Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a

The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel el ectrophoresis followed by ligand blotting with I-125-plasminogen. A 54-kDa protein lost the ability to bind I-125-plasminogen after treatment of intac t cells and was purified by two-dimensional gel electrophoresis and then se quenced by mass spectrometry. Two separate amino acid sequences were obtain ed and were identical to sequences contained within human and rat TIP49a. T he cDNA for the 54-kDa protein matched the human TIP49a sequence, and encod ed a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodie s against rat TIP49a recognized the plasminogen-binding protein on two-dime nsional Western blots of U937 cell membranes. Human I-125-Glu-plasminogen b ound specifically to TIP49a protein, and binding was inhibited by epsilon - aminocaproic acid. A single class of binding sites was detected, and a K-d of 0.57 +/- 0.14 muM was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhan cement mediated by plasmin-treated fibrinogen. These results suggest that T IP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.