Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- α2 -Macroglobulin Interaction

• Published in: Journal of biochemistry (Tokyo) 1998-08, Vol.124 (2), p.274-279
• Main Authors: Sánchez, María C., Chiabrando, Gustavo A., Guglielmone, Hugo A., Bonacci, Gustavo R., Rabinovich, Gabriel A., Vides, Miguel A.
• Format: Article
• Language: English
• Subjects: fibrinolysis; pregnancy zone protein; tissue plasminogen activator; trypsin; α2-macroglobulin
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a022107

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to α2-macroglobulin (α2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and α2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas α2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and α2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with a-Ms-chymotrypsin; (ii) both PZP and α2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85–90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on α2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85–90 kDa. The appearance of free SH-groups of α-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of α2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.