Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA.
The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.
Cheng
X F
XF
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
Brohlin
M
M
Pohl
G
G
Bäck
O
O
Wallén
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D008024:Ligands; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator