A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor
Protein Engineering Design & Selection
anticoagulative agent is heparin; however, more potent compounds such as recombinant hirudin or a synthetic Wolfgang Strassburger 3 thrombin-inhibitory peptide named Hirulog ® are under clinical In order to improve the therapeutic profile of recombinant and 2 Marienhof Laboratorien, Moenchengladbach, Germany 1 To whom correspondence should be addressed single-chain urokinase-type plasminogen activator (rscu-PA), we designed chimeric rscu-PA derivatives with intrinsic In order to design
... to design plasminogen activators with improved thrombin inhibitory activity that showed a higher clot specifithrombolytic properties, bifunctional proteins with both city than prourokinase itself, probably through an increased plasminogen-activating and anticoagulative activity were affinity to the thrombus , constructed by fusing a thrombin-inhibitory moiety to the 1996). These bifunctional chimeras comprise the kringle and carboxy-terminus of a prourokinase derivative lacking protease domain of human rscu-PA fused via the carboxythe growth-factor domain. The thrombin-inhibitory moiety terminus end to a thrombin inhibitory sequence ( Figure 1 ). itself comprises four elements: linker 1, a motif directed The thrombin inhibitory element is built up from the aminoto thrombin's active site, linker 2 and a fragment of hirudin terminus to the carboxy-terminus by four sequence elements: which binds to the fibrinogen-recognition site of thrombin. a pentameric amino acid sequence called linker 1, a sequence In order to improve further the anticoagulative activity, the directed to the thrombin active site, a penta-or hexameric thrombin-inhibitory domain was modified by substituting sequence designated linker 2 and a sequence derived from the linker 2. Introduction of a linker (FLLRNP) from the carboxy-terminal region of hirudin (Dodt et al., 1985 ; Rydel human thrombin receptor conferred about a 10-fold et al., 1990) or from the human thrombin receptor (Vu increase in anticoagulative activity in protein M37 comet al., 1991a), respectively, which both bind to the fibrinogen pared with the parent molecule M23 carrying an aliphatic recognition site (FRS) of thrombin (Figure 2) . In a previous linker. The increase in anticoagulative activity was also report, it was shown that three of the four building blocks reflected in the shift of the K i value from 159 ⍨ 20 nM for contribute to the anticogulative and thrombin-inhibitory activity M23 to 2.0 ⍨ 0.5 nM for M37. The increased thrombinof the chimeric rscu-PA derivatives: the active site-directed inhibitory activity of M37 may be due to the presence of sequence, the FRS-binding sequence and, to a surprisingly an arginine in the linker from the thrombin receptor which high extent, also linker 1 . may interact with one of two glutamic acid residues located The role of linker 2 appeared to be less important. The at the exit of the thrombin substrate binding pocket. This current model of the complex between thrombin and synthetic explanation is supported by the observation that another inhibitory peptides like Hirulog ® which contain an active sitechimera (M35) 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.