CONSTRUCTION AND CHARACTERIZATION OF A CHIMERIC PROTEIN CONSISTING OFTISSUE-TYPE PLASMINOGEN-ACTIVATOR AND THE EPIDERMAL GROWTH FACTOR-LIKE REGIONS OF THROMBOMODULIN

We have constructed and characterized a chimeric anticoagulant and fib rinolytic protein by inserting the portion of thrombomodulin (TM) carr ying protein C (PC) activating cofactor activity into tissue-type plas minogen activator (t-PA). This was done by replacing the epidermal gro wth factor (EGF)-like domain of t-PA with the six EGF-like repeats fro m TM. The 105kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cof actor activity to other soluble derivatives of TM (K-m for Protein C o f 2.2+/-0.4 mu M), as well as amidolytic and plasminogen activating ac tivity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patien ts with thrombosis.