Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor 1

Characterization of a Novel Class of Polyphenolic Inhibitorsof Plasminogen Activator Inhibitor-1*□S

Received for publication, September 18, 2009, and in revised form, January 4, 2010 Published, JBC Papers in Press, January 8, 2010, DOI 10.1074/jbc.M109.067967

Jacqueline M. Cale‡1, Shih-Hon Li‡1, Mark Warnock‡, Enming J. Su‡, Paul R. North§, Karen L. Sanders§,Maria M. Puscau§, Cory D. Emal§, and Daniel A. Lawrence‡2

From the ‡Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan Medical School, AnnArbor, Michigan 48109-0644 and the §Department of Chemistry, Eastern Michigan University, Ypsilanti, Michigan 48197

Plasminogen activator inhibitor type 1, (PAI-1) the primary

inhibitor of the tissue-type (tPA) and urokinase-type (uPA)

plasminogen activators, has been implicated in a wide range of

pathological processes, making it an attractive target for phar-

macologic inhibition. Currently available small-molecule inhib-

itors of PAI-1 bind with relatively low affinity and do not inac-

tivate PAI-1 in the presence of its cofactor, vitronectin. To

search for novel PAI-1 inhibitors with improved potencies and

new mechanisms of action, we screened a library selected to

provide a range of biological activities and structural diversity.

Five potential PAI-1 inhibitors were identified, and all were

polyphenolic compounds including two related, naturally

occurring plant polyphenols that were structurally similar to

compounds previously shown to provide cardiovascular benefit

in vivo. Unique second generation compoundswere synthesized

and characterized, and several showed IC50 values for PAI-1

between 10 and 200 nM. This represents an enhanced potency of

10–1000-fold over previously reportedPAI-1 inactivators. Inhi-

bition of PAI-1 by these compounds was reversible, and their

primarymechanismof actionwas to block the initial association

of PAI-1with a protease. Consistentwith thismechanism and in

contrast to previously described PAI-1 inactivators, these com-

pounds inactivate PAI-1 in the presence of vitronectin. Two of

the compounds showed efficacy in ex vivo plasma and one

blocked PAI-1 activity in vivo in mice. These data describe a

novel family of high affinity PAI-1-inactivating compoundswith

improved characteristics and in vivo efficacy, and suggest that

the known cardiovascular benefits of dietary polyphenols may

derive in part from their inactivation of PAI-1.

Plasminogen activator inhibitor type 1 (PAI-1)3 is the pri-mary physiologic inhibitor of uPA and tPA with a well charac-

terized role in fibrinolysis (1). PAI-1 also plays a role in manyphysiologic processes, including angiogenesis, wound healing,and cell migration (2–6), and has been implicated in fibroticdiseases of the kidney and lung, and in tumormetastasis (7–11).More recently, PAI-1 has been linked to obesity and metabolicsyndrome (12–16), and to the development of vascular diseasessuch as venous thrombosis and atherosclerosis (17–19). Theprospect that PAI-1 may play a direct role in the early develop-ment of a variety of diseases has made it an attractive target fordrug development (20, 21). However, the structural complexityof PAI-1 hasmade the identification and development of PAI-1inhibitors challenging. This is due in part to the metastablestructure of PAI-1, which can adopt several different conforma-tions, including active, latent, cleaved, and protease complexed(1). These different forms of PAI-1 provide conformationalcontrol of PAI-1 interactions and dictate its localization toeither matrix or the cell surface and control its activity in cellsignaling events (22, 23).Active PAI-1 inhibits protease targets and is associated with

vitronectin in plasma or the extracellular matrix. In contrast,PAI-1-protease complexes shift affinity from vitronectin toreceptors of the low density lipoprotein receptor family, trans-ferring PAI-1 from vitronectin to the cell surface (22). ActivePAI-1 is inherently unstable and undergoes a spontaneous con-formational change that results in inactivation of PAI-1 to alatent form that does not bind either vitronectin or low densitylipoprotein receptor familymembers with high affinity (22, 24).The flexible structure of PAI-1, the lack of a rigid active site, andits multiple functions all contribute to the difficulties in identi-fying and designing small-molecule PAI-1 inactivators. Despitethese obstacles, several small-molecule PAI-1 inhibitors havebeen described (25–36); however, each has significant limita-tions that have reduced their potential for further drugdevelopment.One of the best characterized compounds is PAI-039, also

known as tiplaxtinin, which has been shown to reduce physio-logic PAI-1 activity and to be efficacious in animal models ofdisease (3, 37–39). However, PAI-039 has relatively low affinityfor PAI-1, and does not inhibit vitronectin-bound PAI-1 (32,40). To develop better PAI-1 inactivators, we screened a libraryof known compounds for high affinity PAI-1 inhibitors withimproved solubility and activity against vitronectin-boundPAI-1. A high throughput screen of the MicroSource SPEC-TRUM library identified five novel PAI-1 inactivating com-pounds. Two of the molecules identified were related naturalpolyphenolic compounds, which suggested a potential struc-

* This work was supported, in whole or in part, by National Institutes of HealthGrants HL55374, HL54710, and HL089407 (to D. A. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Methods” and Figs. S1–S4.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: 7301 MSRB III, 1150 W.

Medical Center Dr., Ann Arbor MI 48109-0644. Tel.: 734-763-7838; Fax: 734-936-2641; E-mail: [email protected].

3 The abbreviations used are: PAI-1, plasminogen activator inhibitor type 1;uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogenactivator; PAI-1glyco, glycosylated active human PAI-1; mPAI-1, murine PAI-1;CDE, an arbitrary designation based on the initials of one of the authors; SPR,surface plasmon resonance; IVC, inferior vena cava; TA, tannic acid; EGCDG,epigallocatechin-3,5-digallate; EGCG, epigallocatechin monogallate; CCG,Center for Chemical Genomics; PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 11, pp. 7892–7902, March 12, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

7892 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 11 • MARCH 12, 2010

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http://www.jbc.org/content/suppl/2010/01/08/M109.067967.DC1.htmlSupplemental Material can be found at:

ture-activity relationship. Second generation compounds weredesigned and synthesized to probe this structure-activity rela-tionship and tested for their ability to block PAI-1 activity inboth purified systems and in vivo.

EXPERIMENTAL PROCEDURES

Primary Screen—In conjunction with the Center for Chemi-cal Genomics (CCG) at the University of Michigan, we devel-oped a PAI-1 activity assay to screen for compounds with anti-PAI-1 activity in the MicroSource SPECTRUM compoundcollection. This collection consists of knowndrugs, compoundsapproved for agricultural use, natural products, and other bio-active compounds. A chromogenic assay was used with a 2:1molar ratio of PAI-1 to uPA. We selected uPA because it isconsiderably more active toward low molecular weight sub-strates than tPA, allowing for more than 10-fold lower concen-trations of uPA and PAI-1 in this screen (5 nM uPA and 10 nMPAI-1) compared with assays using tPA (70 nM tPA and 140 nMPAI-1) (41). The screen was performed in 384-well microti-ter plates in the CCG lab as follows: recombinant active humanPAI-1 (final 10 nM) was incubated for 60 min at 23 °C eitherwith or without 10 mM of each compound in high throughputscreen buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005%Tween 20), uPA was added (final 5 nM) to each reaction well,and the incubation continued for an additional 30min at 23 °C.Residual uPA activity in each reaction mixture was then deter-mined with p-Glu-Gly-Arg p-nitroanilide chromogenic sub-strate (Sigma) (final 0.25 mM) measured spectrophotometri-cally at 405 nmafter 60min.Compounds that inactivated PAI-1were identified by the restoration of uPA activity. The extent ofuPA activity restoration was determined by comparing eachdrug-containing sample to wells with untreated PAI-1 (100%PAI-1 activity) and to wells with uPA only (0% PAI-1 activity).The data from this screen were then uploaded to the CCGinformatics systemandpositive hitswere identified as any com-pound that increased uPA activity by more than 3 S.D. abovecontrol and compound wells on each plate. Using these selec-tion criteria, the primary screen of 2000 compounds yielded aninitial total of 23 compounds deemed positive hits. Each ofthese hits was then re-assayed by dose-response testing usingthe same chromogenic assay with the compounds at the follow-ing concentrations (0.1, 0.32, 1, 3.2, 10, 32, and 100 mM) induplicate by the CCG. In this secondary analysis 19 of the 23compounds were deemed positive; however, 3 of these com-pounds were known to have significant toxicity and thereforewere not analyzed further. Samples of the 16 remaining com-pounds were then obtained from the CCG for further analysisin our laboratory. These more detailed analyses first investi-gated whether each compound had intrinsic absorbance at 405nm that would give false positive absorbance readings, or wasnot completely soluble in the assay buffer system used becauseinsolubility and compound precipitation could likewise lead tofalse positive absorbance readings. Each compound was alsotested for its ability to directly block PAI-1 complex formationwith uPA by SDS-PAGE analysis. For this latter analysis eachcompound was incubated at 10 mM with 1 mg of PAI-1 for 15min at 23 °C followed by the addition of 1 mg of uPA for anadditional 5 min at 37 °C. Approximately half of the 16 com-

pounds either had intrinsic absorbance at 405 nm or insolubil-ity in the buffer system.Of the remaining compounds, 5 directlyinhibited PAI-1 activity.Enzymatic Assays—Recombinant nonglycosylated or glyco-

sylated active humanPAI-1 (PAI-1 andPAI-1glyco, respectively)or recombinant murine PAI-1 (mPAI-1) was incubated at 2 nMfor 15min at 23 °Cwith increasing concentrations of each com-pound in assay buffer (40 mM HEPES, pH 7.8, 100 mM NaCl,0.005% Tween 20, 0.1% Me2SO), followed by the addition ofuPA (Molecular Innovations) or tPA (Genentech) to 3 nM andfurther incubation for 30min at 23 °C. At each drug concentra-tion, parallel control reactions without PAI-1 were assembled.Residual enzymatic activity was determined by addition of anequal volume of 100 mM Z-Gly-Gly-Arg-AMC (Calbiochem)fluorogenic substrate for uPA or Pefafluor tPA (Centerchem)for tPA, and the rate of AMC release monitored at 23 °C (exci-tation 370 nm and emission 440 nm). The percent change inPAI-1 activity was determined according to Equation 1,

@~E i 2 P i!/E i#/@~E0 2 P0!/E0# (Eq. 1)

where Ei is the enzyme activity at drug concentration i; Pi is theenzyme activity in the presence of PAI-1 at drug concentrationi; E0 is the enzyme activity in the absence of drug; and P0 is theenzyme activity in the presence of PAI-1 in the absence of drug.The effect of the compounds on 2 nM anti-thrombin III in thepresence of 3 units/ml of heparin was also determined using 3nM a-thrombin. The reactions were assembled as above exceptthat 10% Me2SO was included in the assay buffer to ensurecompound solubility at the higher concentrations used. Resid-uala-thrombin activity wasmeasured using an equal volume of100 mM benzoyl-Phe-Val-Arg-AMC (Calbiochem).Synthesis of New Inhibitors—Synthetic procedures and spec-

troscopic data for CDE compounds are provided in supplemen-tal “Methods”.Surface Plasmon Resonance (SPR) Analysis—Direct binding

of PAI-1 treated with vehicle or inhibitor to anhydrotrypsin(Molecular Innovations) was monitored using a Biacore 2000t

optical biosensor. Bovine anhydrotrypsin was immobilized toCM5 SPR chips at a level of ;2000 response units in 10 mM

sodium acetate, pH 5.0. The reference flow cell surface was leftblank to serve as a control. Remaining binding sites wereblocked by 1 M ethanolamine, pH 8.5. All binding reactionswere performed in assay buffer. PAI-1 at 2 nM was first incu-bated with the indicated concentrations of inhibitor in assaybuffer for at least 15min at 23 °C. Binding of PAI-1 to anhydro-trypsin was then monitored at 25 °C at a flow rate of 30 ml/minfor 2.5 min, followed by 2 min of dissociation. Chip surfaceswere regenerated with a 1-min pulse of 10 mM glycine, pH 1.5,followed by a 1-min wash of assay buffer. Injections were per-formed using the Wizard Customized Application program inautomated mode. Binding experiments were performed induplicate and corrected for background and bulk refractiveindex by subtraction of the reference flow cell, and data wereanalyzed with BIAevaluation 3.1 (Biacore) by linear fitting ofthe initial association phase. Compound-induced alterations inPAI-1 binding to anhydrotrypsin were determined by compar-ing the initial slopes of the association phases because there is a

A Novel Class of PAI-1 Inactivating Compounds

MARCH 12, 2010 • VOLUME 285 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7893

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linear relationship between the slope and the concentration ofavailable active PAI-1 (supplemental Fig. S1 and Ref. 32). Thesedata were then fit to an exponential association equation todetermine the apparent affinity between PAI-1 and compound.To monitor the inhibition of vitronectin-bound PAI-1,

human vitronectin purified under non-denaturing conditionswas coupled to a CM5 sensor chip to a surface density of;1000response units (32). Five nM PAI-1 was injected over the chip ata rate of 20 ml/min at 25 °C for 4 min, followed by assay bufferalone or 100 nM CDE-066 in assay buffer for 10 min, and then100 nM uPA for 5 min. After injections of PAI-1 or CDE-066,the chip was washed with assay buffer for ;4 min. Resultswere corrected for background and bulk refractive index inBIAevaluation 3.1.SDS-PAGE/Western Blotting—Human PAI-1 at 2 nM was

incubated with the indicated concentrations of the compoundfor 15 min at 23 °C in assay buffer, followed by a 30-min incu-bation with 3 nM uPA or tPA. Samples were analyzed via reduc-ing SDS-PAGE with 10% Tris-HCl gels (Bio-Rad) and trans-ferred onto polyvinylidene difluoride overnight. PAI-1 wasdetected using polyclonal high-titer sheep anti-human PAI-1antibody (Molecular Innovations), horseradish peroxidase-conjugated donkey anti-sheep IgG (Jackson ImmunoResearchLaboratories), and Pierce ECL Western blotting substrate(Thermo Scientific).Reversibility Assay—The reversibility of PAI-1 inactivation

by the compounds was performed essentially as described (32).PAI-1 (2 nM) was incubatedwith each compound at 3–5-fold itsIC50 value as determined using uPA, followed by serial 2-folddilutions into assay buffer and further incubation to allow dis-sociation of the compounds fromPAI-1. UPA (3.5 nM) was thenadded and PAI-1 activity was determined. The PAI-1 activity incompound-containing samples recovered at each dilution wascalculated as a percentage of the activity in PAI-1 dilutions car-ried out in parallel without compound. Initial concentrations ofcompounds were 150 nM CDE-008, 75 nM CDE-031, 400 nMCDE-034, 300 nM CDE-056, 50 nM CDE-066, and 50 nMCDE-082.Inhibition of mPAI-1 in ex Vivo Plasma—Murine PAI-1 was

added to PAI-1-depleted murine plasma (Molecular Innova-tions) at 5000 pg/ml. Ten microliters of increasing concentra-tions of compound in assay buffer containing 10% Me2SO and10 ml of mPAI-1-reconstituted plasma were incubated for 15min at 23 °C in a filter plate (Millipore), followed by the additionof 25 ml of SeroMAP beads (Luminex) coupled to uPA (2500beads/well), and further incubated in the dark on a microtiterplate shaker for 2 h. The plate was vacuumwashed 3 times withwash buffer (PBS, pH 7.4, 0.05% Tween 20), then 50 ml of PBS,pH 7.4, 1% bovine serum albumin, and 50 ml of 4 mg/ml ofbiotin-labeled rabbit anti-mPAI-1 (Molecular Innovations) wasadded to each well and the plate incubated at 23 °C in the darkon a microtiter plate shaker for 1 h. After vacuum washing 3times, 50ml of PBS, pH7.4, 1% bovine serumalbumin, and 50mlof 4 mg/ml of streptavidin-R-phycoerythrin conjugate (Molec-ular Probes) was added to eachwell and incubatedwith shakingat 23 °C for 30 min in the dark. After washing another 3 times,100ml of sheath fluid (Luminex) was added to eachwell, shakenfor 5 min in the dark at 23 °C, and read on a Luminex100

(mediansetting,50-ml samplesize,100events/bead).Meanfluo-rescence intensities of unknown samples were converted topicograms/ml of base on a standard curve of mPAI-1 in mPAI-1-depleted plasma using a five-parameter regression formula(Masterplex QT version 4.0, Miraibio).PlasmaEnzymatic Assay—Citrated bloodwas collected from

the inferior vena cava (IVC) of C57Bl6J mice that were eitherPAI-1 null or vitronectin/PAI-1 null and plasma were preparedby centrifugation (15 min at 1500 3 g). The plasma was treatedwith 10 mg/ml aprotinin (Roche Applied Science) for 15 min at23 °C before reconstituting with 20 nM PAI-1. Plasma (10 ml,with orwithout PAI-1) was placed inmicrotiter wells with 80mlof CDE-066 or PAI-039, synthesized as described (40) in assaybuffer containing 10% Me2SO and incubated for 15 min at23 °C, followed by addition of 10 ml of 25 nM uPA, and a furtherincubation for 30 min. Residual enzymatic activity was moni-tored as above using the fluorogenic uPA substrate, and PAI-1activity was determined using Equation 1.Inhibition of PAI-1 in Vivo—Transgenic mice heterozygous

formurine PAI-1 overexpression (10) were weighed, then anes-thetizedwith isoflurane for the duration of the experiment. TheIVC was isolated and 50 ml of citrated blood was collected aspre-treatment samples. The syringewas replacedwith a syringecontaining vehicle or CDE-066 (in lactated Ringers) and 100 mlwas injected for doses of 3, 10, and 30mg/kg. After 1 h, 300ml ofcitrated blood was collected via IVC, after which the mice wereeuthanized. Plasma was isolated by centrifugation at 1500 3 gfor 15 min at 23 °C. All animal experiments were approved bythe Institutional Animal Care and Use Committee of Unit forLaboratory AnimalMedicine at the University of Michigan. Todetermine active murine PAI-1 levels in the plasma, 10 ml ofplasma, diluted in PAI-1-depleted murine plasma (MolecularInnovations), 10 ml of buffer (PBS, pH 7.4, 1% bovine serumalbumin), and 25 ml of uPA-coupled SeroMAP beads wereadded to a filter plate and incubated by shaking overnight at4 °C in the dark, and the reactions were analyzed as above in theex vivo plasma assay.Data and Statistical Analysis—Data were analyzed and IC50

values were calculated using Grafit 5. Apparent KD values forthe binding of compounds to PAI-1 were determined usingGraphPad Prism 4. Data were analyzed for significance with aStudent’s t test using non-diluted samples in the reversibilityassays and 0 mg/kg of CDE-066 treatment in the in vivo assaysas the control groups, with p , 0.05 considered significant.

RESULTS

High Throughput Screen—The MicroSource SPECTRUMcompound library was screened under stringent conditionssuch that PAI-1 was present at a 2-fold molar excess over uPA,and each compound was tested at a concentration of 10 mM.The statistical criteria of 3 S.D. above the control and com-pound means on each plate resulted in 23 hits. These com-pounds were further tested by dose-response analysis, and 19remained positive in this secondary screen. Of these, 16 weredeemed safe and subjected to further study including SDS-PAGE analysis of complex formation between PAI-1 and uPA.Based on these analyses, 5 compoundswere confirmed as PAI-1inhibitors in both enzymatic and SDS-PAGE assays, yielding a



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final hit rate of 0.25%. The structures and IC50 values of these 5compounds along with two related compounds are shown inFig. 1.Each of these five compounds contain polyphenolicmoieties,

and three of them, tannic acid (TA), epigallocatechin-3,5-digal-late (EGCDG), and sennoside A, are naturally occurring plantpolyphenols with reported biological activities (42–46). Theformer two compounds, TA and EGCDG, have highly relatedstructures that both contain galloyl or gallo-galloyl moietiessuggesting the possibility of a structure-activity relationshipbetween polyphenols in general, and more specifically gallicacid moieties and PAI-1 inactivation. We therefore examinedtwo additional galloyl-containing compounds, epigallocatechinmonogallate (EGCG) and gallic acid (Fig. 1, B and F). Mono-meric gallic acid was 1000-fold less active toward PAI-1 thanTA, whereas EGCG inhibited PAI-1 only ;10-fold less wellthan TA. Thus, each of the galloyl-containing compounds wasable to inhibit PAI-1, but the efficacy of inhibition appears de-pendent on the number of galloyl units in each compound andtheir relative orientation or context.Synthetic Compounds—The IC50 value obtained with 7 nM

TA was ;1000-fold lower than our previously reported IC50

value for the PAI-1 inactivator, PAI-039 (32), and is markedlylower than any previously reported small-molecule PAI-1 inac-tivating compound (25–36). Likewise the IC50 values obtainedwith EGCG and EGCDG were also significantly better thanPAI-039 andmost other PAI-1 inactivators, suggesting that gal-loyl-containing compounds may represent a potent new familyof PAI-1 inactivating compounds. However, TA is not an idealdrug candidate as its molecular mass of nearly 2000 daltons isconsidered too large, and it was subject to aggregation atmicro-molar concentrations. Therefore, we synthesized a series ofnovel compounds containing different numbers of galloyl moi-eties in different structural configurations and compared theiractivity against PAI-1 to determine a potential structure-activ-ity relationship between galloyl-containing compounds andPAI-1 inhibition. In addition, to make this analysis sensitive toinactivatorswith lownanomolar IC50 values, the PAI-1 concen-tration in the assay was lowered from 10 to 2 nM. Using theseoptimized assay conditions, we were able to accurately deter-mine IC50 values for several novel compounds. Six of thesecompounds, four digallates, one trigallate, and one pentagal-late, are shown in Fig. 2. Comparison of the IC50 values of these6 compounds demonstrated IC50 values ranging from 10 to 174nM for inactivation of PAI-1 (Fig. 2 and Table 1). The activity ofeach compound against glycosylated human PAI-1 (PAI-1glyco)and murine PAI-1 (mPAI-1) was also compared with nongly-cosylated recombinant human PAI-1 (PAI-1) (Table 1). In gen-eral the compounds inhibited PAI-1glyco as well as the nongly-cosylated form; however, most inhibited mPAI-1 less well thanhuman PAI-1. The two exceptions were the pentagallate, CDE-066, and TA, which inhibited all forms of PAI-1 equally well.The inactivation of PAI-1 by the polyphenolic compounds

was specific, because only TA and CDE-082 (IC50 . 10 mM)showed any inhibition of the related serpin anti-thrombin III.Some of the gallate-containing compounds tested did show anapparent inhibition of tPA in assays with a chromogenic orfluorogenic substrate; however, little inhibition of tPA by these

compounds was seen when the physiologic substrate of tPA,plasminogen, was used (supplemental Fig. S2), suggesting thatthe compounds may be interacting with the low molecularweight tPA substrates.It is also apparent from these data that although a single

gallate (gallic acid, 6.6mM) is a relatively poor inhibitor of PAI-1,a minimum of two galloyl units translates into significant anti-PAI-1 activity (20–116 nM, Fig. 2 and Table 1). CompoundCDE-008 was compared with several similar digallates withlinkers of different lengths between the gallate moieties, andCDE-008 was found to have the optimal distance between thegalloyl units (data not shown). To further explore structuralrequirements for digalloyl compound inhibition of PAI-1, weexamined 1,2-disubstituted galloyl units on different ring struc-tures to determine whether cis (CDE-031), trans (CDE-034), orplanar (CDE-056) relationships between galloyl units inhibitedPAI-1 more effectively. All of these compounds were activeagainst PAI-1 with the cis form (CDE-031) being ;2-fold moreactive against PAI-1 than the acyclic CDE-008. These datademonstrate that the relative orientation of the gallates isimportant for anti-PAI-1 activity, with the cis form inhibitingPAI-1 ;4-fold better than the planar form and ;6-fold betterthan the trans form (Table 1).SPR Analysis—To establish binding constants for the drugs

to PAI-1, an indirect approach using SPR was employed. Vary-ing concentrations of each drug were preincubated with PAI-1in solution and then passed over immobilized anhydrotrypsin,and the loss of PAI-1 binding to anhydrotrypsin was quantified.Wehave previously shown for PAI-1 binding to vitronectin (32)that the slope of the association phase of PAI-1 binding to animmobilized ligand has a linear relationship with the concen-tration of available active PAI-1 in solution. This relationship isalso true for PAI-1 binding to immobilized anhydrotrypsin(supplemental Fig. S1). Thus, when the slopes of the associationphase are plotted as a percent of control PAI-1 binding in theabsence of compound versus the concentration of the com-pound, an IC50 can be calculated for the drug-induced inhibi-tion of PAI-1 interaction with anhydrotrypsin (Fig. 3). From atransformation of these data, the apparent KD values for all sixcompounds binding to PAI-1 can be calculated (Table 2). TheapparentKD values range from3.1 to 67 nM and are significantlytighter than the previously reported values for other PAI-1inactivators (28, 32, 33, 40). These values are also similar to theIC50 values calculated in PAI-1 inhibition assays (Table 1).These data indicate that drug binding interferes with the initialassociation of PAI-1 with the protease and can block formationof the PAI-1-protease Michaelis-like complex.SDS-PAGE—Each compound was also tested for its ability to

block complex formation between PAI-1 and PAs, and exam-ples of CDE-008, CDE-066, and CDE-082 are shown in Fig. 4.For these studies each compound was incubated with PAI-1,then either uPA or tPA was added and the formation of SDS-stable complexes was monitored by SDS-PAGE. The concen-trations of PAI-1 and PAs were the same as those used in theenzyme assays (see Fig. 1 and Table 1), and we observed com-parable IC50 values between the two techniques. Inhibition ofcovalent complex formation also closely mirrored inhibition ofPAI-1 binding to anhydrotrypsin (see Fig. 3 and Table 2). An



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increase in PAI-1 cleavage was also noted with each compoundprimarily at compound concentrations just below the IC50;however, this was modest compared with the near completeloss of covalent complex, and much less cleavage was observedat compound concentrations above the IC50. Together with theSPR studies, these data suggest that the principal mechanism ofPAI-1 inactivation by these compounds is the inhibition of thePAI-1 Michaelis-like complex formation with PAs, but that atconcentrations near the IC50 some increase of PAI-1 substratebehavior may be induced.Inactivation of PAI-1 Is Reversible—To test whether the inhi-

bition of PAI-1 by the synthetic polyphenols was reversible,PAI-1 and each synthetic compound was incubated at a con-centration where most of the anti-proteolytic activity of PAI-1was abolished. The mixtures were then serially diluted to

reduce the compound concentra-tions, incubated for an additional 30min, and themixtures tested for res-toration of PAI-1 activity. Fig. 5shows that for each synthetic poly-phenol, PAI-1 activity increasedupon dilution, indicating that PAI-1inactivation by the compounds isreversible. The extent of PAI-1activity recovered with each com-pound was slightly less than pre-dicted, suggesting that the rate ofdissociation between PAI-1 andthese novel compounds is relativelyslow and the samples may not havereached a new equilibrium after 30min. Consistent with this mecha-nism, incubation of PAI-1 for vari-ous times with CDE-066, the mostpotent synthetic compound, dem-onstrated that the IC50 remainedunchanged from15min until termi-nation of the experiment at 4 h (sup-plemental Fig. S3). These data indi-cate that the compounds do notirreversibly modify PAI-1, and areconsistent with a high affinityreversible mechanism of action.Inhibition of mPAI-1 in Plas-

ma—Each of the new compoundswas tested for anti-PAI-1 activity inex vivo plasma. This tests the abilityof the drugs to inhibitmPAI-1 in thepresence of plasma proteins, includ-ing vitronectin. None of the newlygenerated digallate compounds

were active against mPAI-1 in the

plasma activity assay (Fig. 6). This was likely due to high non-

specific protein binding of these digallates in plasma because

the digallates were also ineffective against mPAI-1 in buffers

containing high concentrations of bovine serum albumin (data

not shown). In contrast, all of the compounds with at least 3

galloyl moieties inhibited mPAI-1 in the plasma, including the

trigallate (CDE-082), pentagallate (CDE-066), and TA. Overall,

TA had the lowest IC50 against mPAI-1 in plasma (data not

shown) but it was less specific than the novel polyphenols as it

also inhibited normal plasma clotting, whereas CDE-066 and

CDE-082 did not (supplemental Fig. S4). CDE-066 exhibited

the lowest IC50 of the new compounds in plasma, and was also

significantly more specific than TA, therefore CDE-066 was

used in further studies in plasma and in vivo.

FIGURE 1. IC50 values of PAI-1 inactivating compounds from high throughput screen and related compounds. The two-dimensional structures of the fivehits from the screen (A, C, D, E, and G) and two related compounds (B and F) are shown with IC50 values from the enzyme assays. Recombinant active humanPAI-1 (final 2 nM) was incubated for 15 min at 23 °C with increasing concentrations of each compound in assay buffer. Next uPA (final 3 nM) was added to eachreaction well and incubated for an additional 30 min at 23 °C. Activity of uPA in each reaction mixture was determined with the Z-Gly-Gly-Arg-AMC fluorogenicsubstrate (final 50 mM). UPA activity was measured fluorometrically (excitation 370 nm and emission 440 nm) for 15 min. The IC50 values were calculated usingGrafit IC50 fit and the mean 6 S.E. are based on three independent experiments. The asterisk indicate compounds identified in the original screen, and thedagger indicates related compounds not identified in the original screen.

FIGURE 2. Structures of six synthetic compounds. A, the two-dimensional structures of the 6 syntheticpolyphenolic compounds are pictured. The inhibition curves of each compound against PAI-1 in the presenceof either uPA (B) or tPA (C) are shown. The data were plotted using the Grafit IC50 fit and are based on threeindependent experiments, points represent the mean 6 S.E. For comparison PAI-039 has a reported IC50 in asimilar assay system of ;10 mM (32).



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Our previous studies (32) with PAI-039, the most widelystudied PAI-1 small-molecule inhibitor, indicated that it isunable to inhibit PAI-1 bound to vitronectin, and one of themain objectives of the current studywas to identify compoundsthat could inhibit PAI-1 in the presence of vitronectin. This wasexamined by adding a known amount of PAI-1 to murineplasma from either PAI-1 null mice or from mice doubly nullfor PAI-1 and vitronectin. After incubating the PAI-1 in theseplasmas, samples were incubated with dilutions of either CDE-066 or PAI-039 and then tested for PAI-1 inhibition of uPA. Fig.7 demonstrates that unlike PAI-039, which is only inhibitory inplasma that lacks vitronectin, CDE-066 inhibited PAI-1 equallywell in plasma with or without physiologic vitronectin.

The ability of CDE-066 to inactivate PAI-1 bound to purifiedvitronectin was verified in vitro via BIAcore. To be certain thatthe PAI-1 was in complex with vitronectin, PAI-1 was injectedover immobilized vitronectin and complex formation wasdetected by changes in relative response units. These data dem-onstrate that as expected active PAI-1 binds vitronectin withhigh affinity and dissociates very slowly from immobilizedvitronectin; however, upon reaction with uPA, PAI-1 affinityfor vitronectin is reduced by several orders of magnitude (22,24) and the PAI-1 rapidly dissociates from vitronectin, (Fig. 8,large dots), In contrast, PAI-1 bound to vitronectin and thenexposed to 100 nM CDE-066 does not dissociate from theimmobilized vitronectin following the uPA injection (Fig. 8,small dots). These data indicate that CDE-066 blocks the asso-ciation of uPAwith PAI-1 evenwhen in complexwith vitronec-tin, and are consistent with the hypothesis that the primarymechanism by which CDE-066 inactivates PAI-1 is to preventnon-covalent complex formation with target proteases. Thesedata also demonstrate that CDE-066 is not inducing PAI-1cleavage as a substrate, or latency, because both cleaved andlatent PAI-1 also exhibit low affinity for vitronectin (22, 24), andwould likewise result in loss of PAI-1 signal from the chip.Finally, consistent with the reversibility studies shown in Fig. 5,these data indicate that the dissociation of CDE-066 fromPAI-1 is relatively slowbecause even after 240 s ofwash PAI-1 isstill inhibited by CDE-066.PAI-1 Inactivation in Vivo—Finally, to examine whether

CDE-066 inhibits murine PAI-1 in vivo, mice overexpressingPAI-1 were treated acutely with either vehicle or increasingconcentrations of CDE-066. Plasma samples were removedfromeachmouse before treatment and then 1 h following intra-venous infusion of CDE-066 at the indicated concentrations(Fig. 9). Plasma sampleswere then tested for active PAI-1 levels.Although a small increase in active PAI-1 was observed in thevehicle-treated animals, a dose-dependent decrease in activePAI-1was observed after 1 h of treatmentwithCDE-066. Thesedata indicate that CDE-066 can significantly inhibit PAI-1 in

vivo.

DISCUSSION

PAI-1 is thought to play a role in several chronic “lifestyle”diseases, including cardiovascular and fibrotic diseases, andmetabolic syndrome. These pathologic associations makePAI-1 an ideal drug target; however, its metastable structurehas made it a difficult candidate for drug design and study. Todate most small-molecule inhibitors of PAI-1 lack high affinity

FIGURE 3. Apparent affinity between PAI-1 and synthetic inhibitorsassessed by SPR. Two nM PAI-1 was incubated with the concentrations indi-cated of each synthetic compound and the mixtures injected over an anhy-drotrypsin-conjugated CM5 sensor chip. The compound-dependent changein the initial association rates for PAI-1 binding to anhydrotrypsin, which isdirectly proportional to the amount of free PAI-1 in the analyte, is plottedagainst the compound dose to determine the apparent KD values of eachcompound for PAI-1. Data are based on two independent experiments;points represent the mean 6 S.E. For comparison PAI-039 has a reportedaffinity for PAI-1 of ;15 mM in a direct SPR binding assay system (32).

TABLE 1

The IC50 values of TA and six synthetic compounds against various forms of PAI-1 or anti-thrombin III using the indicated target enzymesIC50 values (nM) 6 S.E. were determined using the Grafit IC50 fit. Values are based on three independent experiments.

CompoundPAI-1 PAI-1glyco mPAI-1 Hep:anti-thrombin IIIa

uPA tPA uPA uPA a-Thrombin

TA 6.6 6 1.1 8.0 6 0.3 4.8 6 1.2 4.1 6 1.2 11,800 6 300CDE-008 44 6 5 53 6 4 28 6 2 162 6 27 .10,000b

CDE-031 20 6 1 28 6 1 18 6 1 132 6 14 .10,000b

CDE-034 116 6 11 174 6 26 169 6 21 644 6 53 .300,000CDE-056 74 6 4 86 6 8 152 6 28 758 6 26 .300,000CDE-066 10 6 1 12 6 2 13 6 2 10 6 1 .300,000CDE-082 14 6 1 18 6 1 56 6 2 79 6 4 15,400 6 4,400

a Values represent measured IC50 values or the highest concentration of compound tested.b ,20% of Hep:anti-thrombin III was inactivated at the highest compound concentration used.

TABLE 2

Affinity between PAI-1 and synthetic compounds as measured bySPRThe data from Fig. 3 were fit to an exponential association curve in GraphPad Prism4 to calculate the apparent KD. Shown are the mean 6 S.E. of two independentexperiments.

Compound Apparent KD

nMCDE-008 23 6 1CDE-031 31 6 2CDE-034 67 6 3CDE-056 51 6 6CDE-066 3.1 6 0.2CDE-082 5.3 6 0.2



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for PAI-1 and are unable to inhibit PAI-1 in the presence of itsplasma binding protein, vitronectin. To identify higher affinityinhibitors with better drug development potential, a high strin-gency screening assay was performed and a class of polyphe-nolic compounds was identified with anti-PAI-1 activity. A

subset of these with the highest anti-PAI-1 activity containedgalloyl moieties, and one, TA, demonstrated the lowest IC50 ofany small-molecule PAI-1 inhibitor yet reported. One otherstudy has identified members of the acylphloroglucinol class ofpolyphenols, sideroxylonals A–C, as potential PAI-1 inactivat-ing compounds (27).However, the reported IC50 values of thesecompounds (3.3–5.3 mM) are 2–3 orders of magnitude higherthan TA and the novel synthetic polyphenols described hereand are comparable with the IC50 of the simplest gallate com-pound in the current study, gallic acid (6.6 mM). This suggests

FIGURE 4. CDE compounds inhibit complex formation between PAI-1 anduPA or tPA. PAI-1 (2 nM) was incubated with 10-fold dilutions of CDE-008 (A),-066 (B), and -082 (C) for 15 min at 23 °C in assay buffer. Then uPA (left panels)or tPA (right panels) was added (3 nM final) and complexes were formed at23 °C for 30 min. Samples were analyzed by reducing SDS-PAGE followed bytransfer to polyvinylidene difluoride membranes and immunoblotting forPAI-1. SDS stable complexes (asterisk), unreacted PAI-1 (open arrowhead), andcleaved PAI-1 (closed arrowhead) were detected.

FIGURE 5. Inactivation of PAI-1 by the synthetic inhibitors is reversible.PAI-1 (2 nM) was incubated with the compounds shown at 3–5-fold excessconcentrations over the IC50 of each compound for 15 min, then seriallydiluted 1:1 three times and further incubated for 30 min. PAI-1 activity wasdetermined as described under “Experimental Procedures” and is shown as apercentage of control activity without compound. The data represent themean 6 S.E. of at least three independent experiments and were evaluatedagainst the activities of the undiluted samples using a Student’s t test (*, p ,0.05; **, p , 0.01).

FIGURE 6. Inhibition of mPAI-1 by synthetic compounds in ex vivo plasma.Murine plasma depleted of PAI-1 was reconstituted with 5000 pg/ml ofmPAI-1 and treated with each compound, and residual active mPAI-1detected by Luminex. Curves were generated with the Grafit IC50 fit and theIC50 6 S.E. are indicated, NI indicates no detectable inhibition. The data arebased on three independent experiments performed in duplicate.

FIGURE 7. CDE-066 but not PAI-039 inhibits PAI-1 in the presence ofvitronectin. Plasma collected from PAI-1 null or PAI-1/vitronectin null micewere reconstituted with 20 nM PAI-1, and then vehicle or PAI-1 inactivators,CDE-066 (A) or PAI-039 (B) were added at the concentrations indicated, andthe samples incubated. Residual PAI-1 activity was determined using uPA andZ-Gly-Gly-Arg-AMC as described under “Experimental Procedures.” The dataare shown as the mean 6 S.E. and are based on three independent experi-ments performed in duplicate.



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thatmany polyphenolic compoundsmay share PAI-1 inactivat-ing activity, but that the galloyl moiety may be a critical deter-minant in polyphenols for potent anti-PAI-1 activity.Despite the low IC50 of TA and its ability to inhibit PAI-1 in

the high protein environment of plasma (data not shown), it isnot an ideal drug candidate due to its molecular mass of nearly2000 daltons and its relative promiscuity, interactingwith otherproteins as well as itself at low- to mid-micromolar concentra-tions. Nonetheless, the inhibition of PAI-1 by TA and othergallate-containing molecules (EGCG, EGCDG, and gallic acid)formed the basis for development of follow-up compoundswith improved properties compared with these naturallyoccurring polyphenols. Smaller di-, tri-, and pentagallates weredesigned with improved solubility in physiologic buffers andgreater specificity toward PAI-1. These studies determined thatalthough two galloyl moieties were sufficient to provide potentanti-PAI-1 activity, aminimumof 3 galloyl groupswas requiredfor efficacy in plasma. This suggests the relationship betweenspecificity for PAI-1 and nonspecific bulk protein binding is

complex and is not dependent on only the number of galloylsubunits.The synthetic polyphenolic derivatives demonstrate clear

advantages over previous pharmacologic inactivators of PAI-1.For example most of the existing PAI-1 inhibitors exhibit IC50

values in the low- to mid-micromolar range in comparable invitro assays, which is several orders of magnitude less potentthan the best novel synthetic polyphenolic derivativesdescribed here (25–27, 29, 30, 32, 34–36). Another class ofPAI-1 inhibitors based on diketopiperazine derivatives havebeen described with in vitro IC50 values reported in the 0.2–1mM range; however, these compounds suffered from consider-able physicochemical problems such as insolubility in physio-logic buffer systems and were not subject to further develop-ment (47). CDE-066, in contrast, is soluble in physiologic salinesolution at concentrations greater than 10 mM without loss ofanti-PAI-1 activity (data not shown). Two other PAI-1 inacti-vators have been described with IC50 values reported in themid-nanomolar range; however, these compounds are ineffec-tive against vitronectin-bound PAI-1, the predominant form ofPAI-1 in plasma and the extracellular matrix (28, 33). Likewiseseveral compounds with micromolar IC50 values are also inef-fective against vitronectin bound PAI-1 (26, 32). The resistanceof vitronectin-bound PAI-1 to these inhibitors is thought to bedue to the location of the binding site for these compounds, in ahydrophobic cavity on PAI-1 that is defined by a-helices D andE and b-strands 1A and 2A, and directly adjacent to thevitronectin-binding site (26, 28, 32). In contrast, the CDE-066compound shows vitronectin-independent anti-PAI-1 activityin a purified system, in ex vivo plasma, and in vivo in PAI-1transgenic mice.The primary mechanism of action by which CDE-066 and

the other synthetic polyphenols inactivate PAI-1 appears to beby binding to PAI-1 in a reversible manner and preventing sta-bilization of the non-covalent Michaelis complex with targetproteases. This is demonstrated in Fig. 3wherein preincubationof PAI-1 with each of the compounds inhibits its binding to theinactive protease, anhydrotrypsin. Identical data were alsoobtained in similar experiments using an inactive mutant oftPA (data not shown), indicating that the effect of the com-pounds on the initial association of PAI-1 with a protease isindependent of the target protease. The SDS-PAGE analysisshown in Fig. 4 suggests that the polyphenolic compounds canalso promote substrate behavior in PAI-1. However, in contrastto the loss of Michaelis complex formation (Fig. 3) and the lossof covalent complex formation (Fig. 4) the extent of cleavageobserved is not dose dependent with the compounds added andvaries with compound and target enzyme. It is possible that theextent of cleavage may be overestimated in these experimentsdue to complex dissociation during SDS-PAGE. Note, forexample, that even in the absence of any compound, cleavedPAI-1 is apparent under experimental conditions where thestoichiometry of inhibition is near 1 (SI 5 1.06, data notshown). Finally, consistent with the primary mechanism ofaction being inhibition of PAI-1:protease association, SPRexperiments demonstrated that no CDE-066-dependent PAI-1cleavage was detected when PAI-1 bound to vitronectin wasreacted with active uPA (Fig. 8). This suggests that the combi-

FIGURE 8. Inactivation of vitronectin-bound PAI-1 by CDE-066 assessedby SPR. PAI-1 (5 nM) was injected over a vitronectin-conjugated CM5 chip,followed by 100 nM CDE-066 (small dots) or vehicle (large dots). Residualvitronectin-bound PAI-1 activity was assessed by injection of 100 nM uPA,with active PAI-1 binding to the uPA and rapidly dissociating from the chipresulting in loss of surface response units, whereas CDE-066-inactivated PAI-1remains on the chip surface after the uPA injection. The starts of injectionsand washes are indicated by black arrows.

FIGURE 9. CDE-066 reduces endogenous active PAI-1 in mouse plasma.Citrated blood was removed via the IVC from mice overexpressing PAI-1before, and 1 h following treatment with the indicated dose of CDE-066.Active murine PAI-1 was measured by Luminex assay and compared withstandards of known murine PAI-1 concentrations. The data are expressed as apercentage of active PAI-1 present in the plasma relative to active PAI-1 attime 0 for each mouse. The data represent the mean 6 S.E., n 5 5 at each dose,and were evaluated against the 0 mg/kg treatment using a Student’s t test (*,p , 0.05; **, p , 0.01).



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nation of compounds and denaturants during SDS-PAGE mayalter how PAI-1 is observed to behave.The identification of naturally occurring polyphenols as a

class of PAI-1 inhibitors is intriguing because such compounds,especially polyphenols derived from teas, fruits, and cocoa, havebeen suggested in recent years to provide benefits againstpathologies such as chronic inflammation, neurodegeneration,cancer, and cardiovascular disease (48–50). Several mecha-nisms of action have been proposed for dietary polyphenols,characterizing these compounds as antioxidants, antiplateletagents, and anti-inflammatory agents. Of particular relevanceto PAI-1 are the proposed mechanisms by which dietary poly-phenols may regulate hemostasis and prevent cardiovasculardisease. In ex vivo and cell culture studies, dietary polyphenolshave been shown to reduce tissue factor expression (51),increase plasminogen activator levels (52), and decrease PAI-1via changes in gene expression (53). These effects are observedat micromolar concentrations of the compounds, a dose rangethat is well within the effective concentrations of the polyphe-nols identified in our study. Thus, it is possible that a previouslyunrecognized direct inactivation of PAI-1 may contribute tothe complex pro-fibrinolytic and cardioprotective effects asso-ciated with dietary polyphenols. Future studies will focus onimproving the specificity and activity of this class of syntheticpolyphenolic compounds against PAI-1 as well as clarifying therole that direct PAI-1 inactivation may play in the healthfulbenefits derived from dietary polyphenols.

Acknowledgments—We thank Martha Larsen and the Center for

Chemical Genomics for drug screening, Dr. Scott Larsen of University

of Michigan College of Pharmacy for the synthesis of PAI-039, and

Nadine El-Ayache for assisting in the synthesis of the CDE inhibitors.

REFERENCES

1. Yepes, M., Loskutoff, D. J., and Lawrence, D. A. (2006) inHemostasis and

Thrombosis: Basic Principles and Clinical Practice (Colman, R. W.,

Marder, V. J., Clowes, A. W., George, J. N., and Goldhaber, S. Z., eds) 5th

Ed., pp. 335–380, Lippincott Williams &Wilkins, Baltimore, MD

2. McMahon, G. A., Petitclerc, E., Stefansson, S., Smith, E., Wong, M. K.,

Westrick, R. J., Ginsburg, D., Brooks, P. C., and Lawrence, D. A. (2001)

J. Biol. Chem. 276, 33964–33968

3. Leik, C. E., Su, E. J., Nambi, P., Crandall, D. L., and Lawrence, D. A. (2006)

J. Thromb. Haemost. 4, 2710–2715

4. Maquerlot, F., Galiacy, S., Malo, M., Guignabert, C., Lawrence, D. A.,

d’Ortho, M. P., and Barlovatz-Meimon, G. (2006) Am. J. Pathol. 169,

1624–1632

5. Stefansson, S., and Lawrence, D. A. (1996) Nature 383, 441–443

6. Cao, C., Lawrence, D. A., Li, Y., Von Arnim, C. A., Herz, J., Su, E. J.,

Makarova, A., Hyman, B. T., Strickland, D. K., and Zhang, L. (2006)EMBO

J. 25, 1860–1870

7. Andreasen, P. A. (2007) Curr. Drug Targets. 8, 1030–1041

8. Huang, Y., Haraguchi, M., Lawrence, D. A., Border, W. A., Yu, L., and

Noble, N. A. (2003) J. Clin. Invest. 112, 379–388

9. Huang, Y., Border, W. A., Yu, L., Zhang, J., Lawrence, D. A., and Noble,

N. A. (2008) J. Am. Soc. Nephrol. 19, 329–338

10. Eitzman, D. T.,McCoy, R. D., Zheng, X., Fay,W. P., Shen, T., Ginsburg, D.,

and Simon, R. H. (1996) J. Clin. Invest. 97, 232–237

11. Pinsky, D. J., Liao, H., Lawson, C. A., Yan, S. F., Chen, J., Carmeliet, P.,

Loskutoff, D. J., and Stern, D. M. (1998) J. Clin. Invest. 102, 919–928

12. Schafer, K., Fujisawa, K., Konstantinides, S., and Loskutoff, D. J. (2001)

FASEB J. 15, 1840–1842

13. Ma, L. J., Mao, S. L., Taylor, K. L., Kanjanabuch, T., Guan, Y., Zhang, Y.,

Brown, N. J., Swift, L. L., McGuinness, O. P., Wasserman, D. H., Vaughan,

D. E., and Fogo, A. B. (2004) Diabetes 53, 336–346

14. De Taeye, B. M., Novitskaya, T., Gleaves, L., Covington, J. W., and

Vaughan, D. E. (2006) J. Biol. Chem. 281, 32796–32805

15. Shah, C., Yang, G., Lee, I., Bielawski, J., Hannun, Y. A., and Samad, F.

(2008) J. Biol. Chem. 283, 13538–13548

16. Lijnen, H. R. (2009) Thromb. Res. 123, Suppl. 4, S46–S49

17. Gohil, R., Peck, G., and Sharma, P. (2009) Thromb. Haemost. 102,

360–370

18. Wiman, B., Andersson, T., Hallqvist, J., Reuterwall, C., Ahlbom, A., and

deFaire, U. (2000) Arterioscler. Thromb. Vasc. Biol. 20, 2019–2023

19. Sobel, B. E., Taatjes, D. J., and Schneider, D. J. (2003)Arterioscler. Thromb.

Vasc. Biol. 23, 1979–1989

20. Wu, Q., and Zhao, Z. (2002) Curr. Drug Targets Cardiovasc. Haematol.

Disord. 2, 27–42

21. Vaughan, D. E., De Taeye, B. M., and Eren, M. (2007) Curr. Drug Targets.

8, 962–970

22. Stefansson, S.,Muhammad, S., Cheng, X. F., Battey, F. D., Strickland,D. K.,

and Lawrence, D. A. (1998) J. Biol. Chem. 273, 6358–6366

23. Webb, D. J., Thomas, K. S., and Gonias, S. L. (2001) J. Cell Biol. 152,

741–752

24. Lawrence, D. A., Palaniappan, S., Stefansson, S., Olson, S. T., Francis-

Chmura, A. M., Shore, J. D., and Ginsburg, D. (1997) J. Biol. Chem. 272,

7676–7680

25. Charlton, P. A., Faint, R. W., Bent, F., Bryans, J., Chicarelli-Robinson, I.,

Mackie, I., Machin, S., and Bevan, P. (1996) Thromb. Haemost. 75,

808–815

26. Bjorquist, P., Ehnebom, J., Inghardt, T., Hansson, L., Lindberg, M., Lin-

schoten, M., Stromqvist, M., and Deinum, J. (1998) Biochemistry 37,

1227–1234

27. Neve, J., Leone, P. A., Carroll, A. R.,Moni, R.W., Paczkowski,N. J., Pierens,

G., Bjorquist, P., Deinum, J., Ehnebom, J., Inghardt, T., Guymer, G., Grim-

shaw, P., and Quinn, R. J. (1999) J. Nat. Prod. 62, 324–326

28. Egelund, R., Einholm, A. P., Pedersen, K. E., Nielsen, R. W., Christensen,

A., Deinum, J., and Andreasen, P. A. (2001) J. Biol. Chem. 276,

13077–13086

29. Gils, A., Stassen, J. M., Nar, H., Kley, J. T., Wienen, W., Ries, U. J., and

Declerck, P. J. (2002) Thromb. Haemost. 88, 137–143

30. Crandall, D. L., Elokdah, H., Di, L., Hennan, J. K., Gorlatova, N. V., and

Lawrence, D. A. (2004) J. Thromb. Haemost. 2, 1422–1428

31. Liang, A., Wu, F., Tran, K., Jones, S. W., Deng, G., Ye, B., Zhao, Z., Snider,

R. M., Dole, W. P., Morser, J., and Wu, Q. (2005) Thromb. Res. 115,

341–350

32. Gorlatova, N. V., Cale, J. M., Elokdah, H., Li, D., Fan, K., Warnock, M.,

Crandall, D. L., and Lawrence, D. A. (2007) J. Biol. Chem. 282, 9288–9296

33. Gardell, S. J., Krueger, J. A., Antrilli, T. A., Elokdah, H., Mayer, S., Orcutt,

S. J., Crandall, D. L., andVlasuk, G. P. (2007)Mol. Pharmacol. 72, 897–906

34. Rupin, A., Gaertner, R., Mennecier, P., Richard, I., Benoist, A., De Nan-

teuil, G., and Verbeuren, T. J. (2008) Thromb. Res. 122, 265–270

35. Izuhara, Y., Takahashi, S., Nangaku, M., Takizawa, S., Ishida, H., Kuro-

kawa, K., van Ypersele de Strihou, C., Hirayama, N., andMiyata, T. (2008)

Arterioscler. Thromb. Vasc. Biol. 28, 672–677

36. Einholm, A. P., Pedersen, K. E., Wind, T., Kulig, P., Overgaard, M. T.,

Jensen, J. K., Bødker, J. S., Christensen, A., Charlton, P., and Andreasen,

P. A. (2003) Biochem. J. 373, 723–732

37. Hennan, J. K., Elokdah, H., Leal, M., Ji, A., Friedrichs, G. S., Morgan, G. A.,

Swillo, R. E., Antrilli, T. M., Hreha, A., and Crandall, D. L. (2005) J. Phar-

macol. Exp. Ther. 314, 710–716

38. Crandall, D. L., Quinet, E. M., El Ayachi, S., Hreha, A. L., Leik, C. E., Savio,

D. A., Juhan-Vague, I., andAlessi,M. C. (2006)Arterioscler. Thromb. Vasc.

Biol. 26, 2209–2215

39. Baxi, S., Crandall, D. L., Meier, T. R., Wrobleski, S., Hawley, A., Farris, D.,

Elokdah, H., Sigler, R., Schaub, R. G., Wakefield, T., and Myers, D. (2008)

Thromb. Haemost. 99, 749–758

40. Elokdah, H., Abou-Gharbia, M., Hennan, J. K., McFarlane, G., Mugford,

C. P., Krishnamurthy, G., and Crandall, D. L. (2004) J. Med. Chem. 47,

3491–3494



by g

uest, o

n J

uly

1, 2

010

ww

w.jb

c.o

rgD

ow

nlo

aded fro

m


41. Gopalsamy, A., Kincaid, S. L., Ellingboe, J. W., Groeling, T. M., Antrilli,

T. M., Krishnamurthy, G., Aulabaugh, A., Friedrichs, G. S., and Crandall,

D. L. (2004) Bioorg. Med. Chem. Lett. 14, 3477–3480

42. Fitzpatrick, D. F., Hirschfield, S. L., and Coffey, R. G. (1993)Am. J. Physiol.

265, H774–H778

43. Chen, X., Beutler, J. A., McCloud, T. G., Loehfelm, A., Yang, L., Dong,

H. F., Chertov, O. Y., Salcedo, R., Oppenheim, J. J., and Howard, O. M.

(2003) Clin. Cancer Res. 9, 3115–3123

44. Liu, X., Kim, J. K., Li, Y., Li, J., Liu, F., and Chen, X. (2005) J. Nutr. 135,

165–171

45. Nakai, M., Fukui, Y., Asami, S., Toyoda-Ono, Y., Iwashita, T., Shibata, H.,

Mitsunaga, T., Hashimoto, F., and Kiso, Y. (2005) J. Agric. Food Chem. 53,

4593–4598

46. van Gorkom, B. A., de Vries, E. G., Karrenbeld, A., and Kleibeuker, J. H.

(1999) Aliment. Pharmacol. Ther. 13, 443–452

47. Folkes, A., Brown, S. D., Canne, L. E., Chan, J., Engelhardt, E., Epshteyn, S.,

Faint, R., Golec, J., Hanel, A., Kearney, P., Leahy, J.W.,Mac,M.,Matthews,

D., Prisbylla,M. P., Sanderson, J., Simon, R. J., Tesfai, Z., Vicker, N.,Wang,

S., Webb, R. R., and Charlton, P. (2002) Bioorg. Med. Chem. Lett. 12,

1063–1066

48. Clement, Y. (2009) Prev. Med. 49, 83–87

49. Saremi, A., and Arora, R. (2008) Am. J. Ther. 15, 265–277

50. Steinberg, F.M., Bearden,M.M., and Keen, C. L. (2003) J. Am. Diet. Assoc.

103, 215–223

51. Di Santo, A., Mezzetti, A., Napoleone, E., Di Tommaso, R., Donati, M. B.,

De Gaetano, G., and Lorenzet, R. (2003) J. Thromb. Haemost. 1,

1089–1095

52. Abou-Agag, L. H., Aikens, M. L., Tabengwa, E. M., Benza, R. L., Shows,

S. R., Grenett, H. E., and Booyse, F. M. (2001) Alcohol Clin. Exp. Res. 25,

155–162

53. Pasten, C., Olave, N. C., Zhou, L., Tabengwa, E. M., Wolkowicz, P. E., and

Grenett, H. E. (2007) Thromb. Res. 121, 59–65



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Technology

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor 1