RECOMBINANT VARIANTS OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR CONTAINING AMINO-ACID SUBSTITUTIONS IN THE FIBRONECTIN FINGER-LIKE DOMAIN AND THEKRINGLE-1 DOMAIN

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent wh ich is used to treat acute myocardial infarction. Pharmacokinetically, t-PA is characterized by a rapid clearance from the circulation. In a previous study, we constructed variant forms of t-PA with genetic mod ifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N 37S.S38V.G39V.R40E. A41F.Q42S had about a 6.0-fold higher plasma half- life in vivo than wild-type t-PA, Two variants with modifications in t he K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improve d kinetic parameters and a 2.2-fold higher plasma half-life in vivo th an wild-type t-PA, respectively. To create a recombinant variant of t- PA with a higher enzymatic activity and a further prolonged half-life in vivo, the genes containing each modifications were joined and expre ssed in animal cells, The two variants, t-PA N37S.S38V.G39V.R40E.A41F. Q42S.G161R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N115P, w ere purified from conditioned media and their biochemical, pharmacokin etic and thrombolytic profiles were investigated. Although the variant I-PA N37S.S38V.G39V.R40E.A41F.Q42S.G161R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild-type t-PA, the half-l ife of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, followi ng intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants. Human plasma clot lysis assa y estimated the Fibrinolytic activity of both variants to be about 2.0 -fold less effective than that of the wild-type t-PA, In the rabbit ju gular vein clot lysis model, doses of 1.0 and 0.0625 mg/kg were requir ed for about 70% lysis in the wild-type tPA and t-PA N37S.S38V.G39V.R4 0E.A41F.Q42S.N115P, respectively. These findings suggested that the va riant in this study can be used at a lower dosage in a single bolus in jection.