osteosarcoma cell line MG-63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusion s are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. I-125- Pg binding to cells was time dependent, saturable, reversible, and spe cific. Binding was characterized by a relatively low affinity (Kd appr oximately 0.9 muM) and a high capacity (approximately 7.5 x 10(6) site s/cell). The binding of I-125-Pg was associated with lysine binding si tes of the plasminogen molecule. Activation of I-125-Pg to I-125-Pm oc curred on the cell surface and was dependent upon cell bound uPA, as d etermined by inhibitory antibodies. Binding of Pg to MG-63 monolayers represented approximately 80% bound specifically to the cell surface a nd the remainder to the surrounding extracellular matrix. Either co-in cubation with uPA or pre-incubation with Pm resulted in increased I-12 5-Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activi ty was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bo und to cells with Pg being activated by endogenous uPA. Plasmin activa ted on the cell surface was partially protected from inhibition by alp ha2-antiPm (requiring Pm lysine binding site interaction) but inhibite d by aprotinin, (interacting directly with the Pm catalytic site). Res istance of cell bound Pm to alpha2-antiPm inhibition suggests that cel l surface proteolysis can occur in the presence of a soluble Pm inhibi tor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicatin g Pm may occur at or near the bone cell surface. (C) 1994 Wiley-Liss, Inc.