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 I-125-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspension s of cultured human umbilical vein EC (HUVEC) or immortalized microvas cular EC (HMEC). By determinations of the concentration-dependent bind ing it was shown that both the A-chain and the B-chain, which were iso lated after partial reduction of two-chain tPA, contain ligands for bi nding 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, respective ly), whereas the number of binding sites was higher for the A-chain th an for the B-chain (Bmax 8x10(5) and 1.2x10(5), respectively). There w ere no cross interactions between the A- and B-chains and their bindin g 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 f ree 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. Bo th tPA and the tPA B-chain were largely protected against inhibition b y an excess plasminogen activator type-1 (PAI-1) when bound to EC, whe reas the same amount of free tPA was totally inactivated. The competit ion studies strongly indicated that an N-terminal segment in the B-cha in, AKHRRSPGER, may be the ligand part of the B-chain. It is interesti ng to note that this polypeptide segment also participates in a bindin g site for PAI-1, necessary for effective inhibition. This implies a p ossible competition between PAI-1 and a tPA-receptor for binding of tP A. High molecular weight urokinase had no quenching effect on the bind ing of the B-chain to EC.