A STRONG THROMBIN-INHIBITORY PROUROKINASE DERIVATIVE WITH SEQUENCE ELEMENTS FROM HIRUDIN AND THE HUMAN THROMBIN RECEPTOR

In order to design plasminogen activators with improved thrombolytic p roperties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibit ory moiety to the carboxy-terminus of a prourokinase derivative lackin g the growth-factor domain. The thrombin-inhibitory moiety itself comp rises four elements: linker 1, a moth directed to thrombin's active si te, linker 2 and a fragment of hirudin which binds to the fibrinogen-r ecognition site of thrombin. In order to improve further the anticoagu lative activity, the thrombin-inhibitory domain was modified by substi tuting linker 2. Introduction of a linker (FLLRNP) from the human thro mbin receptor conferred about a 10-fold increase in anticoagulative ac tivity in protein M37 compared with the parent molecule M23 carrying a n aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the K-i value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the t hrombin receptor which may interact with one of two glutamic acid resi dues located at the exit of the thrombin substrate binding pocket. Thi s explanation is supported by the observation that another chimera (M3 5) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.