High resolution analysis of functional determinants on human tissue-type plasminogen activator.
Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.
Bennett
W F
WF
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
Paoni
N F
NF
Keyt
B A
BA
Botstein
D
D
Jones
A J
AJ
Presta
L
L
Wurm
F M
FM
Zoller
M J
MJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D005337:Fibrin / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D005337:Fibrin; D010959:Tissue Plasminogen Activator