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Originally published In Press as doi:10.1074/jbc.M908079199 on April 10, 2000

J. Biol. Chem., Vol. 275, Issue 26, 19788-19794, June 30, 2000
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Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin*

Thomas J. PodorDagger §||, Cynthia B. Peterson**DaggerDagger, Daniel A. Lawrence§§¶¶, Steingrimur Stefansson§§, Stephen G. ShaughnessyDagger §, Denise M. Foulon§, Martin Butcher§, and Jeffrey I. Weitz§

From the Dagger  Department of Pathology and Molecular Medicine, McMaster University and the § Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada, the §§ American Red Cross J. H. Holland Laboratories, Rockville, Maryland 20855, and the ** Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996

Received for publication, October 1, 1999, and in revised form, April 7, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue-type plasminogen activator (t-PA)1 initiates intravascular fibrinolysis by binding to fibrin, where it activates fibrin-bound plasminogen (1-4). The major inhibitor of t-PA, type 1 plasminogen activator inhibitor (PAI-1), circulates in plasma and is released from platelet alpha -granules during blood clotting (5, 6). PAI-1 accumulates in thrombi, rendering them resistant to t-PA-mediated fibrinolysis (7-14). In purified systems, PAI-1 has been shown to bind directly to fibrin, with a Kd of 3.7 µM (15-18). Consequently, it has been hypothesized that PAI-1 accumulation in thrombi reflects a direct interaction of PAI-1 with fibrin.

PAI-1 circulates in plasma (19, 20) and platelets in complex with vitronectin (21, 22, 23, 24), a glycoprotein that binds PAI-1 with high affinity (25). The vitronectin interaction with PAI-1 stabilizes the inhibitor in its active conformation (26, 27), induces allosteric changes in vitronectin that expose cryptic epitopes (28, 29), and modulates vitronectin-dependent cell adhesion (25, 30, 31). Domain mapping studies using proteolysis, synthetic peptides, monoclonal antibodies, and site-directed mutagenesis have identified two discrete sites on vitronectin that may bind and stabilize PAI-1 (27, 33, 34). Similar approaches have delineated a single vitronectin-binding site on PAI-1 (35, 36).

Recent studies from our laboratories have further characterized the PAI-1-vitronectin interaction. Analytical ultracentrifugation experiments indicate that PAI-1 and native vitronectin form a 320-kDa complex composed of two vitronectin and four PAI-1 molecules, suggesting a 2:1 stoichiometry.2 Binding studies using domain-specific monoclonal antibodies support the concept that there are two PAI-1-binding sites on vitronectin. The reported Mr of circulating PAI-1·vitronectin complexes (19, 20) is consistent with our results and raises the possibility that the complex, rather than the individual proteins, interacts with other macromolecules. Supporting this concept are studies demonstrating that vitronectin can bind PAI-1 and heparin simultaneously, indicating that PAI-1·vitronectin complexes can interact with other molecules (37).

Immunolocalization studies demonstrating both PAI-1 and vitronectin on fibrils of fibrin clots formed in vitro or in vivo (38, 39) are compatible with the notion that the individual proteins interact with fibrin. Although PAI-1 has been reported to bind directly to fibrin, it has yet to be shown that vitronectin or PAI-1·vitronectin complexes interact directly with fibrin. Recently, we demonstrated that vitronectin associates with fibrin in both purified and plasma systems.3 Based on these observations, we hypothesized that fibrin-bound vitronectin supports PAI-1 binding. To explore this possibility, direct binding studies were performed to quantify the interaction between PAI-1 and fibrin-bound vitronectin. In addition, functional PAI-1 assays were used to demonstrate that vitronectin enhances the inhibitory effects of PAI-1 on t-PA-mediated clot lysis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals, Proteins, Reagents-- Human glu-plasminogen, alpha -thrombin, fibronectin, and plasminogen-free fibrinogen was purchased from Enzyme Research Laboratories Inc. (South Bend, IN). Predominantly single-chain recombinant t-PA (Activase®) was obtained from Genentech Inc. (San Francisco, CA). The plasmin-directed chromogenic substrate S2251 was obtained from Chromogenix (Mölndal, Sweden). High binding 96-well microtiter plates were obtained from Costar Science Corp. (Cambridge, MA). Bovine serum albumin (Fraction V), p-nitrophenyl phosphate, alkaline phosphatase-conjugated streptavidin, soybean trypsin inhibitor, and phenylmethylsulfonyl fluoride were purchased from Life Technologies, Inc. Reduced glutathione, Tween-80, ethanolamine, diethanolamine, caprylic acid, and mouse IgG were obtained from Sigma. Affinity-purified sheep anti-human vitronectin IgG (SAHVn) and normal (nonimmune) sheep IgG were obtained from Affinity Biologicals (Hamilton, Ontario, Canada). Monoclonal antibody to PAI-1 (MAI-12) was purchased from Biopool AB (Umeä, Sweden). Plastic inoculation loops were obtained from Fisher Scientific (Nepean, Ontario, Canada). Tween-20, Coomassie Brilliant Blue R-250, urea (electrophoresis grade), acrylamide:bis 37.5:1 (2.6%C), molecular weight markers, glycine, TRIS, SDS, G-25M Sepharose, and gelatin were purchased from Bio-Rad. Specta/Por® CE (cellulose ester) MWCO 15,000 dialysis membrane tubing was purchased from VWR-Canlab (Mississauga, Ontario, Canada).

Preparation of Platelet-poor Plasma (PPP)-- Blood was collected from the antecubital vein of healthy volunteers into plastic syringes prefilled with <FR><NU>1</NU><DE>10</DE></FR> vol of 3.8% trisodium citrate. After mixing, the red blood cells and platelets were sedimented by centrifugation at 1800 × g for 15 min at 4 °C, and PPP was harvested and stored at -70 °C until needed.

Isolation of Vitronectin and Fibrinogen from Human Plasma-- Native vitronectin was purified and characterized as described previously (23). Briefly, vitronectin was isolated from PPP using immunoaffinity purification with the SAHVn IgG coupled to Affi-Gel affinity resin (Bio-Rad) and subjected to PAGE analysis in the presence or absence of SDS. Fibrinogen was rendered vitronectin-free using immunoaffinity chromatography with immobilized SAHVn IgG, which reduced the vitronectin levels from approximately 10 nM down to <10 pM vitronectin per µM fibrinogen.

Preparation of Radiolabeled Fibrinogen and PAI-1-- Fibrinogen was trace radiolabeled with Na125I using Iodobeads (Pierce) to a specific activity of ~100 µCi/mg, and its clotting activity was assessed by measuring its incorporation into fibrin clots (95-98%) (42). Recombinant human PAI-1 containing the 6 residual peptide consensus sequence for heart muscle kinase (HMK) at the amino terminus was constructed using the polymerase chain reaction, and the resulting fusion protein was expressed in Escherichia coli and isolated as described (43). When HMK-rPAI-1 was compared with fully active wild-type rPAI-1, both proteins inhibited t-PA and urokinase-type PA to the same extent, and both bound to vitronectin comparably. HMK-PAI-1 was radiolabeled by incubation with [32P]ATP (ICN Radiochemicals, Oakville, Ontario, Canada) in the presence of purified protein kinase from bovine heart muscle (Sigma), and the labeled protein was isolated by gel filtration on G-25M Sepharose. The specific activity of the 32P-PAI-1 was approximately 5800 cpm/ng.

Immunocytochemistry and Image Analysis-- To examine the spatial distribution of PAI-1 and vitronectin in clots, 150 µl of PPP was placed on APTEX-coated coverslips and clotted by the addition of CaCl2 (final concentration, 10 mM). After incubation at 37 °C for 1 h, the clots were fixed with cold 3% formaldehyde in PBS for 5 min, washed alternately with PBS alone and PBS containing 0.1 mol/liter glycine, and then incubated for 30 min with blocking buffer (PBS containing 0.5% bovine serum albumin and 50 µg/ml normal goat immunoglobulin). Primary antibodies, including a monoclonal anti-PAI-1 IgG (monoclonal antibody MAI-12), SAHVn IgG, and a sheep anti-human fibrinogen IgG were diluted in blocking buffer and incubated with the clots for 1 h at 37 °C. Control clots were stained with each primary antibody separately, stained without primary antibodies, or stained with nonspecific mouse and sheep IgG. After washing, clots were incubated for 1 h at 37 °C with Texas Red rhodamine-conjugated goat anti-sheep or fluorescein isothiocyanate-conjugated goat anti-mouse IgG diluted 1:20 in blocking buffer. The coverslips were washed, mounted on glass slides using Permafluor mounting medium, and then subjected to Z-plane optical sectioning (200 nm/section) using a Zeiss LSM 10 equipped with a 63× planapo oil immersion lens (numerical aperture = 1.4). Dual-wave-length images were acquired using an argon ion laser (488 nm excitation), a helium/neon ion laser (543 nm excitation), and two matched long pass barrier filters for fluorescein isothiocyanate (515-525 nm emission) and Texas Red rhodamine (575-640 nm emission) images. Image processing and three-dimensional volume rendering were performed using Metamorph software (Universal Imaging Inc., Chester, PA). Clots stained with nonspecific primary antibodies were used to threshold for background staining.

Lysis of 125I-Fibrinogen-labeled Plasma and Purified Fibrin Clots-- 125I-Labeled clots were formed around plastic inoculation loops (44) by clotting 250-µl aliquots of PPP or a solution containing purified fibrinogen and glu-plasminogen (final concentrations, 3 and 0.54 µM, respectively) diluted in Tris-buffered saline (TBS) (0.05 M Tris, pH 7.4, containing 0.15 M NaCl, 0.025% Tween 80, 5 nM thrombin, and 25 mM CaCl2). Prior to clotting, these solutions were spiked with 125I-fibrinogen (500,000 cpm/ml). The purified clots were also formed in the presence of various concentrations of PAI-1 and/or vitronectin. All clots were allowed to age for 2 h at 37 °C. After repeated washing with TBS, clots were then incubated with 0.1 nM t-PA or TBS at 37 °C. The extent of t-PA-induced clot lysis was quantified by counting the radioactivity of the residual clots. In addition, aliquots of the bathing buffer were removed at intervals to monitor the time course of the release of 125I-labeled fibrin degradation product. For some experiments, plasma clots were formed in the presence or absence of antibodies directed against PAI-1 (MAI-12) or vitronectin (SAHVn). Treatment of the clots with MAI-12 or SAHVn attenuated PAI-1-mediated inhibition of clot lysis in a dose-dependent manner, with maximal inhibition at 10 and 250 µg/ml, respectively. Previous reports have demonstrated that the monoclonal MAI-12 attenuates PAI-1-mediated inhibition of t-PA-dependent clot lysis (11, 16). The SAHVn IgG attenuates the binding of PAI-1 to vitronectin immobilized on the surface of microtiter wells in a dose-dependent fashion.

The percentage of clot lysis was calculated by subtracting the residual radioactivity of the clots from their initial radioactivity, and expressed this value as a percentage of the initial radioactivity. The vitronectin-dependent inhibition of t-PA-mediated lysis of purified clots was confirmed by solubilizing the residual clots formed in the presence or absence of PAI-1 and/or vitronectin with 1× Laemmli sample buffer in the presence or absence of 5% 2-mercaptoethanol and boiled for 1 h (45). The undissolved portion of the clot was pelleted by centrifugation, 50% of each of the dissolved clots and preclot supernatant samples were subjected to SDS-PAGE (7.5% polyacrylamide gel), and the gels were fixed and stained with Coomassie Blue. The dried gels then exposed to autoradiography film to visualize the distribution of fibrin and fibrin fragments (46).

Effects of Vitronectin and PAI-1 on t-PA-mediated Plasmin Generation on Fibrin Matrices-- To examine the effects of vitronectin on PAI-1 inhibition of t-PA-dependent plasmin generation on fibrin matrices, microtiter wells were first coated with fibrin formed by clotting 50-µl aliquots of a fibrinogen (375 nM) solution containing 0.5 nM vitronectin (or buffer alone) with 30 nM thrombin, 20 nM CaCl2. After incubation for 3 h at room temperature, the plates were stored at 4 °C until used. Previous studies have determined that the addition of 0.5 nM vitronectin to 375 nM fibrinogen prior to clotting resulted in the highest ratio of fibrin-bound versus free vitronectin.3 To block nonspecific binding, 100-µl aliquots of PBS containing 3% bovine serum albumin and 0.05% Tween-20 (blocking buffer) were added to wells for 2 h at 37 °C. The fibrin-coated wells were then preincubated for 1 h at 37 °C with increasing concentrations of PAI-1 (0.01-100 nM) diluted in PBS containing 3% bovine serum albumin, 0.1% Tween-80, 5 mM EDTA, 20 units/ml aprotinin, and 0.05% sodium azide (dilution buffer). For some experiments, fibrin matrices were incubated with PAI-1 in the presence or absence of 10 µg/ml of SAHVn IgG or preimmune sheep IgG. After washing three times with TBS containing 0.1% bovine serum albumin and 0.05% Tween-20 (wash buffer), the wells were then incubated at 37 °C for intervals of up to 24 h. At each time point, 0.48 nM t-PA, 0.54 µM glu-plasminogen, and 1 mM S-2251 were added, and the rate of plasmin generation was quantified by measuring the absorbance at 405 nm (A405 nm) every min for 1 h. Rates of plasmin generation were expressed as a percentage of the maximum rate of plasmin generation in which no PAI-1 was added. Using the residual plasmin activity at each concentration of PAI-1, we calculated the amount of PAI-1 that bound to fibrin in the presence and absence of vitronectin, and the apparent Kd (Kd(app)) was calculated for each condition using regression analysis.

Binding of 32P-HMK-PAI-1 to Fibrin Clots Formed in the Presence or Absence of Vitronectin-- The binding of radiolabeled PAI-1 to fibrin-coated (375 nM) microtiter plate wells was quantified in the presence or absence of 0.5 nM vitronectin. Various concentrations of 32P-HMK-PAI-1 (0.01-22 nM) in dilution buffer were incubated for 1 h at 37 °C with the fibrin matrices that were formed in the presence or absence of vitronectin. After the wells were rinsed three times with wash buffer, the bound and free PAI-1 were quantified by scintillation counting. Data for vitronectin-dependent binding of PAI-1 to fibrin (B in Equation 1) were analyzed by nonlinear least squares analysis according to the Hill equation,
B=(B<SUB><UP>max</UP></SUB>·[S]<SUP>n</SUP>)/((K<SUB>0.5</SUB>)<SUP>n</SUP>+[<UP>S</UP>]<SUP>n</SUP>) (Eq. 1)
in which S is the total PAI-1 concentration, Bmax is the amount of PAI-1 bound at saturation, K0.5 corresponds to the PAI-1 concentration at half-maximal saturation, and n is the Hill coefficient.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Microscopic Colocalization of Plasma Clot-associated PAI-1 and Vitronectin-- Although previous immunohistochemistry studies have demonstrated that PAI-1 and vitronectin are each localized on fibrin (38, 39), the extent to which these proteins colocalize has yet to be determined. Recently, we described the ultrastructural distribution of vitronectin aggregates associated with the surface of fibrin fibrils formed by clotting plasma or purified fibrinogen.3 To determine whether fibrin-associated vitronectin regulates PAI-1 distribution in clots, plasma clots were processed for dual-labeling immunofluorescence and confocal scanning laser microscopic image analysis to examine the distribution of plasma vitronectin and PAI-1 on fibrin fibrils (Fig. 1). Digitalized thresholding of optical sections from clots stained in the absence of a primary antibody or with preimmune IgG was used to establish the background levels of fluorescence. Fibrin-associated vitronectin staining is intense, and is distributed in a punctate, aggregate-like pattern along the fibrin fibrils (Fig. 1A). Specific PAI-1 staining is distributed intermittently along the length of the fibrin fibrils, with intense staining noted at junctions of overlapping fibrin fibrils (Fig. 1B, arrows). Quantitative analysis of the percentage of staining for PAI-1 that overlaps with that for vitronectin reveals that the majority (>87%) of fibrin-bound PAI-1 (Fig. 1C, green) colocalizes with the vitronectin aggregates (red) on the surface of fibrin fibrils (Fig. 1C, yellow). To define the distribution of fibrin fibrils, plasma clots also were stained for fibrin (Fig. 1D).


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  		Fig. 1.   Confocal imaging of vitronectin and PAI-1 colocalization in plasma clots. Pseudo-colored images of optical sections through plasma clots stained with an affinity-purified anti-vitronectin IgG (A) or a monoclonal anti-PAI-1 (B), and each primary was detected with Texas Red rhodamine-conjugated and fluorescein isothiocyanate-conjugated secondary antibodies, respectively. Digital overlap analysis of the vitronectin (red) and PAI-1 (green) images (C) indicates that the majority (>87%) of the PAI-1 distribution overlaps with the vitronectin (yellow) on fibrin fibrils. Staining with anti-fibrinogen IgG (D) illustrates the distribution of fibrin polymers. To facilitate image comparisons, the arrows in A and B provide points of reference where PAI-1 staining is most intense at sites of fibrin fibril overlap. Scale bar, 10 µm.

Influence of Vitronectin on PAI-1-mediated Inhibition of Clot Lysis-- To examine the influence of fibrin-associated vitronectin on PAI-1 activity, we monitored the t-PA-mediated lysis of 125I-fibrinogen-labeled plasma clots (Fig. 2). First, we quantified the t-PA dose- and time-dependent lysis profiles of 125I-fibrinogen-labeled plasma clots in order to determine an optimal t-PA concentration that results in approximately 40-50% lysis after 18-24 h of incubation (Fig. 2A). Based on these results, we used 0.08 nM t-PA to examine the lysis of plasma clots formed in the presence or absence of exogenous 1.0 µg/ml PAI-1. As illustrated in Fig. 2B, approximately 37% of the 125I-fibrin-labeled plasma clots was digested in the absence of exogenous PAI-1, whereas the addition of PAI-1 to the clot significantly (p = 0.001) reduced lysis to only 7%. Formation of clots in the presence of antibodies against PAI-1 (MAI-12) attenuated PAI-1-mediated inhibition of clot lysis, a finding consistent with previous in vitro (11) and in vivo (16) studies with this antibody. In contrast, the anti-vitronectin IgG (SAHVn) completely blocked the inhibitory effects of PAI-1 on clot lysis. Control experiments confirmed that the SAHVn IgG did not contain any plasminogen activator activity, and this antibody had no effect when added after clotting (data not shown). Neither preimmune mouse nor sheep IgG had any effect on clot lysis.


