BIOLOGICAL CHARACTERIZATION OF AMINO-TERMINAL EXON DELETIONS OF T-PA

The secreted form of t-pa is proposed to be a mosaic protein which contains 4 different domain elements based on amino acid homologies with fibronectin finger elements, epidermal growth factor, kringle structures, and the active site of serine proteases. Of the 12 exons which encode these domains only the finger and epidermal growth factor are encoded separately by single exons. To investigate whether a single exon can encode a functional element or domain within a protein, the following precise exon alterations were made by loop-out mutagenesis techniques which deleted either the fibronectin finger, epidermal growth factor, or combination finger/growth factor domain(s). These mutant proteins were expressed in mammalian cells and characterized with respect to affinity for fibrin, fibrinolytic and fibrinogenolytic potential. All mutants demonstrated significantly lower affinity for fibrin with respect to the wild-type protein. We estimate the KD of these mutants for fibrin to be at least 100-fold higher than the wild-type form which we determined to be approximately 0.3 uM. Each mutant retained characteristic activator stimulation by fibrin and were also shown to have the same approximate specific activity as the wild-type form. These mutants were further evaluated in citrated human plasma [125-I]-fibrin clot lysis assays over a range of activator concentrations and shown to behave similarly to wild-type t-pa at therapeutic thrombolytic concentrations. At some lower concentrations, however, reduced fibrinolysis was observed for the mutant forms relative to wild-type. All mutants were evaluated for their fibrinogenolytic potential and demonstrated no significant decrease in coagulable fibrinogen over a five hour period. This was in dramatic contrast to an equivalent activator concentration of urokinase which showed a precipitous decline in coaguable fibrinogen in the first hour.