Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1

Xu, Zhi; Balsara, Rashna D.; Gorlatova, Natalia; Lawrence, Daniel A.; Castellino, Francis J.; Ploplis, Victoria A.

doi:10.1074/jbc.m314197200

Cited by 31 publications

(35 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3E, suggesting that the shift in efficacy is due to a decrease in the affinity of CDE-096 for the PAI-1/SMB complex. To confirm that the shift in IC 50 was not due to a direct effect of SMB on CDE-096, the experiment was repeated with mutant PAI-1 AK (R101A and Q123K), this PAI-1 variant inhibits uPA normally but does not interact with SMB (39). These data showed that there was no difference in inactivation of PAI-1 AK by CDE-096 in the presence (Fig.…”

Section: Resultsmentioning

confidence: 71%

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Reinke

Sanders

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration-approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family.fibrinolysis | thrombolysis | fibrosis | cancer P lasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, chronic fibrotic and inflammatory diseases, and tumor invasion and metastasis (1-6). These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7-16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.PAI-1 is the most potent physiologic inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively) (17). Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19). However, before the protease can dissociate from the serpin, a dramatic conformational change occurs [termed the stressed to relaxed (S to R) transition], in which the cleaved RCL inserts into the central β-sheet A, translocating the covalently-bound protease 70 Å to the base of β-sheet A (20). This conformational change results in distortion of the protease active site (21,22) and thereby prevents the deacylation reaction, inactivating the protease. Should this conformational change be interrupted, the protease can complete its cleavage of the serpin RCL, remaining active and leaving the serpin in a cleaved, inactive, loop-inserted state (23).PAI-1 is unique among serpins in that it readily autoinactivates into a so-called latent form, where the PAI-1 ...

show abstract

Section: Resultsmentioning

confidence: 71%

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Reinke

Sanders

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…In initial experiments, recombinant [wild-type (WT)] PAI-1 or the PAI-1 mutants R76E and PAI-R101A/Q123K, which do not bind to LRP or vitronectin, respectively, were used (37,43). Apoptosis was inhibited to a similar degree by culture of neutrophils with R76E or R101A/Q123K and with WT PAI-1 (Fig.…”

Section: Pai-1-dependent Activation Of Phosphatidylinositol 3-kinase mentioning

confidence: 89%

Inhibition of neutrophil apoptosis by PAI-1

Żmijewski¹,

Bae

Deshane

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

Increased circulating and tissue levels of plasminogen activator inhibitor 1 (PAI-1) are often present in severe inflammatory states associated with neutrophil activation and accumulation and correlate with poor clinical outcome from many of these conditions. The mechanisms by which PAI-1 contributes to inflammation have not been fully delineated. In the present experiments, we found that addition of PAI-1 to neutrophil cultures diminished the rate of spontaneous and TNF-related apoptosis-inducing ligand-induced apoptotic cell death. The effects of PAI-1 on cell viability were associated with activation of antiapoptotic signaling pathways, including upregulation of PKB/Akt, Mcl-1, and Bcl-xL. Although urokinase-plasminogen activator receptor, lipoprotein receptor-related protein, and vitronectin are primary ligands for PAI-1, these molecules were not involved in mediating its antiapoptotic properties. In contrast, blocking pertussis toxin-sensitive G protein-coupled receptors and selective inhibition of phosphatidylinositide 3-kinase reversed the ability of PAI-1 to extend neutrophil viability. The antiapoptotic effects of PAI-1 were also evident under in vivo conditions during LPS-induced acute lung injury, where enhanced apoptosis was present among neutrophils accumulating in the lungs of PAI-1−/−compared with PAI-1+/+mice. These results demonstrate a novel antiapoptotic role for PAI-1 that may contribute to its participation in neutrophil-associated inflammatory responses.

show abstract

“…Although the above experiments indicated that LRP is involved in PAI-1 mediated reduction of neutrophil phagocytosis, they did not establish whether direct interaction of PAI-1 with LRP was required for this effect. To examine this question, and the requirement for PAI-1's protease inhibitory activity, in modulating phagocytosis of viable neutrophils, we preincubated viable neutrophils with the PAI-1 mutants R76E, which is unable to bind to LRP, or R346A, which is incapable of inhibiting protease activity (23), and then determined the phagocytic index. Both of these PAI-1 mutants were as effective as wild-type PAI-1 in inhibiting the enhanced phagocytosis of PAI-1 Ϫ/Ϫ neutrophils or of WT neutrophils preincubated with anti-CD47 antibodies (Fig.…”

Section: Suppression Of Phagocytosis By Pai-1 Does Not Require Proteamentioning

confidence: 99%

PAI-1 inhibits neutrophil efferocytosis

Park

Liu²,

Lorne³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

103

105

View full text Add to dashboard Cite

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical for the resolution of inflammation. Plasma and tissue levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, are elevated in inflammatory conditions, including sepsis and acute lung injury, in which activated neutrophils accumulate in tissues and contribute to organ dysfunction. In this study, we explored the potential involvement of PAI-1 in modulating neutrophil efferocytosis. We found enhanced phagocytosis of viable PAI-1 deficient (PAI-1 ؊/؊ ) and of wild-type neutrophils treated with anti-PAI-1 antibodies. PAI-1 levels were decreased on the surface of apoptotic neutrophils and the enhanced phagocytosis of apoptotic wild-type neutrophils or of viable PAI-1 ؊/؊ neutrophils was diminished by preincubation with PAI-1. The increased phagocytosis associated with PAI-1 deficiency or blockade depended on both the lipoprotein receptor-related protein (LRP) and its ligand, calreticulin (CRT), because the LRP-mediated increase in phagocytosis of viable neutrophils induced by blockade of CD 47 was abrogated by PAI-1. CRT levels are increased on viable PAI-1 ؊/؊ neutrophils. While CRT colocalizes with PAI-1 on viable neutrophils, markedly diminished colocalization of PAI-1 and CRT was present on apoptotic neutrophils. Our data therefore indicate that PAI-1 serves as a novel ''don't eat me'' signal for viable and apoptotic neutrophils.calreticulin ͉ phagocytosis ͉ plasminogen activator inhibitor-1

show abstract

Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1

Cited by 31 publications

References 38 publications

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Inhibition of neutrophil apoptosis by PAI-1

PAI-1 inhibits neutrophil efferocytosis

Contact Info

Product

Resources

About