Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA-  2 -Macroglobulin Interaction

Abstract

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-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 alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-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 alpha2-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 alpha-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 alpha2-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.

Low‐density lipoprotein receptor‐related protein mediates in PC12 cell cultures the inhibition of nerve growth factor‐promoted neurite outgrowth by pregnancy zone protein and α2‐macroglobulin

“…We and others have previously reported that PZP and human α 2 M show different abilities to interact with proteinases (Arbelaez et al, 1997; Sánchez et al, 1998; Saidi et al, 1999; Ramos et al, 2002). In the present study, we show that both human αMs have similar or complementary functions on NGF‐promoted neurite outgrowth in PC12 cells, but, in rats, activated α 1 M exhibits a neurostimulatory effect (Lee and Koo, 2000), which is clearly different from the neuroinhibitory activity of activated α 2 M (Lee and Koo, 1999).…”

“…The bait region sequence is markedly different in PZP compared with human α 2 M as well as α 1 M and rat α 2 M, and it is, in fact, the region with the lowest similarity, suggesting different or complementary functions for the proteinase inhibition exerted by these αMs (Garton et al, 1991). In this sense, recently we demonstrated that PZP interacts more quickly than human α 2 M with tissue‐type plasminogen activator (t‐PA), promoting its clearance via the main αM receptor known as low‐density lipoprotein (LDL) receptor‐related protein (LRP) (Sánchez et al, 1998; 2001) and suggesting that PZP, and not α 2 M, may contribute to the control of the activity of proteinases derived from the fibrinolytic system during pregnancy.…”

Molecular Circuits of Resolution: Formation and Actions of Resolvins and Protectins

“…S100A9 is a cytosolic PMN protein that can be secreted and exhibits potent actions on inflammatory cell recruitment (35). In addition, ␣ 1 - macroglobulin, similar to ␣ 2 -macroglobulin, binds several proteinases including tissue-plasminogen activator and promotes their clearance (36). Thus, it is possible that ␣ 1 -macroglobulin, like ␣ 2 -macroglobulin, also plays a role in the removal of proinflammatory cytokines in the exudate and/or lowering these effective local concentrations before the onset of resolution (37).…”

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

“…However, a 2 M Ã only recognizes LRP1 and not any other LDL receptor members [Hussain et al, 1999]. In addition, a 2 M Ã is internalized by LRP1, implying that both molecules are involved in the modulation of the extracellular activity of several proteases [Sanchez et al, 1998;Herz and Strickland, 2001;Ramos et al, 2002]. LRP1 is a cell surface glycoprotein synthesized as a 600-kDa proreceptor and post-translationally processed into 515-kDa a-subunit and 85-kDa b-subunit that remain associated through non-covalent interactions [Herz and Strickland, 2001].…”