Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

Summary

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

• REFERENCES

• 1 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: Correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 1984; 81: 5355-5359
• 2 Matsuo O, Rijken DC, Collen D. Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus. Nature 1981; 291: 590-591
• 3 Matsuo O, Rijken DC, Collen D. Comparison of the relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb Haemostas 1981; 45: 225-229
• 4 Tiefenbrunn AJ, Sobel BE. Thrombolysis and myocardial infarction. Fibrinolysis 1991; 5: 01-15
• 5 Nilsson S, Einarsson M, Ekvarn S, Haggroth L, Mattsson Ch. Turnover of tissue plasminogen activator in normal and hepatec-tomized rabbits. Thromb Res 1985; 39: 511-521
• 6 Verstraete M, Su CAPF, Tanswell P, Feuerer W, Collen D. Pharmacokinetics and effects on fibrinolytic and coagulation parameters of two doses of recombinant tissue-type plasminogen activator in healthy volunteers. Thromb Haemostas 1986; 56: 1-5
• 7 Kalyan NK, Lee SG, Wilhelm J, Fu KP, Hum W-T, Rappaport R, Hartzell RW, Urbano C, Hung PP. Structure-function analysis with tissue-type plasminogen activator. J Biol Chem 1988; 263: 3971-3978
• 8 Lau D, Kuzma G, Wei CM, Livingston DJ, Hsiung N. A modified human tissue plasminogen activator with extended half-life in vivo. Bio/technology 1987; 5: 953-958
• 9 Browne MJ, Chapman CG, Dodd I, Reavy B, Esmail AF, Robinson JH. The role of tissue-type plasminogen activator A-chain domains in plasma clearance. Fibrinolysis 1989; 3: 207-214
• 10 Langer-Safer PR, Ahern TJ, Angus LB, Barone KM, Brenner MJ, Horgan PG, Morris GE, Stoudemire JB, Timony GA, Larsen GR. Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. J Biol Chem 1991; 266: 3715-3723
• 11 Bennett WF, Paoni NF, Keyt BA, Botstein D, Jones AJS, Presta L, Wurm FM, Zoller MJ. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem 1991; 266: 5191-5210
• 12 Larsen GR, Timony GA, Horgan PG, Barone KM, Henson KS, Angus LB, Stoudemire JB. Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase. J Biol Chem 1991; 226: 8156-8161
• 13 Sorbel BE, Sarnoff SJ, Nachowiak DA. Augmented and sustained plasma concentrations after intramuscular injection of molecular variants and deglycosylated forms of tissue-type plasminogen activators. Circulation 1990; 81: 1362-1373
• 14 Banyai L, Varadi A, Patthy L. Coaanyai L, Varadi A, Patthy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
• 15 Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY: 1982
• 16 Yamashita K, Ikenaka Y, Kakutani T, Kawaharada H, Watanabe K. Comparison of human lymphotoxin gene expression in CHO cells directed by genomic DNA or cDNA sequences. Agric Biol Chem 1990; 54: 2801-2809
• 17 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV. Cloning and expression of human tissue type plasminogen activator cDNA in E. Coli . Nature 1983; 214-221
• 18 Chu G, Sharp PA. SV40 DNA transfection of cells in suspension: Analysis of the efficiency of transcription and translation of T-antigen. Gene 1981; 13: 197-202
• 19 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 20 Collen D, Stassen J, Larsen GR. Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits. Blood 1988; 71: 216-219
• 21 Yamaoka K, Nakagawa T, Uno T. Statistical moments in pharmacokinetics. J Pharmacokinet Biopharm 1978; 6: 547-558
• 22 Matsuo O, Bando H, Okada K, Tanaka K, Tsukada M, Iga Y, Arimura H. Thrombolytic effect of single-chain pro-urokinase in a rabbit jugular vein thrombosis model. Thromb Res 1986; 42: 187-194
• 23 Rijken DC, Emeis JJ. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. Biochem J 1986; 238: 643-646
• 24 Kuiper J, Otter M, Rijken DC, van Berkel TJC. Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells. J Biol Chem 1988; 263: 18220-18224
• 25 Pannekoek H, de Vries C, van Zonneveld A-J. Mutants of human tissue-type plasminogen activator (t-PA): Structural aspects and functional properties. Fibrinolysis 1988; 2: 123-132
• 26 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. J Biol Chem 1988; 263: 1023-1029
• 27 Ahern TJ, Morris GE, Barone KM, Horgan PG, Timony GA, Angus LB, Henson KS, Stoudemire JB, Langer-Safer PR, Larsen GR. Site-directed mutagenesis in human tissue-plasminogen activator. J Biol Chem 1990; 265: 5540-5545