Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4–P3′ residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 β-sheet B, and the 147-loop directly contacts PAI-1 β-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.     

The contribution of the exosite residues of plasminogen activator inhibitor-1 to proteinase inhibition

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.     

Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes

Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.     

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13–800-fold increase in K_d values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.     

急性脑梗死患者介入治疗的疗效及对患者纤溶系统的影响

目的:研究急性脑梗死患者脑血管球囊成形支架置入术治疗的临床疗效及其对患者纤溶系统的影响。方法:选择我院收治的急性脑梗死患者68例,随机分为观察1组和观察2组,各34例,观察1组给予尿激酶100万U静脉溶栓治疗;观察2组给予脑血管球囊成形支架置入术治疗,术后口服氯吡格雷和阿司匹林。观察两组患者治疗前、治疗后1d、7d组织型纤溶酶原激活物(t PA)、血浆血管性假血友病因子(v WF)、纤溶酶原激活物特异性抑制物(PAI-1)水平,并选择同期体检健康者30例作为对照组。结果:治疗后观察2组血流再通明显高于观察1组(P0.05);治疗前所有患者v WF、PAI-1、t PA明显高于对照组,t PA/PAI-1明显低于对照组(P0.05),但观察1组和观察2组比较无统计学差异(P0.05);治疗后1d观察1组、观察2组t PA、t PA/PAI-1明显升高,PAI-1明显降低(P0.05),治疗后7d,观察1组t PA、t PA/PAI-1明显降低,观察2组v WF明显升高(P0.05)。结论:脑血管球囊成形支架置入术治疗急性脑梗死可使梗死的血管再次通畅,术后采用抗凝及抗血小板治疗,效果显著,且对体内纤溶系统无明显影响,相比静脉溶栓治疗临床效果更加显著。     

Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator: CUTTING EDGE AND CLEAVAGE SITES*

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at (¹⁷⁷GR↓KR¹⁸⁰) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically “silent”) channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.     

Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin

Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescence changes in P1'-Cys and P9-Cys PAI-1 variants labeled with the fluorophore, NBD, indicative of a substrate-like interaction of the serpin reactive loop with the proteinase active-site, with the P1' label but not the P9 label perturbing the interactions by 10-60-fold. Rapid kinetic analyses of the labeled PAI-1-inactive enzyme interactions were consistent with a single-step reversible binding process involving no conformational change. Blocking of PAI-1 reactive loop-beta-sheet A interactions through mutation of the P14 Thr --> Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding of the inactive enzymes, suggesting that loop-sheet interactions were unlikely to be induced by the binding. Only active trypsin and tPA induced the characteristic fluorescence changes in the labeled PAI-1 variants previously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinase reaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of the P1'-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling the noncovalent complex on the path to the covalent complex. Indistinguishable K(M) and k(lim) values of approximately 20 microM and 80-90 s(-1) for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limited association of PAI-1 and trypsin and rate-limiting acylation step, insensitive to the effects of labeling, controlled covalent complex formation. By contrast, differing values of K(M) of 1.7 and 0.1 microM and of k(lim) of 17 and 2.6 s(-1) for tPA reactions with P1' and P9-labeled PAI-1s, respectively, suggested that tPA-PAI-1 exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 and tPA and reversible formation of an acyl-enzyme complex but impeded a rate-limiting burial of the reactive loop leading to trapping of the acyl-enzyme complex. Together, the results suggest a kinetic pathway for formation of the covalent complex between PAI-1 and proteinases involving the initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalent complex.     

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial.     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.     

Rational Design of Complex Formation between Plasminogen Activator Inhibitor-1 and Its Target Proteinases

Considerable progress in understanding the mechanism of inhibition of proteinases by serpins has been obtained from different biochemical studies. These studies reveal that stable serpin/proteinase complex formation involves insertion of the reactive-site loop of the serpin and occurs at the acyl-enzyme stage. Even though no three-dimensional structure of a serpin/proteinase complex is resolved, structural information is available on some of the individual compounds. Molecular modeling techniques combined with recently acquired biochemical/biophysical data were used to provide insight into the stable complex formation between plasminogen activator inhibitor-1 (PAI-1) and the target proteinases: tissue-type plasminogen activator, urokinase-type plasminogen activator, and thrombin. This study reveals that PAI-1 initially interacts with its target proteinase when its reactive-site loop is solvent exposed and thereby accessible for the proteinase. Stable complex formation, however, involves the insertion of the reactive-site loop up to P7 and results in a tight binding geometry between PAI-1 and its target proteinase. The influence of different biologically relevant molecules on PAI-1/proteinase complex formation and the differences in inhibition rate constants observed for the different proteinases can be explained from these models.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

The variable region-1 from tissue-type plasminogen activator confers specificity for plasminogen activator inhibitor-1 to thrombin by facilitating catalysis: release of a kinetic block by a heterologous protein surface loop.

Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer PAI-1 specificity to a heterologous serine protease. A structural and kinetical approach to establish the function of the VR1 loop of t-PA in the context of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombin structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active site was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI-1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inactive Ser195-->Ala variants of thrombin and thrombin-VR1(tPA) is only increased fivefold, i.e. KD is 652 and 128 nM for thrombin-S195A and thrombin-S195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate reaction between the proteases and PAI-1 was largely unaffected in any variant studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kinetic modeling demonstrated that acylation is the rate-limiting step in thrombin inhibition by PAI-1 (k approximately 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k>1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop of PAI-1, thereby enhancing the rate of acylation in the Michaelis complex between thrombin-VR1(t-PA) and PAI-1 by more than two orders of magnitude.     

叶下珠有效部位的溶栓作用及其对PAI-1和tPA活性的影响

采用改良的Charlton和Tomihisa等方法评价叶下珠植物(Phyllanthus urinaria)含corilagin的水溶性有效部位(代号PUW)对电刺激大鼠颈动脉血栓的溶栓作用;应用发色底物方法测定PUW在体内外对血浆tPA、血浆或血小板释放的PAl-1活性的影响。结果显示,5mg／kg的PUW,其再通率为50％,再栓率为60％;10mg／kg PUW的血管再通率为60％,其再栓率为33．3％,低于2万U／kg尿激酶42．9%的再栓率。再通后1h内,血管开放状态表现为,2万U／kg尿激酶组的血管开放状态与5mg／kg PUW组的相似;10mg／kg PUW组,其持续再通率高于2万U／kg的尿激酶组。PUW在体外或静脉注射均明显降低血浆PAI—1活性,同时提高血浆tPA的活性;PUW静注还明显抑制血小板释放的PA1-1活性。本实验结果提示,PUW静脉注射显著提高闭塞颈动脉的再通率,同时降低再通后颈动脉的再栓率;抑制PAI—1活性,同时提高tPA的活性可能是PUW具有较好溶栓作用的分子机制。     

Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis

The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA−/− MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA−/− MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis.     

The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the mammalian fibrinolytic/plasminogen system (29, 37). The fibrinolytic system comprises the serine protease zymogen plasminogen, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), PAI-1, and plasmin inhibitor α₂-antiplasmin (α₂AP) (for a review, see reference 52). Plasminogen is converted to plasmin, which is a broad-spectrum serine protease that dissolves fibrin in blood clots, degrades laminin of basement membranes, and activates matrix metalloproteinases that degrade collagens and gelatins in tissue barriers. Herewith, plasmin controls physiological processes such as fibrinolysis/coagulation, cell migration and invasion, and tumor metastasis (29, 37). PAI-1 maintains normal hemostasis by inhibiting the function of the plasminogen activators tPA and uPA, which are serine proteases and highly specific for cleavage of the plasminogen molecule. tPA binds to fibrin and is associated with plasmin-mediated breakdown of fibrin clots, whereas uPA has low affinity for fibrin and associates with cell surface proteolysis, cellular migration, and damage of tissue barriers (52).The mammalian fibrinolytic and coagulation systems are targeted by invasive bacterial pathogens during infection (reviewed in references 6, 11, 34, and 61). In bacterial sepsis, increased production of fibrin clots at a damaged endothelium results from enhanced thrombin-catalyzed fibrin generation and from an increased serum level of PAI-1. Coagulation can protect the host by activating immune systems or by physically restraining the bacteria (6, 15, 25, 41). On the other hand, several invasive bacterial pathogens enhance fibrinolysis either through direct plasminogen activation or by immobilizing plasminogen/plasmin on the surface (6, 34, 61). Activation of the plasminogen system by bacteria enhances bacterial dissemination and invasiveness through release of bacteria from fibrin deposits and through degradation of tissue barriers. Bacterial plasminogen activators and receptors have been under extensive structural and functional studies, but much less is known about interactions of bacteria with the regulatory proteins of fibrinolysis.PAI-1 is present in a large variety of tissues and is secreted by several human cells (37). In healthy individuals, the level of PAI-1 antigen in human plasma is low (6 to 85 ng/ml), but synthesis and secretion of PAI-1 are strongly elevated in disease states and induced by, e.g., inflammatory cytokines and endotoxin of Gram-negative bacteria (37). PAI-1 is a serine protease inhibitor (serpin), which exists in two forms. In its active form, PAI-1 rapidly inactivates both tPA and uPA by forming a covalent bond between the hydroxyl group of a catalytic serine residue of tPA/uPA and the carboxyl group of the residue R346 at the reactive center loop (RCL) of PAI-1 (52). The RCL of PAI-1 is a 19-amino-acid-long flexible loop which inserts into the catalytic center of tPA/uPA and contains the “bait” residues R346 and M347, which mimic the normal target of tPA/uPA. PAI-1 induces distortion of the active site of tPA/uPA, which prevents completion of the catalytic cycle (70). The active form of PAI-1 is unstable, with a half-life of 2 to 3 h at 37°C, and it changes spontaneously and irreversibly into a latent form, where the RCL is incorporated into a central β-sheet of the PAI-1 molecule and therefore cannot react with tPA or uPA. This conformational change takes place also after proteolytic cleavage of PAI-1 at the R346-M347 bond. The active form of PAI-1 binds with high affinity to vitronectin (Vn), and PAI-1/Vn complex formation increases the half-life of PAI-1 2- to 4-fold (10, 46, 69). Most circulating PAI-1 is thought to be in a complex with Vn, and the complex serves as the reservoir of physiologically active PAI-1 (44).Plague disease caused by Yersinia pestis is associated with imbalance of the fibrinolytic system, and decreased fibrin(ogen) deposition has been observed in both bubonic and pneumonic plague (11, 36). The plasminogen activator Pla, which is encoded by a Y. pestis-specific 9.5-kb virulence plasmid, pPCP1 (59), does not degrade fibrin directly but mimics the action of tPA and uPA in converting plasminogen to plasmin by cleavage at R561-V562. Pla also degrades the serpin α₂AP and thus creates uncontrolled plasmin activity (32, 60). Pla belongs to the omptin superfamily of bacterial β-barrel outer membrane proteases (for reviews of omptins, see references 21 and 23). The omptins share molecular size and transmembrane fold but differ markedly in their substrate selectivities. In their catalytic centers, omptins combine structural features of aspartic and serine proteases (66).Increased fibrinolysis observed in plague led us to investigate whether Y. pestis increases plasminogen activation also indirectly by controlling the activity of PAI-1. We compared Y. pestis to Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis, and the study also included omptins of other enterobacterial species.     

Differential Expression of Urokinase-Type Plasminogen Activator (uPA), Its Receptor (uPA-R), and Inhibitor Type-2 (PAI-2) during Differentiation of Keratinocytes in an Organotypic Coculture System

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-I stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.     

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

Background

We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE).

Methods

Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α₂-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl₂, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405).

Results

Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT.

Conclusion

The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.     

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.     

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC₅₀ value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC₅₀ of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K_D of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.