Effect of plasminogen activators on human recombinant apolipoprotein(a) having the plasminogen activation cleavage site.
The serine-proteinase domain in human apolipoprotein(a) [apo(a)] and plasminogen exhibit 89% sequence identity including the catalytic triad. Cleavage of the Arg(561)-Val(562) activation site in plasminogen by either tissue- or urokinase-type plasminogen activator results in formation of the fibrinolytic enzyme plasmin. Apo(a) does not contain measurable amidolytic activity nor can it be activated by plasminogen activators. It has been suggested that the latter finding might be explained by the substitution of the plasminogen Arg-Val activation site by Ser-Ile in apo(a). To investigate if introduction of the Arg-Val activation site in apo(a) might result in sensitivity towards plasminogen activators, we expressed wild-type and Arg-Val mutant recombinant apo(a) [r-apo(a)] in human embryonic kidney and hepatocyte cell lines. Free r-apo(a) and lipoprotein-like particles [r-Lp(a)] were obtained in the culture supernatants of transfected 293 and HepG2 cells, respectively. Incubation of mutant r-apo(a)/r-Lp(a) with plasminogen activators produced neither plasmin-like activity nor cleavage at the Arg-Val activation site, even in the presence of various stimulators of plasminogen activation. Our data suggest that the high selectivity of activators for plasminogen activation requires interactions with regions in plasminogen distant from the activation disulfide loop which are not present in apo(a).
Hervio
L
L
Scripps Research Institute, TSRI-VB3, La Jolla, CA, USA.
Brunner
C
C
Sorell
L
L
Kang
C
C
Müller
H
H
Anglés-Cano
E
E
D000595:Amino Acid Sequence; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018076:DNA, Complementary / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D018082:Kringles; D017270:Lipoprotein(a) / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D010958:Plasminogen / Q000737:chemistry; D010960:Plasminogen Activators / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry
D001054:Apolipoproteins A; D018076:DNA, Complementary; D017270:Lipoprotein(a); D011994:Recombinant Proteins; D010958:Plasminogen; D010960:Plasminogen Activators; D012697:Serine Endopeptidases