Characterisation of a mutant of recombinant human single chain urokinase-type plasminogen activator (scu-PA), obtained by substitution of arginine 156 and lysine 158 with threonine

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA)

“…We also demonstrated that once the proteins (whether mutant or WT) were cleaved at position 158, they became active and were able to promote autoactivation as well as tcuPA WT. Previous authors had mutated K158 to Gly, Glu, Thr, Val, and Met and found that these mutants did have some activity ( 51 , 52 , 53 , 54 , 55 ). It is important to note that the new N terminus, which is required for ordering the catalytic triad, would be the same regardless of the side chain occupying position 158.…”

“…It is important to note that the new N terminus, which is required for ordering the catalytic triad, would be the same regardless of the side chain occupying position 158. Interestingly, some of the previous articles show the SDS-PAGE of their “single-chain” mutant protein, and it is very evident that there is a band at ∼36 kDa that could correspond to some version of the heavy chain of uPA ( 53 , 54 ) suggesting that the observed “intrinsic activity” was most likely because of the cleavage of even these mutants at position 158.…”

The Nature of Synergy between Tissue-Type and Single Chain Urokinase-Type Plasminogen Activators

“…Although initial studies suggested that conversion of scu-PA to the two-chain form of urokinase was required for plasminogen activation (21, 22,32), subsequent studies indicate that scu-PA is much less efficient in activating plasminogen and that scu-PA is likely a true pro-enzyme which must be activated to some extent in order to promote activation of plasminogen (33,34). We (unpublished observations) and others (35,36) have been able to demonstrate only limited activation of plasminogen in vitro with molecular variants of scu-PA in which the plasmin cleavage site has been altered by substitution mutation to prevent conversion of scu-PA to tcu-PA. If tcu-PA activates plasminogen much more efficiently than scu-PA does, then accelerated conversion of scu-PA to tcu-PA would be expected to increase the extent and rapidity of thrombolysis.…”

Strategies for the Improvement of Thrombolytic Agents

“…Because clot lysis with scu-PA is associated with a higher degree of fibrin-specificity as compared to tcu-PA, attempts were made, using recombinant DNA technology, to prevent its conversion to a two chain molecule. Several investigators have constructed mutants of scu-PA with the aim to destroy the plasmin cleavage site by site-specific mutagenesis of Lysl58 or Ilel59 (167,173,(188)(189)(190). Alternatively, scu-PA has been rendered less sensitive to cleavage by both thrombin and plasmin by substitution of both Argl56 and Lysl58 by Thr (190), or by introduction of a nega tive charge near the cleavage sites (substitution of Phel57 by Asp) (191).…”