The p11 Subunit of the Annexin II Tetramer Plays a Key Role in the Stimulation of t-PA-Dependent Plasminogen Activation

Abstract

Annexin II tetramer (AIIt) is an important endothelial cell surface protein receptor for plasminogen and t-PA. AIIt, a heterotetramer, is composed of two p36 subunits (called annexin II) and two p11 subunits. In this report, we have compared the ability of the isolated p36 and p11 subunits to stimulate t-PA-dependent [Glu]plasminogen activation. The fluid-phase recombinant p11 subunit stimulated the rate of t-PA-dependent activation of [Glu]plasminogen about 46-fold compared to an approximate stimulation of 2-fold by the recombinant p36 subunit and 77-fold by recombinant AIIt. The stimulation of t-PA-dependent activation of [Glu]plasminogen by the p11 subunit was Ca2+-independent and inhibited by epsilon-aminocaproic acid. [Glu]Plasminogen bound to a p11 subunit affinity column and could be eluted with epsilon-aminocaproic acid. Both AIIt and the p11 subunit protected t-PA and plasmin from inactivation by PAI-1 and alpha2-antiplasmin, respectively. A peptide to the C terminus of the p11 subunit (85-Y-F-V-V-H-M-K-Q-K-G-K-K-96) inhibited the p11-dependent stimulation of t-PA-dependent plasminogen activation. In addition, a deletion mutant of the p11 subunit, missing the last two C-terminal lysine residues, retained only about 15% of the activity of the wild-type p11 subunit. Similarly, a mutant AIIt composed of the wild-type p36 subunit and the p11 subunit deletion mutant possessed about 12% of the wild-type activity. These results, therefore, suggest that the C-terminal lysine residues of the p11 subunit bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen by AIIt.

RNA Interference-mediated Silencing of the S100A10 Gene Attenuates Plasmin Generation and Invasiveness of Colo 222 Colorectal Cancer Cells

“…62). Candidate plasminogen receptors possessing carboxyl-terminal lysines include S100A10 (31,33,46), cytokeratin-8 (47-49), TIP49a (50), and ␣-enolase (51)(52)(53). Although the identity of the one or more plasminogen receptors that participate in plasminogen activation is at present controversial, our observation that plasminogen binding was reduced by 45% in S100A10 knock-down cells establishes S100A10 as a key plasminogen receptor of colorectal cells.…”

“…We originally reported that recombinant S100A10 stimulated plasmin formation about 46-fold (31,33,39). Because S100A10 possesses two lysines at its carboxyl terminus, we postulated that the carboxyl-terminal lysines of S100A10 played a key role in the stimulatory activity of AIIt (33).…”

“…40 -45). Both plasminogen and uPA interact with specific cellsurface receptors: plasminogen binds to receptors such as S100A10 (31,33,35,46), cytokeratin-8 (47-49), TIP49a (50), and ␣-enolase (51-53), whereas uPA binds to its receptor, uPAR. As shown in Fig.…”

“…In addition to an intracellular distribution, it has also been established that the heterotetrameric form of S100A10 is present on the extracellular surface of many cells (26 -32). Extracellularly, the S100A10 subunit functions as a plasminogen receptor (33,34). The penultimate and ultimate carboxyl-terminal lysines of this subunit bind tPA and plasminogen (35) and regulate the stimulation of tPA-dependent plasminogen activation (36).…”

The C Terminus of Annexin II Mediates Binding to F-actin

“…Purification of Recombinant p11-The production of the recombinant p11 construct and the subsequent protein purification scheme have been previously reported (27,28). Briefly, bacteria were grown and induced with isopropyl-1-thio-␤ -D-galactopyranoside as described above for annexin II.…”