Polymerization of Plasminogen Activator Inhibitor-1

Abstract

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into ␤-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and ␤-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-Asheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving ␤-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet. Plasminogen activator inhibitor type 1 (PAI-1)1 is a member of the serine proteinase inhibitor or serpin superfamily (1, 2). Serpins play an important role in the control of proteinases involved in blood coagulation, complement activation, and inflammation and are distinguished functionally from other types of protein inhibitors by their ability to form SDS stable complexes with target proteinases. Crystal structures have shown that members of the family share a highly conserved tertiary fold consisting of three large ␤-sheets surrounded by nine ␣-helices. This scaffold presents the reactive loop as a pseudosubstrate that binds to and inhibits the target proteinase (3). The target proteinases of PAI-1 are urokinase-type plasminogen activator and tissue-type plasminogen activator (tPA) (4) and as such it is an important modulator of events of extracellular proteolysis in fibrinolysis and in the turnover of extracellular matrix (5).One of the most striking features of serpins is their ability to undergo a dramatic conformational rearrangement with the N-terminal portion of the reactive loop inserting into ␤-sheet A (6). This transition with cleavage of the loop is central to the formation of a stable inhibitory complex (7-10), but it can also occur spontaneously in vivo, without cleavage of the loop, to form an inactive latent conformation (11-14). Moreover, antithrombin and ␣ 1 -antitrypsin can be induced to adopt a latent conformation by heating in stabilizing concentrations of sodium citrate (15-17). Serpins are also able to link their reactive loop to a ␤-sheet of another molecule to form loop-sheet polymers, one form of which (18) has recently been crystallized (19,20). These polymers are of considerable importance because they underlie the deficiency of ␣...

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

“…This antiparallel ␤-sheet interaction between strands 1 of sheet C (orange) results in a continuous sheet C from molecule A to molecule B. The stability of such an interface has been demonstrated by the observation of similar interactions in crystals and in solution for AT (7,36) and plasminogen activator inhibitor-1 (37,38). The interface between the two monomers also corresponds to the position of the fragment of the acidic tail observed in the structure.…”

Mutant Neuroserpin (S49P) That Causes Familial Encephalopathy with Neuroserpin Inclusion Bodies Is a Poor Proteinase Inhibitor and Readily Forms Polymers in Vitro

“…The bound protein was eluted with a NaCl gradient (0 -1 M), dialyzed against 50 mM Tris-HCl, 50 mM KCl, pH 7.4, concentrated, and then stored at Ϫ70°C. Purified neuroserpin migrated as a single band on SDS-PAGE and Ͼ90% was in a monomeric form when assessed by nondenaturing and transverse urea gradient PAGE (39).…”

Inhibitory Activity of a Heterochromatin-associated Serpin (MENT) against Papain-like Cysteine Proteinases Affects Chromatin Structure and Blocks Cell Proliferation

“…Native Protein Gel Electrophoresis-Complexes between MENT and cathepsins L or V were visualized using a modified version of a previously described native acid gel protocol (25), in which the stacking gel, running gel, and tank buffers were 90 mM acetate, pH 6; 260 mM acetate, pH 5; and 40 mM ␤-alanine, pH 5, respectively. Reaction mixtures with a final volume of 20 l containing 4 M MENT and 0 -4 and 8 M cathepsin V or 4 M human cathepsin L in pH 5.5 buffer were incubated at room temperature for 5 min prior to electrophoresis.…”