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  		Fig. 2.   Neutralization of PAI-1-dependent inhibition of plasma clot lysis by vitronectin antibodies. A, effects of varying the t-PA concentration on the time-dependent lysis of clots formed using plasma containing 100,000 cpm of 125I-fibrinogen. After clotting, samples were washed with PBS and then incubated at 37 °C for various times in 1 ml of PBS containing buffer alone (open circle ) or different doses of t-PA: 0.02 nM (black-square), 0.08 nM (black-triangle), 0.2 nM (black-down-triangle ), 0.4 nM (black-diamond ), and 0.8 nM (×). At each time point, clots and clot supernatants were then separated and counted. Data are expressed as percentage of clot lysis and represent the mean (n = 3) ± S.D. of one of five experiments. A, effects of antibodies directed against PAI-1 and vitronectin (Vn) on the lysis of plasma clots. Clots were formed using plasma containing 100,000 cpm of 125I-fibrinogen in the absence (NO PAI-1) or presence of 1.0 µg/ml (22 nM) PAI-1 alone (NO IgG) or PAI-1 plus anti-PAI-1 IgG (10 µg/ml) (ANTI-PAI-1), anti-Vn IgG (250 µg/ml) (ANTI Vn), or equivalent concentrations of nonimmune mouse or nonimmune sheep IgG (NM/NS). After clotting, samples were washed with PBS and then incubated at 37 °C for 18 h in 1 ml of PBS containing 0.08 nM t-PA. Clots and clot supernatants were then separated and counted. Data are expressed as percentage of clot lysis and represent the mean (n = 3) ± S.D. of one of five experiments.

Fibrin-bound Vitronectin Potentiates the PAI-1-mediated Inhibition of Fibrinolysis-- To confirm the results with SAHVn IgG in plasma, 125I-fibrinogen-labeled clots were formed from 3.0 µM purified fibrinogen, 22 nM PAI-1, 0.54 µM plasminogen, and various doses of vitronectin. These clots were then exposed to t-PA (Fig. 3). In control experiments, clots formed in the presence of increasing concentrations of vitronectin, but in the absence of PAI-1, no significant difference in t-PA-induced clot lysis was demonstrated. Indeed, >95% of the fibrin was solubilized (Fig. 3, open triangles). In the absence of vitronectin, PAI-1 had only a modest inhibitory effect on clot lysis, with 85% of the fibrin clot degraded (Fig. 3, open circle on left axis). When PAI-1 was added in conjunction with increasing concentrations of vitronectin there was a dose-dependent potentiation of PAI-1-mediated inhibition of clot lysis (Fig. 3, closed circles), and <10% of the clot was degraded with vitronectin doses >14 nM. These results suggest that preformed PAI-1·vitronectin complexes are incorporated into fibrin clots during coagulation. To determine whether the potentiation of PAI-1-mediated inhibition of clot lysis is due to direct interactions of vitronectin with fibrin, and to determine whether fibrin-bound vitronectin can bind PAI-1, clots were first formed in the presence of increasing doses of native vitronectin. After extensive washing, the clots were then incubated with PAI-1 for 1 h prior to t-PA exposure (Fig. 3, open circles). These studies indicate that PAI-1 inhibition of clot lysis is less efficient when vitronectin is first prebound to the fibrin clot during coagulation. Although these findings may simply reflect impaired diffusion of PAI-1 into a preformed clot, the results nonetheless demonstrate that fibrin-bound vitronectin can still potentiate PAI-1-mediated inhibition of t-PA. Furthermore, the results confirm that the incorporation of vitronectin into the clot is not PAI-1-dependent.


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  		Fig. 3.   PAI-1 inhibition of 125I-fibrin clot lysis is dependent on vitronectin. Purified fibrin clots were formed using 3 µM fibrinogen containing 100,000 cpm of 125I-fibrinogen per clot, 30 nM thrombin, 10 mM CaCl2, 0.54 µM glu-plasminogen, and increasing concentrations of (i) vitronectin alone (triangle ), (ii) vitronectin plus 1.0 µg/ml (22 nM) PAI-1 (), or (iii) vitronectin alone followed by 1.0 µg/ml PAI-1 in the bathing buffer postclotting (open circle ). Samples were clotted for 2 h at 37 °C, washed and then incubated for 18 h at 37 °C in 1 ml of TBS buffer containing 0.08 nM t-PA. Clots and clot supernatants were then separated and counted. Data are expressed as percentage of clot lysis and represents the mean (n = 3) ± S.D. of one of five experiments.

In order to confirm that purified vitronectin potentiates the PAI-1-mediated inhibition of fibrinolysis, purified 125I-fibrinogen-labeled clots that had been subjected to t-PA-mediated fibrinolysis were solubilized. The soluble extracts were fractionated using SDS-PAGE under reduced and nonreduced conditions in order to evaluate changes in fibrin degradation (Fig. 4). As demonstrated in Fig. 3, the presence of PAI-1 or vitronectin alone had no significant effects on the degradation of fibrin (Fig. 4). However, in the presence of PAI-1 and vitronectin doses >4.0 µg/ml, the fibrin degradation pattern on nonreduced gels illustrates that vitronectin markedly inhibits the degradation of fibrin polymers with a molecular mass of >250 kDa (Fig. 4, arrows a), and lower molecular mass forms of covalently linked polymers of fragments D, Y, and X (arrows b). The vitronectin-dependent potentiation of PAI-1-mediated inhibition of fibrinolysis was further noted following reduction of the clot lysate samples as indicated by the increased amounts of gamma -gamma dimers (Fig. 4, arrows c) and beta -chain fragments (arrows d).


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  		Fig. 4.   SDS-PAGE analysis of vitronectin and PAI-1 effects on fibrin degradation. Purified 125I-fibrinogen-labeled clots formed in the presence or absence of vitronectin and/or PAI-1 and subjected to t-PA-mediated fibrinolysis were solubilized, and the extracts were fractionated using SDS-PAGE under reduced and nonreduced conditions, followed by Coomassie Blue staining and autoradiography in order to visualize changes in fibrin degradation. Numbers under the lanes correspond to the concentrations of vitronectin (Vn) and PAI-1 added together to the fibrinogen mixture prior to clotting. Arrows: a, uncleaved fibrin polymers; b, fibrin fragments D, Y, and X; c, gamma -gamma dimers; d, beta -chain fragments.

Vitronectin Mediates the Binding and Stabilization of PAI-1 on Fibrin-- To examine the effects of vitronectin on the binding of active PAI-1 to fibrin, we used a functional assay as an index of active PAI-1 bound to fibrin (Fig. 5). Fibrin clots formed in the presence or absence of 0.5 nM vitronectin were incubated for 1 h with increasing concentrations of PAI-1. After washing, the clots were then incubated for a further 1-24 h at 37 °C prior to measuring the fibrin-bound PAI-1 activity using an amidolytic substrate assay. From this information, the concentration of functionally active PAI-1 bound to fibrin was estimated. Regression analysis of the data was used to calculate Kd(app) for the interaction between active PAI-1 and fibrin. In the absence of vitronectin, very little active PAI-1 bound to fibrin after 1 h of incubation, as the interaction is very weak (Kd(app) ~ 3.5 µM). Moreover, there was no fibrin-associated PAI-1 activity detectable after 24 h. In contrast, in the presence of vitronectin, the Kd(app) values at 1 and 24 h were 0.7 and 0.9 nM, respectively. These findings suggest that the fibrin-bound PAI-1 remains functionally active over a 24-h period when in complex with vitronectin. Furthermore, co-incubation of the PAI-1 dilutions with 10 µg/ml of the SAHVn IgG increased the Kd(app) value up to 60 nM after 24 h (data not shown), thereby confirming the observed pro-fibrinolytic effect of this antibody in the lysis of plasma clots (Fig. 2B).


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  		Fig. 5.   Vitronectin-dependent stabilization of PAI-1 activity on fibrin clots. Increasing doses of PAI-1 were incubated for 1 h at 37 °C in wells coated with fibrin (0.375 µM) formed in the presence (circles) or absence (triangles) of vitronectin (0.5 nM), the unbound PAI-1 was washed away, and samples were further incubated at 37 °C for either 1 h (open symbols) or 24 h (closed symbols). After the specified time points, the plasmin generation on the fibrin surface was monitored by adding t-PA, plasminogen, and S-2251 to each well, and samples were monitored every 1 min at 405 nm for 1 h. The rate of plasmin generation was calculated for each condition, and data are plotted as percentage of plasmin activity of control samples (no added PAI-1).

We next directly quantified the specific binding of radiolabeled PAI-1 to purified fibrin clots formed in the presence or absence of vitronectin (Fig. 6). The results indicate that in the absence of vitronectin (Fig. 6A, squares), there is only low affinity binding of PAI-1 to fibrin, with an estimated micromolar affinity similar to that calculated from the functional assays in Fig. 5. In contrast, in the presence of vitronectin (Fig. 6A, circles), there is saturable binding at much lower concentrations. Evaluating the vitronectin-specific component of the binding of PAI-1 by subtracting the contribution of PAI-1 binding to fibrin alone yields a sigmoidal binding isotherm (Fig. 6B) that can be fit to the Hill equation with a Hill coefficient of 1.5 and maximal binding equal to 1 nM concentrations of PAI-1. The Hill number is indicative of cooperativity between PAI-1-binding sites on vitronectin. It is noteworthy that the binding stoichiometry almost exactly equals 2 PAI-1 molecules per vitronectin molecule. This result is consistent with our recent report from biophysical analyses that indicates that higher order complexes between vitronectin and PAI-1 are formed with a molar binding ratio of 2:1 (PAI-1:vitronectin).2 Fig. 6C shows a Scatchard analysis of the PAI-1 binding data that is specific to vitronectin. The downward curvature of the plot at low concentrations of bound ligand is indicative of cooperative binding. The Scatchard plot is extremely sensitive to cooperative binding and may be diagnostic in cases in which cooperativity is less apparent from the binding isotherms of the type shown in Fig. 6B. This cooperative, bivalent binding of PAI-1 to fibrin-associated vitronectin suggests that the initial binding of one PAI-1 molecule induces conformational changes in fibrin-bound vitronectin such that the second PAI-1-binding site on vitronectin is more readily available for interacting with a second PAI-1 molecule.


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  		Fig. 6.   Dose-response of PAI-1 binding to fibrin formed in the presence or absence of vitronectin. Increasing doses of 32P-HMK-PAI-1 were incubated for 1 h at 37 °C in wells coated with fibrin (0.375 µM) formed in the absence or presence of vitronectin (0.5 nM). A shows total binding to fibrin matrices formed in the presence (circles) or absence (squares) of vitronectin. The data in B, which represent binding to the fibrin matrix that is strictly vitronectin-dependent, were generated by subtracting the amount of PAI-1 bound to fibrin alone from the amount of PAI-1 bound to vitronectin-containing clots for the corresponding amounts of total PAI-1 added in the experiment. The fit to the data is a nonlinear fit to the Hill equation, yielding a value for the Hill coefficient of 1.5 and a maximum amount of PAI-1 bound at saturating equal to 1 nM. The K0.5, or average binding affinity for PAI-1 to fibrin-bound vitronectin, is approximately 1.5 nM from the fit. C shows a treatment of the data for PAI-1 binding that is specific for fibrin-bound vitronectin using a Scatchard analysis. The downward curvature of the plot at lower concentrations of bound PAI-1 is indicative of cooperative binding.

Although these results support the concept of two PAI-1-binding sites, the cooperative nature of these binding interactions make it virtually impossible to measure the dissociation constants for each interaction using these experimental approaches. Furthermore, we cannot elucidate the mechanisms regulating the binding preferences, or the communication between sites from these data alone. An estimate of the average binding affinity, K0.5 in the Hill equation, using these data gives a value of 1.5 nM, consistent with previously reported high affinity measurements for PAI-1-vitronectin binding interactions that were derived using an oversimplified 1:1 binding site model (25, 31, 33-37). Although not equivalent to a true binding constant, this value of K0.5 reflects a high affinity interaction that is in the range to give saturable binding of approximately nanomolar concentrations of PAI-1 under the conditions of this experiment, in which the vitronectin concentration was fixed at 0.5 nM.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The studies presented in this report provide the first evidence that vitronectin plays a critical role in the regulation of PAI-1 binding to fibrin. Under a variety of experimental conditions, we have demonstrated that fibrin-associated vitronectin influences fibrinolysis by serving as an intermolecular bridge to support high affinity binding of PAI-1 to fibrin. In this work, the importance of vitronectin as the mediator of fibrin-associated PAI-1 activity is underscored by the finding that PAI-1-mediated inhibition of clot lysis is vitronectin-dependent in both purified fibrin and plasma clots. The concept that vitronectin binds to fibrin and PAI-1 binds to fibrin via vitronectin explains why both of these proteins have been immunolocalized on the surface of fibrin polymers (38, 39) and provides a more plausible explanation for the mechanism by which active PAI-1 binds to fibrin clots. Although our binding studies confirm previous reports of a low affinity binding interaction of PAI-1 to fibrin, the physiological relevance of these interactions is questionable. Given that plasma concentrations of PAI-1 are in the nanomolar range, whereas the concentration of vitronectin is in the micromolar range, all active plasma PAI-1 would be expected to be complexed with vitronectin. Moreover, the observation that the reported low affinity fibrin-binding site on PAI-1 overlaps with the vitronectin-binding domain (16, 35, 40) implies that the fibrin-binding site on PAI-1 would be masked when PAI-1 is in complex with vitronectin.

A New Model for the PAI-1-dependent Inhibition of Fibrinolysis-- Our recent studies indicate that there are a limited number of specific vitronectin-binding sites available on fibrin. Vitronectin binds saturably to these sites with an estimated stoichiometry as high as 1 mol of vitronectin bound for every 20-70 mol of fibrin.3 When native vitronectin is in excess, self-association of vitronectin on the fibrin surface is observed. It is likely that this reflects cooperative binding interactions of additional fluid-phase vitronectin subunits to the fibrin-bound vitronectin multimers. In light of the high concentration of vitronectin incorporated into purified or platelet-poor plasma clots, it is not surprising that fibrin-associated vitronectin has a major impact on PAI-1 binding to fibrin. For example, the incorporation of 0.5 µM plasma vitronectin into clots may represent up to 1.0 µM PAI-1 binding capacity, an amount that is significantly greater than the highest reported concentration of plasma PAI-1 in any pathophysiological state.

Quantitative immunofluorescence confocal microscopy confirmed that >87% of the plasma PAI-1 colocalizes with vitronectin aggregates along fibrin fibrils (Fig. 1). Moreover, the functional importance of plasma vitronectin for PAI-1 interactions with plasma clots was put to the test by examining the effects of neutralizing antibodies directed against PAI-1 and vitronectin on the t-PA-mediated lysis. The 125I-fibrinogen-labeled plasma clots were formed in the presence of 22 nM PAI-1, a concentration of PAI-1 at the upper limit of its circulating levels (Fig. 2). The monoclonal anti-PAI-1 IgG partially attenuated the PAI-1 inhibitory effects on t-PA but did not completely block its activity consistent with previous reports with this antibody (11, 16). In contrast, affinity-purified anti-vitronectin IgG completely blocked the PAI-1 effects on clot lysis, raising the possibility that inhibiting the vitronectin-dependent incorporation of PAI-1·vitronectin complexes into clots may be a novel strategy to enhance in situ thrombolysis.

Fibrin-bound Vitronectin Mediates the Binding and Stabilization of PAI-1 to Clots-- The functional consequences of fibrin-bound vitronectin on fibrin-bound PAI-1 activity was examined with clots formed in the presence or absence of vitronectin and then preincubated with PAI-1 for various times prior to measuring t-PA-mediated plasmin generation on the fibrin surface using an amidolytic assay (Fig. 5). The results confirmed the presence of low affinity fibrin-binding sites for PAI-1 and demonstrated that fibrin-bound vitronectin significantly increases the level of stable fibrin-bound PAI-1 activity. In order to further quantify the effects of vitronectin on PAI-1 binding to fibrin, we incubated clots formed in the presence or absence of vitronectin with increasing concentrations of 32P-HMK-PAI-1 (± excess unlabeled PAI-1), and measured the specific radioactivity that was bound (Fig. 6). The results confirm previous reports of the presence of relatively low affinity binding sites for PAI-1 on fibrin (15-18). Furthermore, these data indicate that PAI-1 binds cooperatively to fibrin-bound vitronectin, with a stoichiometry of 2 mol of PAI-1 bound per mol of fibrin-bound vitronectin.

Some controversy has surfaced over the last few years regarding the issue of PAI-1-binding sites on vitronectin. Indeed, PAI-1-binding sites have been localized to two distinct regions of vitronectin. However, most of the binding data reported to date for PAI-1 and vitronectin have been interpreted assuming a single binding site model. This is despite the fact that early binding experiments with one or the other reactant adsorbed to a microtiter plate surface indicated ratios varying from 1:1 (vitronectin:PAI-1) with immobilized PAI-1 to a ratio of 1:3 (PAI-1:vitronectin) using immobilized vitronectin (32). The PAI-1 binding studies in this study utilize vitronectin that is co-localized with fibrin during coagulation, providing a surface that likely displays vitronectin in a physiologically relevant conformation. As noted above, this conformation of vitronectin appears to bind two molecules of PAI-1. Our recent work using analytical ultracentrifugation and monoclonal antibodies specific for different epitopes on vitronectin consistently provided evidence for a 1:2 (vitronectin:PAI-1) stoichiometry of the complex formed by the two proteins in solution.2 Taken together with the results from this study on fibrin-associated vitronectin, the data support the notion that both of the vitronectin regions appear to be functional PAI-1-binding sites.

Our results support the concept that vitronectin is the mediator of PAI-1 binding to fibrin clots. Moreover, these results confirm our previous report of a bivalent interaction between PAI-1 and vitronectin2 and provide the first evidence that the PAI-1·vitronectin complex is the physiological form that interacts with fibrin. Many questions remaining regarding the mechanism regulating PAI-1·vitronectin complex binding interactions with fibrin remain. It is unlikely that Factor XIII transglutaminase cross-linking of these proteins is involved, because the binding of vitronectin to fibrin and the binding of PAI-1 or PAI-1·vitronectin complexes to fibrin are independent of cross-linking activity (not shown). These results are consistent with the recent report that monocyte-derived PAI-2, but not PAI-1, is cross-linked to fibrin (41). This difference implies that these two inhibitors bind to thrombi through distinct mechanisms. Clots formed in the presence of platelets also show co-distribution of PAI-1·vitronectin complexes on fibrin (39). These findings raise the possibility that the binding of platelet PAI-1 to fibrin may also be vitronectin-dependent. Future studies will be aimed at further defining the mechanism regulating fibrin interactions with vitronectin and with PAI-1·vitronectin complexes and examining the role of fibrin-bound PAI-1·vitronectin complexes in the organization of thrombi and in angiogenesis.

    FOOTNOTES

* This work was funded by an operating grant from the Medical Research Council of Canada (to T. J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Career Investigators of the Heart and Stroke Foundation of Canada.

|| To whom correspondence should be addressed: Hamilton Civic Hospitals Research Centre, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.: 905-527-2299, ext. 2630; Fax: 905-575-2646; E-mail: podort@fhs.csu.mcmaster.ca.

Dagger Dagger Supported by National Institutes of Health Grant HL50676 and by an Established Investigator Award from the American Heart Association.

¶¶ Supported by National Institutes of Health Grants HL 55374, HL55747, and CA 83090.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M908079199

2 Podor, T. J., Shaughnessy, S. G., Blackburn, M., and Peterson, C. (May 19, 2000) J. Biol. Chem. 10.1074/jbc.M000362200.

3 T. J. Podor, J. Weitz, and C. Peterson, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: t-PA, tissue-type plasminogen activator; PAI-1, type 1 plasminogen activator inhibitor; PBS, phosphate-buffered saline; PPP, platelet-poor plasma; SAHVn, affinity-purified sheep anti-vitronectin IgG; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; HMK, heart muscle kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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