Vimentin Exposed on Activated Platelets and PlateletMicroparticles Localizes Vitronectin and Plasminogen ActivatorInhibitor Complexes on Their Surface*

Received for publication, October 5, 2001, and in revised form, December 14, 2001Published, JBC Papers in Press, December 14, 2001, DOI 10.1074/jbc.M109675200

Thomas J. Podor‡§¶, Davindra Singh‡§, Paul Chindemi‡§, Denise M. Foulon‡§, Robert McKelvie§,Jeffrey I. Weitz§, Richard Austin‡§, Ghislain Boudreau�, and Richard Davies**

From the Departments of ‡Pathology and Molecular Medicine, and §Medicine, McMaster University and the HamiltonCivic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada, �Pfizer Pharmaceuticals, Montreal, QuebecH9J 2M5, Canada, and the **Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

Type 1 plasminogen activator inhibitor (PAI-1), theprimary inhibitor of tissue-type plasminogen activator(t-PA), is found in plasma and platelets. PAI-1 circulatesin complex with vitronectin (Vn), an interaction thatstabilizes PAI-1 in its active conform. In this study, weexamined the binding of platelet-derived Vn and PAI-1to the surface of isolated platelets. Flow cytometry indi-cate that, like P-selectin, PAI-1, and Vn are found on thesurface of thrombin- or calcium ionophore-activatedplatelets and platelet microparticles. The binding ofPAI-1 to the activated platelet surface is Vn-dependent.Vn mediates the binding of PAI-1 to platelet surfacesthrough a high affinity (Kd of 80 nM) binding interactionwith the NH2 terminus of vimentin, and this Vn-bindingdomain is expressed on the surface of activated plateletsand platelet microparticles. Immunological and func-tional assays indicate that only �5% of the total PAI-1 inplatelet releasates is functionally active, and it co-pre-cipitates with Vn, and the vimentin-enriched cytoskele-ton fraction of activated platelet debris. The remainingplatelet PAI-1 is inactive, and does not associate withthe cytoskeletal debris of activated platelets. Confocalmicroscopic analysis of platelet-rich plasma clots con-firm the co-localization of PAI-1 with Vn and vimentinon the surface of activated platelets, and platelet micro-particles. These findings suggest that platelet vimentinmay regulate fibrinolysis in plasma and thrombi bybinding platelet-derived Vn�PAI-1 complexes.

Most acute coronary syndromes are caused by thrombosissuperimposed on disrupted atherosclerotic plaque (1). Thrombiformed at sites of arterial injury are comprised of platelets andfibrin. The extent of fibrin formation at these sites depends onthe dynamic balance between the coagulation and fibrinolyticpathways. Intravascular fibrinolysis is initiated by tissue-typeplasminogen activator (t-PA)1 that converts plasminogen to

plasmin. The process is regulated by type 1 plasminogen acti-vator inhibitor (PAI-1), the physiological inhibitor of t-PA (2).PAI-1 circulates in two distinct pools (3). Only 10% of circulat-ing PAI-1 is in the plasma where it is bound to vitronectin (Vn)(4–6), an interaction that stabilizes PAI-1 in its active confor-mation (7, 8). The remaining PAI-1 is stored in platelet �-gran-ules (3, 9, 10) and released by platelet activation. The observa-tion that only a small proportion of platelet PAI-1 isfunctionally active (9–11) is difficult to reconcile with the factthat levels of platelet-derived PAI-1 in thrombi determines thesusceptibility of these thrombi to lysis by t-PA (12, 14–20).Although Vn may contribute to thrombogenesis by its interac-tion with platelet integrins (21, 22), its exact role in thrombosisand thrombolysis is less clear.

Recent studies with PAI-1 and Vn-deficient mice indicatethat the incorporation of both PAI-1 and Vn into thrombiformed at sites of carotid artery injury prevent prematurethrombolysis (23, 24). Interestingly, the initial thrombotic re-sponse of Vn-deficient mice to arterial injury was similar tothat of wild-type controls, but their thrombi were unstable andfrequently embolized (24). Consequently, the patency rate ofthe injured arteries 30 min after injury were as high in Vn-deficient mice as in PAI-1-deficient mice, which demonstrateprogressive thrombolysis, and significantly greater than wild-type mice. These findings may reflect an effect of Vn on theregulation of platelet PAI-1 activity, or the Vn-dependent bind-ing of PAI-1 to other proteins such as fibrin (25).

Platelet �-granule PAI-1 is derived from de novo megakaryo-cyte biosynthesis (26, 27). In contrast, Vn internalized fromplasma is targeted to platelet �-granules (27). Although theplatelet storage pool of PAI-1 is reportedly stabilized by itsinteractions with calcium, and other �-granule proteins, it isnot clear if �-granule Vn regulates platelet PAI-1 function (28).Based on size fractionation and immunoblot analysis, somePAI-1 released by activated platelets is complexed with highmolecular weight forms of Vn (13, 28, 29), and almost half ofthe platelet Vn remains cell-associated after platelet activation(30). Immunogold electron microscopic studies have revealedVn and PAI-1 localized within the surface connected system,and on the surface of resting (31) and activated platelets (32,

* This work was supported by an operating grant from the CanadianInstitutes of Health Research (to T. J. P), Career Investigator Awardsfrom the Heart and Stroke Foundation of Canada (to T. J. P. andJ. I. W.), and a grant from the Medical Research Council of Canada/University-Industry (Pfizer Pharmaceuticals) (to T. J. P., R. M., andR. D.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

¶ To whom correspondence should be addressed: Hamilton Civic Hos-pitals Research Centre, 711 Concession St., Hamilton, Ontario L8V1C3, Canada. Tel.: 905-527-2299 (ext. 42630); Fax: 905-575-2646;E-mail: podort@mcmaster.ca.

1 The abbreviations used are: t-PA, tissue-type plasminogen activa-

tor; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate;HMK, heart muscle kinase; mAb, monoclonal antibody; PAI-1, type 1plasminogen activator inhibitor; PBS, phosphate-buffered saline; PE,phycoerythrin; PPP, platelet-poor plasma; PRP, platelet-rich plasma;SAH-Vn, affinity-purified sheep anti-vitronectin IgG; SAH-VIM133, af-finity purified sheep anti-VIM133 IgG; TRAP, thrombin receptor acti-vating peptide; VIM133, recombinant 133 residue NH2-terminal vimen-tin peptide; Vn, vitronectin; CAD, coronary artery disease.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 9, Issue of March 1, pp. 7529–7539, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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33). Although these finding suggest that Vn and PAI-1 bind tothe surface of activated platelets, such interactions have notbeen defined.

Previously, we have demonstrated that PAI-1 and Vn co-localize with the Triton X-100-insoluble vimentin intermediatefilaments of damaged cells (34). Because vimentin also is acomponent of the platelet cytoskeleton (35, 36), we hypothe-sized that vimentin exposed upon platelet activation may serveto localize Vn�PAI-1 complexes on the platelet surface. Usingrecombinant vimentin fragments, we have (a) identified a highaffinity Vn-binding site on the amino-terminal domain of vi-mentin, and (b) by developing an antibody against this frag-ment, have demonstrated that the expression of this bindingsite on activated platelets and platelet microparticles serves tolocalize Vn�PAI-1 complexes to their surface. Our results arethe first demonstration that the active form of platelet PAI-1 isselectively bound to the surface of activated platelets, and thatflow cytometry is a useful method for measuring its surfaceexpression in vitro and in vivo.

MATERIALS AND METHODS

Chemicals and Reagents—Alkaline phosphatase-conjugated strepta-vidin, p-nitrophenyl phosphate, soybean trypsin inhibitor, and phenyl-methylsulfonyl fluoride were purchased from Invitrogen. Bovine vimen-tin was obtained from Roche Molecular Biochemicals. Human PAI-1derived from human HT 1080 fibrosarcoma cells was obtained fromAmerican Diagnostica. Human �-thrombin was purchased from En-zyme Research Laboratories. Bovine serum albumin (BSA), casein,Triton X-100, reduced glutathione, Tween 80, ethanolamine, diethano-lamine, caprylic acid, bovine protein kinase, thrombin receptor activat-ing peptide (TRAP), prostaglandin E1, apyrase, normal goat immuno-globulin (IgG), normal rabbit IgG, and ascitic fluids for monoclonalantibodies (mAbs) to vimentin (clone V.9) and isotype-matched, non-specific mouse IgG were obtained from Sigma. Phycoerythrin (PE)-conjugated mAb directed against CD42b (GPIb-�), fluorescein isothio-cyante (FITC)-conjugated mAb directed against CD62P (P-selectin),and FITC-conjugated streptavidin were purchased from ImmunotechInc. Monospecific antiserum directed against bovine vimentin wasraised in rabbits as described (37). Affinity purified sheep anti-humanVitronectin IgG (SAH-Vitronectin), anti-human vimentin IgG (SAH-VIM133), and normal sheep IgG were obtained from Affinity Biologi-cals. Monoclonal antibody to PAI-1 (MAI-12) was purchased fromBiopool AB. Purified vitronectin, PAI-1, and the various antibodieswere biotinylated with biotinyl-�-amino caproic-N-hydroxysuccinimideester (Roche Molecular Biochemicals) (27, 37). Rabbit polyclonal anti-body directed against residues 35–50 in the amino-terminal domain ofmurine vimentin (Rab 484) was kindly provided by Dr. Wally Ip (Uni-versity of Cincinnati). High binding 96-well microtiter plates wereobtained from Costar Science Corp. Tween 20, Coomassie Brilliant BlueR-250, urea (electrophoresis grade), acrylamide:bis 37.5:1, molecularweight markers, glycine, TRIS, SDS, G-25M Sepharose, and gelatinwere purchased from Bio-Rad.

Preparation of Washed Platelets—Blood was collected from the ante-cubital vein of healthy volunteers into one-sixth volume of acid citratedextrose. After centrifugation at 200 � g for 15 min at 22°C, platelet-rich plasma (PRP) was harvested. Platelets were then pelleted bysubjecting PRP to centrifugation at 1,000 � g for 10 min at 22 °C. Theplatelet pellet was washed twice with calcium-free Tyrodes buffer, pH7.4, containing 0.35% BSA, 10 nM prostaglandin E1, 0.1 mg/ml apyrase,and one-tenth volume of Diatube H vacutainer tube solution (BectonDickenson) containing citrate, theophylline, adenosine, and dipyri-damole. Platelets were then resuspended at 109 platelets/ml in calcium-free Tyrodes buffer containing 0.01 mg/ml apyrase and stored at 23 °Cuntil used.

Flow Cytometry—Immunofluorescence flow cytometry was used todetect platelet-associated PAI-1, vitronectin, and vimentin. Washedplatelets (108/ml) were incubated for 10 min in the absence or presenceof thrombin, A23187, or TRAP prior to analysis using a Coulter EPICSElite ESP flow cytometer with light scatter and fluorescence channelsset at logarithmic gain. Platelets and platelet-derived microparticleswere distinguished from background light scatter by gating acquisitionto include only those particles staining positive for PE-conjugated anti-von Willebrand factor receptor (GPIb-�) IgG (CD42b) within the FL2fluorescence gate. To identify P-selectin, PAI-1, Vn, or vimentin on thesurface of platelets and platelet microparticles, FITC-conjugated anti-

CD62P, or biotinylated preparations of MAI-12, SAH-Vn IgG, SAH-VIM133 IgG, followed by FITC-conjugated streptavidin, were used. Foreach analysis, at least 10,000 PE-positive particles were analyzed forforward and side angle light scatter, and for relative FITC and PEfluorescence intensities. Platelets and platelet microparticles wereidentified by their characteristic light scatter profiles on dot plots offorward light scatter versus right angle side light scatter. Flow cytom-etry data were analyzed using WinMDI software version 2.8.

Isolation and Labeling of Vn from Plasma—Vn was isolated fromnormal platelet-poor plasma by nondenaturing heparin-Sepharose andimmunoaffinity chromatography (27). Oligomeric Vn preparations wereobtained by incubating native Vn with 6 M urea in PBS for 1 h at 37 °C,followed by extensive dialysis against PBS. Isolated Vn was character-ized using SDS-PAGE, and native-PAGE and by its affinity for heparinand the conformation-sensitive antibody, mAb8E6. Total Vn proteinwas quantified using a Vn-specific enzyme-linked immunosorbent as-say (38). For some experiments, Vn was radiolabeled using 125I-Bolton-Hunter reagent (22) to a specific activity of 800 cpm/ng.

Vimentin Binding Assays—To demonstrate the Vn dependence ofPAI-1 binding to vimentin, 96-well microtiter plates were coated over-night at 4 °C with various concentrations of purified bovine vimentindiluted in PBS, pH 7.4. After blocking with 3% BSA, the washed wellswere incubated with 20 nM biotinylated PAI-1 (bt-PAI-1) diluted in PBScontaining 3% BSA, 0.1% Tween 80, 5 mM EDTA, and 20 units/mlaprotinin in the presence of increasing concentrations of purified Vn.Binding of bt-PAI-1 was measured by monitoring the absorbance at 405nm after the addition of streptavidin-conjugated alkaline phosphatase/p-nitrophenyl phosphate substrate, and subtracting binding to BSA-coated wells. To examine the interaction of Vn with vimentin or recom-binant vimentin peptide fragments, 96-well microtiter plate wellscoated with bovine vimentin, vimentin peptides, or BSA were incubatedfor 1 h at 37 °C with varying concentrations of 125I-labeled native orurea-treated Vn in the absence or presence of a 20-fold molar excess ofunlabeled ligand. In each case, specifically bound Vn was determined bysubtracting the radioactivity bound to BSA-coated wells.

Construction of Recombinant Human Vimentin Peptide Plasmids—Human umbilical vein endothelial cells were isolated as described (39),and total RNA isolated using a single-step acid guanidinium thiocya-nate-phenol-chloroform method (40). A 0.5-kb cDNA fragment encodingthe amino terminus head domain and a portion of helix 1A (133 aminoacids) of human vimentin was generated using the reverse transcrip-tion-PCR. Denatured RNA was reverse transcribed using InvitrogensSuperscript II7 RNase H reverse transcriptase, and the PCR productwas then gel purified using the QIAEX7 DNA Gel Extraction kit,subcloned into T-ended pBLUESCRIPT, and used to transform DH5Escherichia coli. Plasmid DNA was isolated as previously described (41)and digested with XhoI. This produced a 460-bp fragment that wassubcloned into the XhoI site of pFL-1-(HMK) heart muscle kinase(HMK) recognition site-modified vector. After digesting the pFL-1-HMK-VIM133 plasmid with HindIII/XhoI to release the HMK-VIM133DNA insert, the fragment was then ligated into the HindIII/XhoI site ofpET-21b to yield the pET-21b-NT-VIM133 plasmid. This plasmid wasthen used to transform competent BL21(DE3) E. coli. Likewise, a 1.1-kbcDNA fragment encoding the central �-helical rod domain and nonhe-lical carboxyl-terminal tail domain (367 amino acids) of human vimen-tin was amplified by standard reverse transcription as described above(42, 43). This cDNA was subcloned into the EcoRI site of a pET-21cvector to produce the pET-21c-CT-VIM367 plasmid that was used totransform BL21(DE3) competent E. coli. Recombinant VIM133 andVIM367 fusion proteins were isolated under denaturing conditions us-ing a Ni2�-HIS column system (Novagen), and then dialyzed into TBSbuffer, pH 7.4, containing 6 M urea. For some studies, the pFL-1-HMK-VIM133 fusion peptide was radiolabeled with 50 units of protein kinaseand 50 �Ci of [32P]ATP (ICN Biomedicals) to a specific activity of 950cpm/ng.

Flow Cytometry Detection of in Situ Platelet Activation—To demon-strate a clinical example of vimentin-mediated expression of PAI-1 onactivated platelets in situ (44, 45), nine patients with documentedcoronary artery disease (CAD) were recruited from the Hamilton Gen-eral Hospital Cardiac Rehabilitation Program. CAD patients were ha-bitually sedentary, and all had a clinical history of CAD, and electro-cardiographically positive treadmill tests documenting exercise-induced ischemic heart disease. The control group consisted of fourage-matched control subjects with no documented history of CAD, aswell as the absence of electrocardiographic or chest pain symptomsduring a symptom limited exercise treadmill test. Patients were receiv-ing enteric-coated aspirin 325 mg daily, and had been withdrawn fromall anti-anginal medications. Written informed consent was obtained

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from all subjects, and all protocols were approved by the HamiltonHealth Sciences Human Ethics Committee. All subjects were instructedto avoid vigorous physical activity and meals for at least 2 h on themorning of the testing. A pre-exercise blood sample was obtained froman antecubital vein saline lock 20 min prior to beginning the treadmillexercise test. Patients then performed a symptom limited treadmillexercise test using the ACIP protocol (46), with repeat blood samples

being drawn immediately after peak exercise, and 3 h post-exercise. Forwhole blood flow cytometry, blood samples were collected into Dia-tube-H vacutainer tubes, incubated with saturating concentrations offluorescent-labeled, or biotinylated antibodies plus streptavidin-FITC,and then briefly fixed with 1% formalin, diluted with HEPES-Tyrodesbuffer, and processed for flow cytometry as described above. The ex-pression of Vn on the surface of platelets was not evaluated in whole

FIG. 1. Vitronectin-dependent expression of PAI-1 on the surface of activated platelets and platelet microparticles. Washed, resting(panel A) human platelets were activated with thrombin (0.5 units/ml) for 10 min (panel B), briefly fixed with paraformaldehyde, washed, and thenincubated with fluorescently labeled antibodies directed against PAI-1 (MAI-12), vitronectin (SAHVn IgG); or P-selectin (CD62P), plus GPIb-�(CD42b) prior to their analysis by fluorescence flow cytometry. Platelets were gated on GPIb-� positive events, and by forward and side scatter forsize and shape. Histogram depiction of the relative fluorescence intensity (RFI) of the various antibodies on: panel C, resting platelets; panel D,thrombin-activated platelets; and panel E, the RFI for MAI-12-FITC binding to platelets activated in the presence or absence of SAHVn IgG, orpreimmune sheep IgG. Histograms depicting the RFI of the anti-P-selectin (panel F) and anti-PAI-1 (panel G) binding to platelets microparticlesthat were detected by gating on GPIb-� positive events within the smaller forward and side scatter for size and shape (lower left quadrants inpanels A and B).

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blood flow cytometry because of the interference from the micromolarconcentrations of plasma Vn.

Analysis of Vn, PAI-1, and Vimentin in Platelet Releasate—Restingand activated platelet releasates were subjected to differential centrif-ugation and Western blot analysis to examine the distribution of Vnand PAI-1 with the Triton X-100 insoluble vimentin. Washed plateletswere incubated for 10 min at 37 °C in the absence or presence ofthrombin (2 units/ml). After centrifugation at 12,000 � g for 10 min, the

supernatant was subjected to centrifugation at 100,000 � g for 3 h toensure complete precipitation of platelet debris. Resultant pellets werelysed with lysis buffer (PBS, pH 7.4, containing 100 mM NaCl, 300 mM

sucrose, 10 mM benzamidine, 5 mM EDTA, 10 units/ml aprotinin, 10 mM

phenylmethylsulfonyl fluoride, 0.05% sodium azide, and 0.5% TritonX-100). The Triton-insoluble pellet was then extracted with lysis buffercontaining 2% SDS, and the SDS neutralized by the addition of 2%Triton X-100. For immunoblot analysis, samples were solubilized in

FIG. 2. Vitronectin-dependent binding of PAI-1 to purified bovine vimentin. Panel A, 96-well plates microtiter were coated with variousconcentrations of purified vimentin, and then incubated with 20 nM bt-PAI-1 in the presence of increasing concentrations of Vn, and the boundbt-PAI-1 was determined by measuring the change in A405 nm after the addition of streptavidin-conjugated alkaline phosphatase/p-nitrophenylphosphate substrate. Specific binding to vimentin was calculated by subtracting the background bt-PAI-1 binding to BSA-coated wells; panel B,microtiter plates were coated with various concentrations of purified vimentin, and then incubated with 250 nM 125I-Vn alone (green circles), orequimolar concentrations of both Vn and PAI-1 (red triangles). After washing, the bound radioactivity was determined, and corrected forradioactivity bound to BSA-coated wells.

FIG. 3. Construction of human vimentin plasmids and identification of the Vn-binding site on the amino-terminal vimentinfragment. Panel A, schematic representation of full-length human vimentin amino acid domain structures and overlapping pET-21b-NT-VIM133and pET-21c-CT-VIM367 vimentin fusion peptides; panel B, Characterization of Vn binding to native bovine vimentin (lanes 1), pET-21b-NT-VIM133 (lanes 2), and pET-21c-CT-VIM367 (lanes 3). Coomassie Blue stain of reduced SDS-PAGE illustrates the three different forms of purifiedvimentin migrate at 57, 20, and 41 kDa, respectively. Western blot analysis confirmed the identity of the different forms of vimentin using: (i) rabbitantibody directed against the full-length native 57-kDa bovine vimentin (Rab371); (ii) murine anti-vimentin (mAb V.9) directed against an epitopein the carboxyl-terminal tail domain; or (iii) rabbit antibody directed against the amino-terminal head domain (Rab484). Ligand blot analysis usingbt-Vn illustrates that Vn only binds to native vimentin and the pET-21b-NT-VIM133 peptide; panel C, nondenaturing PAGE and autoradiographicanalysis of diluted normal plasma (1:5 with PBS) containing 125I-labeled native Vn and various concentrations of either VIM133 or VIM367peptide. Control, buffer alone (lanes 1), 1.8 �M peptide (lanes 2), 45 �M peptide (lanes 3), 90 �M peptide (lanes 4). The arrows indicate the mobilityof Vn multimers in the stacking gel (arrows, a); at the stacking/separating gel interface (arrows, b); and native Vn (arrows, c). Panel D, 96-wellmicrotiter plates were coated with purified VIM133 peptide (100 nM coating concentration) and incubated for 1 h at 37 °C with variousconcentrations of 125I-labeled native Vn or urea-treated oligomeric Vn in the presence or absence of 20-fold excess cold, unlabeled Vn. The plot onthe left is the Vn specifically bound to the VIM133 peptide after subtracting the background radioactivity bound to BSA-coated wells in the presenceof 20-fold excess unlabeled Vn. The Scatchard plot on the right represents the specific oligomeric Vn bound to a single class of binding sites for Vnon the immobilized VIM133.

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Laemmli sample buffer (47) (50 mM Tris-HCl, pH 6.8, 2% SDS, 10%glycerol and 0.001% bromphenol blue) containing 5% 2-mercaptoetha-nol, subjected to boiling water for 1 h, and then fractionated by SDS-PAGE using 7.5% slab gels. Separated proteins were transferred tonitrocellulose membranes, and after blocking with blotting buffer (PBS,pH 7.4, containing 1% casein and 0.05% Tween 20), the membraneswere incubated for 1 h with 5 �g/ml SAH-VIM133, SAH-Vn, or SAH-PAI-1 IgG. After washing with blotting buffer, membranes were incu-bated with a 1:3000 dilution of horseradish peroxidase-conjugated rab-bit anti-sheep IgG for 1 h. The blots were washed again, immersed inchemiluminescence reagent (ICN Biomedicals), and briefly exposed toautoradiography film.

The quantity of PAI-1 in the Triton-soluble and insoluble extracts(109 platelets/sample) was determined immunologically using a sand-wich enzyme-linked immunosorbent assay (39), and functionally usinga two-step method in which immobilized t-PA was first used to bindactive PAI-1, and the bound PAI-1 then quantified with an affinitypurified, horseradish peroxidase-conjugated sheep anti-human PAI-1(48).

Immunocytochemistry and Confocal Microscopic Image Analysis—Sample preparation for immunofluorescence confocal microscopic im-aging of the distribution of PAI-1, Vn, and vimentin in PRP clots waspreviously described for platelet-poor clots (25). Briefly, 150 �l of PRPwas placed on APTEX-coated coverslips and clotted by the addition ofthrombin (2 units/ml) and CaCl2 (10 mM, final concentration). Afterincubation at 37 °C for 1 h, the clots were fixed with cold 3% formalde-hyde in PBS for 5 min, washed alternately with PBS and PBS contain-ing 0.1 mol/liter of glycine, and then incubated for 30 min with blockingbuffer (PBS containing 0.5% BSA and 50 �g/ml normal goat IgG).Primary antibodies, including a monoclonal anti-PAI-1 IgG (MAI-12),SAH-Vn IgG, SAH-VIM133 IgG, and SAH-vWf IgG, were diluted inblocking buffer and incubated with the clots for 1 h at 37 °C. Controlclots were stained with each primary antibody separately, stained with-out primary antibodies, or stained with nonspecific mouse and sheep

IgG. After washing, clots were incubated for 1 h at 37 °C with Texas Redrhodamine-conjugated goat anti-sheep or FITC-conjugated goat anti-mouse IgG diluted 1:20 in blocking buffer. The coverslips were washed,mounted on glass slides using Permafluor mounting medium, and thensubjected to Z-plane optical sectioning (200 nm/section) using a ZeissLSM 10 and Metamorph software (Universal Imaging). Clots stainedwith nonspecific primary antibodies were used to threshold for back-ground fluorescence intensity.

RESULTS

Vn-mediated PAI-1 Expression on the Surface of ActivatedPlatelets and Microparticles—Flow cytometry studies have pre-viously shown that activated platelets release plasma mem-brane-bound vesicles, called microparticles, which have a highdensity of prothrombotic proteins on their surface, includingthe adhesive receptor P-selectin (44, 45). We used immunoflu-orescence flow cytometry to determine whether the endogenousplatelet PAI-1 and Vn are expressed on the surface of throm-bin-activated platelets and platelet microparticles. Analysis ofunstimulated platelets using the PE-CD42b (GPIb-�) reveals asingle population of particles characterized by an ovoid forwardand side light-scattering pattern (Fig. 1A). The unstimulatedplatelets also exhibited relatively low fluorescence intensitystaining with FITC-MAI-12 (PAI-1), FITC-CD62P (P-selectin),SAHVn (vitronectin), and normal (preimmune) sheep IgG (Fig.1C). Thrombin activation significantly alters the forward andside light-scattering characteristics of the GPIb-� positiveplatelets, and results in the generation of smaller plateletmicroparticles that are evident within the lower left quadrantof the dot plot (Fig. 1B). Platelet activation increases the rela-tive fluorescence intensity of the FITC immunolabeling forP-selectin, PAI-1, and Vn (Fig. 1D). Activation also generatesP-selectin and PAI-1 positive microparticles that were identi-fied by virtue of their lower forward and side light scattervalues, and their positive co-staining for GPIb-� (Fig. 1, F andG). Moreover, the presence of anti-Vn IgG during platelet ac-tivation virtually eliminates the binding of MAI-12 IgG to theactivated platelet surfaces (Fig. 1E), and thereby indicates thatVn mediates the binding of PAI-1 to activated platelet surfaces.

Vimentin Binds PAI-1 in a Vn-dependent Manner—Buildingon our previous observation that PAI-1 and Vn co-localize withvimentin of damaged cells, we set out to determine whetherPAI-1 and/or Vn bind to bovine vimentin. Hypothesizing thatPAI-1 binding to vimentin would be Vn-dependent, we incu-bated vimentin-coated wells with a fixed concentration of bt-PAI-1 in the absence or presence of increasing concentrations ofVn, and then monitored PAI-1 binding. The binding of bt-PAI-1to vimentin is negligible in the absence of Vn, but increases asa function of the Vn concentration (Fig. 2A). These resultsindicate that bt-PAI-1 binding to vimentin is Vn-dependent.Next, we examined the impact of PAI-1 on the binding of125I-Vn to vimentin-coated plates (Fig. 2B). PAI-1 potentiatesVn binding to vimentin 2-fold, possibly reflecting the previ-ously described formation of Vn dimers within a higher orderVn�PAI-1 complex (49).

Identification and Characterization of the Vn-binding Do-main on the Amino Terminus of Vimentin—To identify theVn-binding domain on vimentin, two overlapping recombinanthuman vimentin peptides were expressed (Fig. 3A). The first, a133-amino acid peptide, is comprised of the amino-terminalhead domain (residues 1–95) and 36 residues of the coil 1Awithin the central rod domain (residues 96–131). The secondpeptide is a 367-amino acid peptide analogue composed of thecentral rod domain, and the carboxyl-terminal rod domain.These peptides were designated VIM133 and VIM367, respec-tively. The VIM133 fusion protein migrated as a 20-kDa bandon 12% SDS-polyacrylamide gels (Fig. 3B, lanes 2), and wasdetectable with a polyclonal antibody directed against bovine

FIG. 4. Western and ligand blot analysis of thrombin and en-doproteinase Lys-C proteolytic digests of the rVIM133 peptide.Samples were fractionated by SDS-PAGE on reduced 20% gels, andeither stained with Coomassie Blue or electrophoretically transferredonto nitrocellulose membranes, and then processed for: panel A, West-ern blotting using Rab 484, or panel B, ligand blotting using bt-Vn.VIM133 peptide (1.4 �g) standard (lanes 1); VIM133 peptide (1.4 �g)digested with thrombin for 30 min (lanes 2); VIM133 peptide (1.4 �g)digested with endo Lys-C for 40 min (lanes 3); native bovine vimentinprotein standard (1 �g) (lanes 4). Arrows indicate electrophoretic mo-bility of: arrow a, native bovine vimentin at 57 kDa; arrow b, thrombinat 37.5 kDa; arrow c, rVIM133 peptide at 20 kDa; arrow d, endo Lys-Cproteolytic cleavage peptide at 12 kDa;, arrow e, thrombin cleavagepeptide at 10 kDa; and arrow f, 9-kDa endoproteinase Lys-C cleavagepeptide.

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vimentin (Rab371) and one directed against the vimentin headdomain residues 35–50 (Rab484). In contrast, VIM133 did notstain with the monoclonal V.9 IgG that is directed against thecarboxyl-terminal tail domain (Fig. 3B, lanes 2). VIM367 mi-grates as a 41-kDa band that stained with a polyclonal anti-body against bovine vimentin and V.9 IgG, but not with Rab484IgG, the antibody directed against the head domain of vimentin(Fig. 3B, lanes 3). Ligand blot analysis was used to identify theVn-binding domain on vimentin. Native bovine vimentin andVIM133 bind bt-Vn (Fig. 3B, lanes 1 and 2), whereas VIM367does not. These data reveal that the Vn-binding site resides inthe amino-terminal region of vimentin.

We next examined the interaction of the VIM133 peptidewith normal platelet-poor plasma that contains micromolarquantities of plasma Vn. First, varying doses of VIM 133, or thecontrol VIM367 peptide were preincubated for 15 min withplatelet-poor plasma containing trace amounts of 125I-labelednative Vn, and then subjected to nondenaturing native PAGE,and autoradiography to visualize changes in the molecularweight of Vn (Fig. 3C). The results indicate that in the absenceof VIM peptide (Fig. 3C, lanes 1), plasma Vn predominantlymigrates as the monomeric form (arrow c), with lesser quanti-ties of Vn multimers apparent (arrow b). However, the VIM133peptide induces formation of plasma Vn multimers in a dose-dependent manner (Fig. 3C, arrow a, lanes 2–4). In contrast,the VIM367 peptide did not effect the electrophoretic mobilityof Vn. Western blot analysis of the plasma samples containing

VIM133 indicate that the vimentin peptide co-migrates withthe Vn multimers, and these multimers are resistant to SDStreatment (not shown).

To quantify the binding of Vn to the amino terminus ofvimentin, plate microtiter surfaces coated with VIM133 or BSAwere incubated with 125I-labeled native or urea-treated oligo-meric Vn in the absence or presence of a 20-fold molar excess ofthe unlabeled ligand. Both native and oligomeric 125I-Vn bindsto VIM133 in a dose-dependent fashion (Fig. 3D). Whereasbinding of urea-denatured oligomeric 125I-Vn is saturable, thebinding of native 125I-Vn is not saturable at the concentrationsused in these experiments. Scatchard analysis of the specificbinding of urea-denatured Vn reveals the presence of a singleclass of high affinity binding sites with a Kd of 80 nM. Prein-cubation of the 125I-Vn with PAI-1 increases the binding of Vnto VIM133 by �2-fold. Oligomeric Vn binds bovine vimentinwith a Kd similar to that for VIM133.

To further define the Vn-binding domain on VIM133, thepeptide was subjected to limited proteolysis with thrombin andendoproteinase Lys-C, and ligand blot analysis was used toidentify the Vn-binding fragments. Thrombin, which cleavesthe head domain of vimentin at Arg78 (50), generates a frag-ment that was recognized by Rab484 (Fig. 4A, lane 2, arrow e),an antibody directed against residues 35–50. This fragmentbinds bt-Vn (Fig. 4B, lane 2, arrow e). Bt-Vn also binds tothrombin within the digestion mixture (Fig. 4B, lane 2, arrowB). Endoproteinase Lys-C cleavages at residues Lys97 and

FIG. 5. Co-Expression of vimentin with PAI-1, and P-selectin on the surface of thrombin-activated platelets and microparticles.Panel A, washed platelets were activated with thrombin (0.5 units/ml), calcium ionophore A23187 (5 �M), or TRAP (5 �M) for 10 min, briefly fixedwith paraformaldehyde, and then incubated with antibodies directed against GPIb-� (CD42b) and normal, preimmune sheep IgG (NS IgG), oranti-VIM133 IgG prior to their analysis by fluorescence flow cytometry. Platelets were detected by gating on GPIb-� positive events, and by forwardand side scatter for size and shape; panel B, histogram depiction of the relative fluorescence intensity (RFI) for the binding of anti-VIM 133 FITCto microparticle-gated events from samples analyzed in panel A, and illustrating response of resting, unstimulated platelets (red filled), andplatelets activated with thrombin (blue line), TRAP (green line), and A23187 (black line); panel C, bar graph indicating that number of CD62 andvimentin-positive platelets increases as a function of the concentration of thrombin used for platelet activation; panel D, histogram depiction of theRFI for the binding of MAI-12 to platelets activated in the presence or absence of anti-VIM133 IgG, or preimmune normal sheep (NS) IgG.

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Lys104 of VIM133, and generates two bands (51) that are bothrecognized by the Rab 484 antibody (Fig. 4A, lane 3, arrows dand f), and bind to bt-Vn (Fig. 4B, lane 3, arrows d and f). Takentogether, these data suggest that the Vn-binding domain lieswithin residues 1–78 of vimentin.

Vimentin-dependent Expression of Active PAI-1 on the Sur-face of Activated Platelets and Platelet Microparticles—We nextgenerated a sheep polyclonal antisera directed against the re-combinant VIM133 peptide, and prepared affinity-purified, bi-otinylated anti-VIM133 IgG for flow cytometry analysis of vi-mentin expression on the surface of platelets that were

activated with various agonists including thrombin, A23187,and TRAP (Fig. 5A). As with the antibodies directed againstP-selectin, the anti-VIM133 IgG exhibits minimal or no bindingto resting platelets. Activation of platelets with thrombin,A23187, and TRAP increases the expression of the VIM133sequence on the surface of whole platelets (Fig. 5A), and plate-let microparticles (Fig. 5B). The anti-VIM133 IgG binding toplatelet surfaces increases rapidly (5–10 min) after thrombinstimulation, and the intensity of binding depends on the con-centration of thrombin used to activate the platelets (Fig. 5C).Moreover, activating platelets in the presence of the anti-

FIG. 6. Exercise-induced increase in P-selectin, vimentin, and PAI-1 expression on the surface of circulating activated plateletsand microparticles in a CAD patient. Panel A, subject underwent a standardized treadmill testing as described under “Materials and Methods,”and blood samples were drawn into Diatube H vials before exercise (baseline), immediately after treadmill testing (1 min post-exercise), and afterresting for 3 h (3 h post-exercise). Flow cytometry analysis was performed on the whole blood diluted in HEPES-Tyrodes buffer and incubated withsaturating concentrations of antibodies directed against GPIb-� (CD42b), and CD62, anti-VIM133 IgG, or MAI-12 IgG. The samples were thenfixed in 1% paraformaldehyde, diluted, and analyzed by flow cytometry with gating on GPIb-� positive events, and by forward and side scatter forsize and shape for total platelets in: panels A, whole blood; panel B, for platelet microparticles.

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VIM133 IgG significantly attenuates PAI-1 expression (Fig.5D) thereby confirming that vimentin mediates the binding ofVn�PAI-1 complexes on activated platelet surfaces.

Previous flow cytometry studies have demonstrated that inmany patients with CAD, unaccustomed strenuous exercisecan induce platelet activation and P-selectin expression, eventsthat may contribute to exercise-induced myocardial ischemia(45). We performed flow cytometry analysis on blood samplestaken from a small group of healthy individuals and CADpatients undergoing acute exercise stress to provide a clinicallyrelevant example of elevated levels of vimentin and PAI-1expression on activated surface of platelets activated in situ.Using P-selectin expression again as an index of platelet acti-vation, there was no evidence of exercise-induced platelet acti-vation in any of the four controls following exercise. In contrast,there was modest evidence of exercise-induced platelet activa-tion in four of nine CAD patients, with the percentage of acti-vated platelets increasing from �1% pre-exercise, up to 2.2–6.8% at the 1 min post-exercise time point, and returned backto baseline levels after 3 h. However, in one CAD patient, theplatelet activation response was significantly more pronounced(Fig. 6). As in the healthy controls, the pre-exercise flow cytom-etry dot plots reveal that �1% of the gated events were positivefor P-selectin, vimentin, and PAI-1 (Fig. 6A, first row panels).In contrast, 1 min after exercise, the percentage of circulatingplatelets that were positive for surface P-selectin, vimentin,and PAI-1 increased to �15, 35, and 10% of the total events,respectively (Fig. 6A, second row panels). After resting for 3 hfollowing the exercise test, the number of P-selectin and PAI-1positive platelets had returned to baseline levels. However, thenumber of vimentin-positive events remained elevated (Fig.6A, third row panels). Analysis of the microparticles present inthe PRP prepared from these blood samples confirmed thetransient increase in P-selectin and PAI-1, and the sustainedelevation in the number of vimentin-positive platelet micropar-ticles (Fig. 6B).

Selective Expression of Active PAI-1 on the Surface of Acti-vated Platelets—The use of the inhibitory antibody MAI-12 inour flow cytometry demonstration of the Vn-dependent ex-pression of platelet PAI-1 supports the hypothesis that activePAI-1 is associated with the vimentin cytoskeleton on the

surface of activated platelets and platelet microparticles. Tofurther investigate this issue, we isolated the cellular debrisfrom activated platelet releasates using differential centrif-ugation, and then analyzed the supernatants and pellets forthe presence of PAI-1, Vn, and vimentin using Western blotanalysis, and immunoassays for quantifying PAI-1 antigenand activity. The Western blots presented in Fig. 7 revealsthat, in contrast to resting platelets, the PAI-1, Vn, andvimentin associated with activated platelet releasates re-main in the low speed supernatant, and are then distributedbetween both the supernatant and Triton X-100-insolubleplatelet pellet fractions after a second high speed (100,00 � g,3 h) centrifugation step. Quantification of the specific activityof the PAI-1 in the high speed supernatant and pellet frac-tions reveals that �96% of the total platelet PAI-1 antigen inthe platelet releasate remains in the supernatant, and is notfunctionally active (Table I). In contrast, �5% of the PAI-1antigen released from activated platelets is precipitated inthe Triton-insoluble platelet pellet fraction, and virtually allof this PAI-1 is functionally active.

Incorporation of Platelet PAI-1 and Vitronectin with theVimentin Cytoskeleton of Platelets in PRP Clots—We con-ducted dual-labeling immunofluorescence confocal micros-copy of fixed, and unfixed PRP clots to examine whetherplatelet vimentin also regulates the deposition of plateletVn�PAI-1 complexes in platelet-rich thrombi. The distribu-

FIG. 7. Association of Vn and PAI-1with the Triton X-100-insoluble vi-mentin cytoskeleton of thrombin-ac-tivated platelets and microparticles.Resting and thrombin-activated plateletswere first subjected to a low speed centrif-ugation (12,000 � g; 10 min), and theresulting supernatants were then furtherfractionated by high speed centrifugationat 100,000 � g for 3 h. The pellets wereextracted with Laemmli sample buffer,and equal proportions (10 �g of protein/lane) of the supernatants and pellet ex-tracts were electrophoretically fraction-ated by SDS-PAGE (reduced), theproteins transferred to nitrocellulose, andthen probed with antibodies directedagainst vimentin, Vn, and PAI-1.

TABLE IPAI-1 activity and antigen in thrombin-activated platelet releasatesSuspended washed platelets (109 platelets/sample) were incubated in

the presence or absence of human thrombin (2 NIH units/ml) for 10 minat 37 °C, the thrombin neutralized with D-phenyl-Pro-Arg-chloromethylketone, and the platelets centrifuged at 12,000 � g for 10 min asdescribed. The supernatants were then re-centrifuged at 100,000 � gfor 3 h to precipitate smaller debris and microparticles, and the con-centrations of PAI-1 antigen and activity in the platelet supernatantand pellet fractions quantified as described under “Experimental Pro-cedures.” The data represents the average (�S.D.) results from one ofthree representative experiments performed in triplicate.

Platelet fraction PAI-1 antigen Active PAI-1 % Active PAI-1

100,000 � g; supernatant 1,080 (�186) �0.6 �1%100,000 � g; pellet 183 (�42) 247 (�42) 100%

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tion of von Willebrand factor-positive platelets and plateletmicroparticles in PRP clots is evident in Fig. 8A. PAI-1 co-localizes with Vn on the surface of activated platelets and thesmaller platelet microparticles that are associated with fibrinfibrils (Fig. 8, B–D). Similar images indicative of co-localiza-tion were obtained with PRP clots stained for PAI-1 andvimentin (Fig. 8, E–G), or Vn and vimentin (Fig. 8, H–J). Apseudo-colored image of an optical section through an unfixedPRP clot illustrates the two distinct staining patterns for

fibrin clot-associated Vn. First, there is relatively low levelsof Vn distributed along the length of fibrin fibrils (Fig. 8K,lower arrow), and reflects the proportion of plasma Vn whichbinds to fibrin during coagulation (67). Second, there is themore intense, focal accumulation of platelet-derived Vn thatco-distributes with the exposed platelet vimentin cytoskele-ton (Fig. 8, K and L, upper arrow). The high resolutionvolume rendering of the overlaid optical sections through theindicated platelet (Fig. 8L, arrow) within the clot reveals the

FIG. 8. Immunofluorescence confocal microscopic localization of PAI-1, Vn, and vimentin in PRP clots. PRP clots were stained withprimary antibodies directed against vWf, followed by FITC (green)-conjugated secondary antibodies (panel A). Alternatively, PRP clots weredual-labeled with primary antibodies directed against PAI-1 and Vn (panels B–D), PAI-1 and vimentin (panels E–G), or Vn and vimentin (panelsH–L), and each primary detected with FITC (green) or Texas Red rhodamine (red), respectively. Digital overlay of green/red images (panels D, G,J, and inset L), indicates co-localization of fluorochromes (yellow). Panels K and L are higher magnification pseudo-colored images of clot-associatedVn and vimentin from clots in panels H and I. The staining pattern in panel K illustrates the two distinct distributions of Vn within clots (arrows),namely fibrin-associated plasma Vn and platelet-derived Vn. Panel L demonstrates the punctuate distribution of platelet-associated vimentin, andthe inset illustrates the overlapping distribution (yellow) of the platelet Vn and vimentin (platelet indicated by arrows in panels K and L) on theinvaginated surface of the activated platelet surface (scale bar in panels A–J, 20 �m).

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significant co-distribution (yellow) of Vn (FITC; green) andvimentin (Texas Red rhodamine; red) over the invaginatedsurface of the activated platelet (Fig. 8L, inset).

DISCUSSION

Recently, we demonstrated that Vn mediates PAI-1 bindingto fibrin (25). In this study, we provide evidence that Vn alsomediates the binding of PAI-1 to the vimentin intermediatefilament cytoskeleton that is exposed on the surface of acti-vated platelets. Platelets activated by various agonists or byexercise generate platelet microparticles enriched in Vn�PAI-1complexes. Moreover, confocal image analysis visually confirmsthat the Vn�PAI-1 complexes co-localize with the platelet vi-mentin cytoskeleton on the surface platelets in platelet-richplasma clots suggesting vimentin also regulates the incorpora-tion of active platelet PAI-1 into thrombi. Our evidence that Vncan bind PAI-1 simultaneously with other macromoleculessuch as vimentin and fibrin (25, 67) further supports our pro-posal that Vn�PAI-1 complexes bind to macromolecules ratherthan the individual proteins themselves, and that it is theVn within these complexes that mediates these bindinginteractions.

A New Paradigm for Platelet PAI-1 Function—Platelet acti-vation triggers cytoskeletal rearrangement that can lead toshape change, exocytosis, microparticle generation, adhesion,aggregation, and retraction (52). Using washed, resting andactivated platelets, we measured surface expression of PAI-1and Vn. PAI-1 was identified with the monoclonal antibodyMAI-12, which preferentially binds to the active conform. Ourstudies suggest that only �5% of the PAI-1 in platelet releas-ates is active, a concept supported by previous work (9–11).Only the active PAI-1 is associated with Vn and vimentin onthe surface of activated platelets and platelet microparticles.We postulate that active PAI-1 in platelets reflects preformedVn�PAI-1 complexes that are stored in the �-granule, and arereleased upon platelet activation. These complexes are boundto vimentin that is exposed during cytoskeleton rearrange-ment, membrane blebbing, and exocytosis. Given that Vn sta-bilizes PAI-1 in its active conformation, it is tempting to spec-ulate that PAI-1-bearing platelets or microparticles that aregenerated in situ may have the potential to inhibit fibrinolysisfor prolonged periods of time in the circulation, or at sites ofvascular injury and thrombosis. Our findings are also consist-ent with the recent studies with arterial injury models inVn-deficient mice that suggest that Vn promotes thrombosis atsites of arterial injury (23, 24).

Platelet activation in response to both weak and potent ago-nists results in the expression of Vn�PAI-1 complexes on thesurface of activated platelets and platelet microparticles. Be-cause we used washed platelets in these studies, the com-ponents of these surface-bound complexes are likely to beplatelet-derived. This concept supports previous reports dem-onstrating the release of high molecular weight Vn�PAI-1 com-plexes from activated platelets (13), and the immunolocaliza-tion of these proteins on the surface of activated platelet andfibrin (31–33).

Vimentin-type Intermediate Filaments—Vimentin is the ma-jor type III intermediate filaments expressed in cells of mesen-chymal (e.g. endothelium, fibroblasts, megakaryocytes), andmyogenic origin (53). Vimentin, a minor component of theplatelet cytoskeleton (52), is associated with the Triton X-100insoluble fraction of human platelets (35, 36), the same fractionthat contains the active PAI-1 in activated platelet releasates.When examined by electron microscopy, vimentin forms a net-work of 10-nm intermediate filaments that form a ring close tothe cell membrane, as well as a network in the body of cells.Our flow cytometry data indicate that this vimentin network is

exposed when platelets are activated with thrombin or otheragonists. Using recombinant vimentin fragments, we identifieda high affinity Vn-binding site on the amino-terminal headdomain of vimentin. The binding site is localized to the first 78amino acids, a region that contains a non-�-helical domain withtwo motifs crucial for filament assembly: a nona-peptide withthe sequence SSYRRXFGG (53) and an arginine and proline-containing domain known as the RP-Box (54). The two arginineresidues within the nona-peptide sequence are critical for fila-ment assembly (50, 51, 54, 55). Likewise, the highly conservedRP-box sequence, which also is found in type III intermediatefilaments, such as desmin, peripherin, and glial fibrillary acidicprotein, contains serine phosphorylation sites, and arginineresidues that also are critical for filament assembly (53–55).The amino terminus of vimentin interacts with negativelycharged phospholipid bilayers (56), thereby rationalizing itslocation near the cell membrane. Our studies reveal a novelrole for the amino terminus of vimentin; namely, as a highaffinity binding site for Vn.

The observation that the VIM133 peptide induces plasma Vnmultimerization raises the possibility that vimentin exposureon the surface of activated platelets, or other cells may not onlybind Vn, but may also modulate the structure and function oflocalized Vn polymers. Furthermore, our current findingsrepresent a plausible explanation for our previous demonstra-tion of increased expression of PAI-1 on the surface of endo-thelium exposed to bacterial endotoxin and other inflammatorymediators in vitro (57) and in vivo (34). Vimentin also maymodulate inflammatory responses and atherosclerosis (58)through its interactions with plasma proteins such ascomplement (59, 60), IgG (61, 62), and fibrinogen (62). Cyto-keratin-type intermediate filaments that are also exposed onthe surface of various cells interact with various hemostasisfactors including kininogen (63, 64), plasminogen (65), andthrombin�antithrombin�Vn complexes (66). The potentialclinical significance of our findings is underscored by the evi-dence of exercise-induced expression of vimentin and PAI-1 onthe surface of circulating platelets and platelet microparticlesin a small number of high-risk patients with CAD. Futurestudies are required to better understand the mechanisms reg-ulating vimentin interactions with Vn, and the role of plateletsurface-bound PAI-1 in fibrinolysis and in various pro-thrombotic states.

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Vitronectin Mediates PAI-1 Binding to Platelet Vimentin 7539

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Jeffrey I. Weitz, Richard Austin, Ghislain Boudreau and Richard DaviesThomas J. Podor, Davindra Singh, Paul Chindemi, Denise M. Foulon, Robert McKelvie,

Vitronectin and Plasminogen Activator Inhibitor Complexes on Their SurfaceVimentin Exposed on Activated Platelets and Platelet Microparticles Localizes

doi: 10.1074/jbc.M109675200 originally published online December 14, 20012002, 277:7529-7539.J. Biol. Chem. 

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