Production in Eukaryotic Cells and Characterization of Four Hybrids of Tissue-Type and Urokinase-Type Plasminogen Activators

Abstract

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.

Thrombolytic and pharmacokinetic properties of chimeric tissue-type and urokinase-type plasminogen activators.

“…These recombinant products had intact catalytic activities of scu-PA but had not acquired fibrin affinity of t-PA or an improved in vitro fibrin selectivity compared with scu-PA. 20,25,26 In a further development, two chimeric plasminogen activators (t-PA/u-PA-s and t-PA/u-PA-e) consisting of the NH2-terminal part of t-PA (amino acids 1-263 and 1-274, respectively) and the catalytic domain of scu-PA (amino acids 144-411 and 138-411, respectively) were obtained. These compounds retained the specific activity, catalytic efficiency, and in vitro fibrinolytic potency of u-PA (either in their single-chain or their two-chain forms) and partially acquired the fibrin affinity and fibrin stimulation of t-PA. '8,27 Intravenous infusion of t-PA/ scu-PA-s, t-PA/scu-PA-e, or scu-PA in a rabbit jugular vein thrombosis model resulted in very similar dose-response curves of clot lysis, indicating that combination of the fibrin-binding domains of t-PA with the catalytic domain of scu-PA, which endows the chimeric molecules with significant fibrin affinity, is not associated with an increased thrombolytic potency in vivo.…”

Thrombolytic Activity of Two Chimeric Recombinant Plasminogen Activators (FK2tu-PA and K2tu-PA) in Rabbits

“…Several groups have reported on the construction of chimeric plasminogen activators combining favourable characteristics of t-PA and scu-PA, fibrin binding, fibrin stimulation and catalytic efficacy into single molecules (23)(24)(25)(26)(27)(28)(29)(30). The t-PA/scu-PA hybrid proteins have been analysed in vitro and shown to combine, to some extent, the affinity for fibrin and the ability to be activated by fibrin of t-PA with the enzymatic properties of two-chain scu-PA.…”

Strategies for the Improvement of Thrombolytic Agents

“…Substitution of the F-and E-domains of t-PA by the E-and K-domains of u-PA, however, resulted in a strongly decreased fibrin-binding (197). Similarly, a chimera in which K2 of t-PA (amino acids 173 to 262) was inserted between amino acids 130 and 139 and u-PA, the same chimera in which the F domain of t-PA (amino acids 1 to 50) was exchanged for amino acids 1 to 10 of u-PA, or a chimera con sisting of the F-domain of t-PA and of amino acids 10 to 411 of u-PA with replacement of Lysl35 and Lysl36 by Gin, did not have enhanced fibrin-binding (200,201). de Vries et al (196) have constructed two chain chimeras containing amino acids Serl to Ser262 of t-PA and either residues Gin 147 to Leu411 or Gin 134 to Leu411 of u-PA; both chimeras showed decreased fibrin-binding.…”