Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the fibronectin finger-like domain and the kringle 1 domain.
Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which 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 modifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N37S.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 the K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improved kinetic parameters and a 2.2-fold higher plasma half-life in vivo than 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 expressed in animal cells. The two variants, t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N11 5P, were purified from conditioned media and their biochemical, pharmacokinetic and thrombolytic profiles were investigated. Although the variant t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild-type t-PA, the half-life of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, following intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants.(ABSTRACT TRUNCATED AT 250 WORDS)
Yahara
H
H
Biochemical Research Laboratories, Kanegafuchi Chemical Industry Co. Ltd., Takasago, Japan.
Matsumoto
K
K
Maruyama
H
H
Nagaoka
T
T
Ikenaka
Y
Y
Yajima
K
K
Fukao
H
H
Ueshima
S
S
Matsuo
O
O
D000595:Amino Acid Sequence; D000818:Animals; D004195:Disease Models, Animal; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D014644:Genetic Variation; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D018082:Kringles / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
D005353:Fibronectins; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator