Characterization of human tissue-type plasminogen activator variants with amino acid mutations in the kringle 1 domain.
Blood Coagul Fibrinolysis
; 3(4): 381-7, 1992 Aug.
Article
em En
| MEDLINE
| ID: mdl-1330023
ABSTRACT
Recombinant variants of tissue-type plasminogen activator (t-PA) were constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Five variants were designed to improve the function of t-PA by mutagenesis in the kringle 1 (K1) domain. The amino acids were replaced with the corresponding residues present in the kringle 2 (K2) domain of native t-PA. The t-PA mutants expressed were as follows variant E94V.D95G with point mutations in Glu94----Val and Asp95----Gly; variant N115P.S119M, Asn115----Pro and Ser119----Met; variant P125A.R129Q.R13OS, Pro125----Ala, Arg129----Gln and Arg130----Ser; variant G161R.K162R.-S165W, Gly161----Arg, Lys162----Arg and Ser165----Trp; and variant N115P, Asn115----Pro, respectively. The half-life following intravenous bolus injection in rabbits was prolonged in all variants except P125A.R130S. This was particularly true for N115P.S119M. The kinetic parameters for plasminogen activation were improved in t-PA G161R.K162R.S165W which showed a 0.6-fold decrease in Km, and a 1.8-fold increase in Vmax, thus promoting a 2.7-fold increase in kcat/Km compared to native t-PA. For a similar degree of thrombolysis in the rabbit jugular vein thrombosis model, the thrombolytic activity of G161R.K162R.S165W, at the dose tested, was four-fold greater than that of native t-PA. Thus, the substitution of the amino acids in the K1 domain with those corresponding in the K2 domain significantly enhanced the enzymatic activity of t-PA and improved the plasma survival.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
/
Mutagênese Sítio-Dirigida
/
Ativador de Plasminogênio Tecidual
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Blood Coagul Fibrinolysis
Assunto da revista:
ANGIOLOGIA
/
HEMATOLOGIA
Ano de publicação:
1992
Tipo de documento:
Article
País de afiliação:
Japão