THE ACTIVATION OF TYPE-1 AND TYPE-2 PLASMINOGEN BY TYPE-I AND TYPE-II TISSUE-PLASMINOGEN ACTIVATOR

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogem and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D* (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the K-m value for activation by all tPA variants; N-glycosylation of type I tPA at Asn(184) decreased the k(cat) (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D* tPA showed the highest turnover rate, In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.