Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site.
Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 x 10(6) and 1.0 x 10(6) t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 microgram/ml for K2tu-PA and 1 microgram/ml for FK2tu-PA, as compared to 0.5 microgram/ml and > 2 micrograms/ml for t-PA and scu-PA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Colucci
M
M
Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Bari, Italy.
Cavallo
L G
LG
Agnelli
G
G
Mele
A
A
Bürgi
R
R
Heim
J
J
Semeraro
N
N
D015195:Drug Design; D005337:Fibrin / Q000494:pharmacology; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000494:pharmacology*
D005343:Fibrinolytic Agents; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator