title: Site-directed mutagenesis in human tissue-plasminogen activator. Distinguishing sites in the amino-terminal region required for full fibrinolytic activity and rapid clearance from the circulation.
abstract: Recombinant variants of tissue plasminogen activator (t-PA) containing either substitutions or deletions of amino acids within the fibronectin finger-like domain (residues 6-50) were found to exhibit widely varying in vivo clearance profiles in rats and fibrinolytic activity in 125I-fibrin clot lysis assays. Clearance was not significantly affected by changes in the densely charged region of amino acid residues 7-10. Deletions or substitutions of amino acids in the region 14-32 decreased both fibrinolytic activity and the clearance of the enzyme. Modifications within the predicted omega loop of residues 37-41 affected clearance only to a small degree, whereas amino acid alterations in the region of residues 42-49 resulted in as much as a 6-fold decrease in the rate of clearance with only relatively minor decreases in the fibrinolytic activity of the variants. The cumulative results distinguish discrete sections of the NH2-terminal region of the enzyme as determinants of in vivo clearance and fibrinolytic activity of t-PA. In addition, the fibrinolytic activity of a variant containing the substitutions Gln42----Asn, His44----Glu, and Asn117----Gln, when compared with wild-type t-PA in an in vivo rabbit venous clot lysis model, was found to have similar lytic efficacy at approximately one-fourth the dose. We conclude that decreases in the in vivo clearance of t-PA can result in more potent thrombolytic agents in vivo, even though the in vitro fibrinolytic activity of the enzyme may be somewhat impaired.
authors.affiliation: Genetics Institute Inc., Cambridge, Massachusetts 02140.
authors.forename: T J
authors.initials: TJ
authors.lastname: Ahern
authors.forename: G E
authors.initials: GE
authors.lastname: Morris
authors.forename: K M
authors.initials: KM
authors.lastname: Barone
authors.forename: P G
authors.initials: PG
authors.lastname: Horgan
authors.forename: G A
authors.initials: GA
authors.lastname: Timony
authors.forename: L B
authors.initials: LB
authors.lastname: Angus
authors.forename: K S
authors.initials: KS
authors.lastname: Henson
authors.forename: J B
authors.initials: JB
authors.lastname: Stoudemire
authors.forename: P R
authors.initials: PR
authors.lastname: Langer-Safer
authors.forename: G R
authors.initials: GR
authors.lastname: Larsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005342:Fibrinolysis; D006801:Humans; D008297:Male; D008657:Metabolic Clearance Rate; D008969:Molecular Sequence Data; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000097:blood* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator