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.
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.
Ahern
T J
TJ
Genetics Institute Inc., Cambridge, Massachusetts 02140.
Morris
G E
GE
Barone
K M
KM
Horgan
P G
PG
Timony
G A
GA
Angus
L B
LB
Henson
K S
KS
Stoudemire
J B
JB
Langer-Safer
P R
PR
Larsen
G R
GR
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
D011994:Recombinant Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator