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The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable and active conformation, with a solvent-accessible reactive center loop (RCL), to a stable, inactive, or latent conformation, with the RCL inserted into the central β-sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin, which hinders the latency transition rate approximately twofold. The molecular mechanisms leading to this rate change are unclear. Here, we investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central β-sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition; this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site, thus blocking transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that were previously identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin-bound state, corresponding to a cryptic drug-binding site identified by crystallography. Overall, these results shed new light on PAI-1 latency transition regulation by vitronectin and illustrate the potential of path sampling simulations for understanding functional protein conformational changes and for facilitating drug discovery., Competing Interests: Conflict of interest This article was prepared while A.G. was associated with the University of Massachusetts Amherst. P. F and G. S. are cofounders of Sibylla Biotech (www.sibyllabiotech.it), a company involved in early stage drug discovery. P. F., G. S., L. T., and A. B. are shareholders of Sibylla Biotech. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Factors associated with postthrombotic syndrome are known clinically, but the underlying cellular processes at the vein wall are not well delineated. Prior work suggests that vein wall damage does not correlate with thrombus resolution but rather with plasminogen activator-1 (PAI-1) and matrix metalloproteinase (MMP) activity., Objective: We hypothesized that PAI-1 would confer post venous thrombosis (VT) vein wall protection via a vitronectin (Vn)-dependent mechanism., Methods: A stasis model of VT was used with harvest over 2 weeks, in wild-type, Vn(-/-) , and PAI-1-overexpressing mice (PAI-1 Tg)., Results: PAI-1 Tg mice had larger VT at 6 and 14 days, compared to controls, but Vn(-/-) mice had no alteration of VT resolution. Gene deletion of Vn resulted in an increase in, rather than the expected decrease in, circulating PAI-1 activity. While both Vn(-/-) and PAI-1 Tg had attenuated intimal fibrosis, PAI-1 Tg had significantly less vein wall collagen and a compensatory increase in collagen III gene expression. Both Vn(-/-) and PAI-1 Tg vein wall had less monocyte chemotactic factor-1 and fewer macrophages (F4/80), with significantly less MMP-2 activity and decreased TIMP-1 antigen. Ex vivo assessment of transforming growth factor β-mediated fibrotic response showed that PAI-1 Tg vein walls had increased profibrotic gene expression (collagens I and III, MMP-2, and α-smooth muscle actin) compared with controls, opposite of the in vivo response., Conclusions: The absence of Vn increases circulating PAI-1, which positively modulates vein wall fibrosis in a dose-dependent manner. Translationally, PAI-1 elevation may decrease vein wall damage after deep vein thrombosis, perhaps by decreasing macrophage-mediated activities., (© 2014 International Society on Thrombosis and Haemostasis.)

Plasminogen activator inhibitor-1 (PAI-1) promotes or abates fibrotic processes occurring in different organs. Binding of PAI-1 to vitronectin, an extracellular matrix component, may inhibit vitronectin-integrin complex-mediated cellular responses in pathophysiological conditions. To investigate the importance of plasmin suppression vs vitronectin-binding pathways of PAI-1 in cardiac fibrosis, we studied uninephrectomized mice fed a high salt diet and infused with angiotensin II (Ang II) together with different PAI-1 variants, including PAI-1AK (AK) that inhibits plasminogen activators but does not bind vitronectin, PAI-1RR (RR) that binds vitronectin but does not have protease inhibitory effects or control PAI-1 (CPAI), the control mutant that has similar molecular backbone and half-life as AK and RR while retaining all functions of native PAI-1. Compared with RR and CPAI, non-vitronectin-binding AK significantly increased expression of cardiac fibroblast marker, periostin (Ang+AK 8.40±3.55 vs Ang+RR 2.23±0.44 and Ang+CPAI 2.33±0.12% positive area, both P<0.05) and cardiac fibrosis (Ang+AK 1.79±0.26% vs Ang+RR 0.91±0.18% and Ang+CPAI 0.81±0.12% fibrotic area, both P<0.05), as well as Col1 mRNA (Ang+AK 12.81±1.84 vs Ang+RR 4.04±1.06 and Ang+CPAI 5.23±1.21 fold increase, both P<0.05). To elucidate mechanisms underlying the protective effects of vitronectin-binding PAI-1 against fibrosis, fibroblasts from normal adult human ventricles were stimulated with Ang and different PAI-1 variants. Protease inhibitory AK and CPAI increased supernatant fibronectin, while decreasing plasminogen activator/plasmin activities and matrix metalloproteinase. RR and CPAI variants significantly reduced fibroblast expression of integrin β3, vitronectin level in the supernatant and fibroblast adhesion to vitronectin compared with the non-vitronectin-binding AK. Further, RR and CPAI preserved apoptotic, decreased anti-apoptotic and proliferative activities in fibroblasts. Thus, PAI-1 promotes or protects against development of cardiac fibrosis differentially through the protease inhibitory pathway or through its binding to vitronectin.

Purpose: Vitronectin (VN) in provisional extracellular matrix (ECM) promotes cell migration. Fibrotic ECM also includes VN and, paradoxically, strongly adherent myofibroblasts (Mfs). Because fibrotic Mfs secrete elevated amounts of urokinase plasminogen activator (uPA), we tested whether increased extracellular uPA promotes the persistence of Mfs on VN., Methods: Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free medium on VN or collagen (CL) with 1 ng/mL transforming growth factor β1 (TGFβ1). Adherent cells were quantified using crystal violet. Protein expression was measured by Western blotting and flow cytometry. Transfection of short interfering RNAs was performed by nucleofection. Mfs were identified by α-smooth muscle actin (α-SMA) stress fibers. Plasminogen activator inhibitor (PAI-1) levels were quantified by ELISA., Results: TGFβ1-treated HCFs secreted PAI-1 (0.5 uM) that bound to VN, competing with αvβ3/αvβ5 integrin/VN binding, thus promoting cell detachment from VN. However, addition of uPA to cells on VN increased Mf differentiation (9.7-fold), cell-adhesion (2.2-fold), and binding by the VN integrins αvβ3 and -β5 (2.2-fold). Plasmin activity was not involved in promoting these changes, as treatment with the plasmin inhibitor aprotinin had no effect. A dominant negative PAI-1 mutant (PAI-1R) that binds to VN but does not inhibit uPA prevented the increase in uPA-stimulated cell adhesion and reduced uPA-stimulated integrin αvβ3/αvβ5 binding to VN by 73%., Conclusions: uPA induction of TGFβ1-dependent Mf differentiation on VN supports the hypothesis that elevated secretion of uPA in fibrotic tissue may promote cell adhesion and the persistence of Mfs. By blocking uPA-stimulated cell adhesion, PAI-1R may be a useful agent in combating corneal scarring.

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

Background: Vascular smooth muscle cell (VSMC) migration is a critical process in arterial remodeling. Purified plasminogen activator inhibitor-1 (PAI-1) is reported to both promote and inhibit VSMC migration on two-dimensional (D) surfaces., Objective: To determine the effects of PAI-1 and vitronectin (VN) expressed by VSMC themselves on migration through physiological collagen matrices., Methods: We studied migration of wild-type (WT), PAI-1-deficient, VN-deficient, PAI-1/VN doubly-deficient (DKO) and PAI-1-transgenic (Tg) VSMC through three-D collagen gels., Results: WT VSMC migrated significantly slower than PAI-1- and VN-deficient VSMC, but significantly faster than DKO VSMC. Experiments with recombinant PAI-1 suggested that basal VSMC PAI-1 expression inhibits migration by binding VN, which is secreted by VSMC and binds collagen. However, PAI-1-over-expressing Tg VSMC migrated faster than WT VSMC. Reconstitution experiments with recombinant PAI-1 mutants suggested that the pro-migratory effect of PAI-1 over-expression required its anti-plasminogen activator (PA) and LDL receptor-related protein (LRP) binding functions, but not VN binding. While promoting VSMC migration in the absence of PAI-1, VN inhibited the pro-migratory effect of active PAI-1., Conclusions: In isolation, VN and PAI-1 are each pro-migratory. However, via formation of a high-affinity, non-motogenic complex, PAI-1 and VN each buffers the other's pro-migratory effect. The level of PAI-1 expression by VSMC and the concentration of VN in extracellular matrix are critical determinants of whether PAI-1 and VN promote or inhibit migration. These findings help to rectify previously conflicting reports and suggest that PAI-1/VN stoichiometry plays an important role in VSMC migration and vascular remodeling., (© 2010 International Society on Thrombosis and Haemostasis.)

Objective: Plasminogen activator inhibitor-1 (PAI-1) overexpression is implicated in vascular disease. However, the effects of a primary increase in PAI-1 expression on arterial remodeling are poorly defined. We tested the hypothesis that recombinant PAI-1 inhibits intimal hyperplasia after vascular injury., Methods and Results: Rats underwent carotid artery injury and received intraperitoneal injections of saline or mutant forms of PAI-1 for 14 days, including an active stable mutant (PAI-1-14-1b), a mutant lacking anti-PA activity (PAI-1-R), or a mutant defective in vitronectin (VN) binding (PAI-1-K). All forms of PAI-1 significantly inhibited neointima formation, whereas elastase-cleaved PAI-1, which lacks both anti-PA and VN-binding functions, did not. Similar effects were observed in a murine model. However, the antiproliferative effect of PAI-1-R was lost in Vn(-/-) mice, suggesting that PAI-1 can inhibit intimal hyperplasia in vivo by a VN-dependent pathway not involving direct inhibition of proteases. In vitro, recombinant PAI-1 inhibited wild-type vascular smooth muscle cell (VSMC) proliferation, promoted apoptosis, and inhibited migration. These effects were lost in VN-deficient VSMCs., Conclusions: Recombinant PAI-1 inhibits intimal hyperplasia by inhibiting proteases and binding VN. VN is a key determinant of the antiproliferative effect of PAI-1 overexpression. PAI-1-R has therapeutic potential to inhibit vascular restenosis without promoting thrombosis.

Administration of a mutant, noninhibitory PAI-1 (PAI-1R), reduces disease in experimental glomerulonephritis. Here we investigated the importance of vitronectin (Vn) binding, PAI-1 stability and protease binding in this therapeutic effect using a panel of PAI-1 mutants differing in half-life, protease binding, and Vn binding. PAI-1R binds Vn normally but does not inhibit proteases. PAI-1AK has a complete defect in Vn binding but retains full inhibitory activity, with a short half-life similar to wild-type (wt)-PAI-1. Mutant 14-lb is identical to wt-PAI-1 but with a longer half-life. PAI-1K has defective Vn binding, inhibits proteases normally, and has a long half-life. In vitro wt-PAI-1 dramatically inhibited degradation of mesangial cell ECM while the AK mutant had much less effect. Mutants 14-1b and PAI-1K, like wt-PAI-1, inhibited matrix degradation but PAI-1R failed to reverse this inhibition although PAI-1R reversed the wt-PAI-1-induced inhibition of ECM degradation in a plasmin-, time-, and dose-dependent manner. Thus the ability of PAI-1 to inhibit ECM degradation is dependent both on its antiproteinase activity and on maintaining an active conformation achieved either by Vn binding or mutation to a stable form. Administration of these PAI-1 mutants to nephritic rats confirmed the in vitro data; only PAI-1R showed therapeutic effects. PAI-1K did not bind to nephritic kidney, indicating that Vn binding is essential to the therapeutic action of PAI-1R. The ability of PAI-1R to remain bound to Vn even in a high-protease environment is very likely the key to its therapeutic efficacy. Furthermore, because both PAI-1R and 14-1b bound to the nephritic kidney in the same pattern and differ only in their ability to bind proteases, lack of protease inhibition is also keyed to PAI-1R's therapeutic action.

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into beta-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with beta-anhydrotrypsin, which does not undergo rearrangement of beta-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to beta-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1.plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.

During wound healing, migrating cells increase expression of both the vitronectin receptor (VNR) integrins and plasminogen activators. Here we report that vitronectin significantly enhances the migration of smooth muscle cells (SMCs), and that the specific VNR alpha V beta 3 is required for cell motility. We also show that the alpha V beta 3 attachment site on vitronectin overlaps with the binding site for plasminogen activator inhibitor (PAI)-1, and that the active conformation of PAI-1 blocks SMC migration. This effect requires high-affinity binding to vitronectin, and is not dependent on the ability of PAI-1 to inhibit plasminogen activators. Formation of a complex between PAI-1 and plasminogen activators results in loss of PAI-1 affinity for vitronectin and restores cell migration. These data demonstrate a direct link between plasminogen activators and integrin-mediated cell migration, and show that PAI-1 can control cell-matrix interactions by regulating the accessibility of specific cell-attachment sites. This indicates that the localization of plasminogen activators at sites of focal contact does not initiate a proteolytic cascade leading to generalized matrix destruction, but instead is required to expose cryptic cell-attachment sites necessary for SMC migration.

Thrombin is a multifunctional protein that has both proteinase and growth factor-like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha2-macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN). In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, 125I-thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-proteinase inhibitor, or d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I-thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of 125I-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury.