Characterization of human tissuetype plasminogen activator variants with amino acid mutations in the kringle 1 domain

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.R129Q.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 inKm, and a 1.8-fold increase inVmax, thus promoting a 2.7-fold increase inkcat/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.