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Binding and activation of plasminogen on the surface of osteosarcoma cells
Author(s) -
Campbell Phil G.,
Wines Karen,
Yanosick Thomas B.,
Novak Josef F.
Publication year - 1994
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041590102
Subject(s) - plasmin , zymogen , cell , chemistry , serine protease , biochemistry , plasminogen activator , microbiology and biotechnology , binding site , cell growth , cell culture , protease , biology , enzyme , endocrinology , genetics
Abstract Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane‐bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG‐63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125 I‐Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd ∼0.9 μM) and a high capacity (∼7.5 x 10 6 sites/cell). The binding of 125 I‐Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125 I‐Pg to 125 I‐Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG‐63 monolayers represented ∼80% bound specifically to the cell surface and the remainder to the surrounding extracellular matrix. Either co‐incubation with uPA or pre‐incubation with Pm resulted in increased 125 I‐Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by α2‐antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to α2‐antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface. © 1994 wiley‐Liss, Inc.