Published online before print October 6, 2005, doi: 10.1161/01.ATV.0000189305.84297.8b
© 2005 American Heart Association, Inc.
Thrombosis |
Fibrinogen Contains Cryptic PAI-1 Binding Sites That Are Exposed on Binding to Solid Surfaces or Limited Proteolysis
From the Center of Medical Biology (K.S., J.B., C.S.C.), Polish Academy of Sciences, Lodz; the Department of Molecular and Medical Biophysics (J.S., C.S.C.), Medical University in Lodz, Poland; and the Laboratory for Pharmaceutical Biology and Phytopharmacology (A.G.), Katholieke Universiteit Leuven, Belgium.
Correspondence to Czeslaw S. Cierniewski, PhD, Department of Medical and Molecular Biophysics, Medical University in Lodz, 92-213 Lodz, 6/8 Mazowiecka St. E-mail cciern{at}zdn.am.lodz.pl
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Abstract |
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Objective— In this work, we identified the fibrinogen sequence that on exposure serves as the primary binding site for functionally active PAI-1 and to a lesser extent for its latent form. In contrast, this site only weakly interacts with PAI-1 substrate.
Methods and Results— The binding site is located in the N-terminal 
(20-88) segment of fibrinogen, in the region exposed on (1) adsorption of fibrinogen to solid surfaces; (2) the release of fibrinopeptide A during thrombin conversion of fibrinogen to fibrin; and (3) plasmin degradation of fibrinogen. This region was first identified by the yeast 2-hybrid system, then its binding characteristics were evaluated using the recombinant (16-120) fragment and its shorter version, the (20-88) fragment, in a solid phase binding assay and plasmon surface resonance measurements. Because fibrinogen fragment E does not bind PAI-1, it suggests that sequences of A chain interacting with PAI-1 are located in the N-terminal part of the (20-88) segment.
Conclusions— Therefore, PAI-1 directly bound to the (20-88) and thus concentrated in fibrinogen/fibrin, particularly at sites of injury and inflammation, may account for the recent observations that both its active and latent forms stimulate cell migration and wound healing.
We showed that PAI-1 is directly bound to the (20-88) and thus its concentration on fibrinogen/fibrin, particularly at sites of injury and inflammation, may account for the recent observations that both its active and latent forms stimulate cell migration and wound healing.
Key Words: conformational changes • fibrinogen • PAI-1 binding sites • proteolysis
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Introduction |
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PAI type-1 (PAI-1) is mainly identified as the primary physiological inhibitor of both urokinase-type (urokinase plasminogen activator) and tissue-type (tPA) plasminogen activators, and plays an important role in the regulation of the fibrinolytic system as well as in extracellular matrix remodeling. Such a broad range of biological activities of PAI-1 results from its unusual properties, ie, its inherent ability to self-inactivate 1,2 and harboring interaction sites for a number of proteins.3–6 Under normal conditions, PAI-1 is present in plasma at low concentrations, although high levels are observed in a variety of clinical settings.7 The interaction of PAI-1 with fibrin has been extensively studied and is predominantly defined by reversible low-affinity binding.8,9 Initially, it was proposed that PAI-1 binds directly to fibrin,10 accumulates within human thrombi and protects fibrin clots from premature dissolution.11 However, recent studies provided the evidence that PAI-1 not only circulates in a complex with vitronectin12 but binds to fibrin via vitronectin which provides an intermolecular bridge facilitating high affinity interactions between PAI-1 and the fibrin network.13 (please see supplement I at http://atvb.ahajournals.org).
However, this concept does not take into consideration recent experimental observations. Local concentrations of PAI-1 can be much higher than those measured in blood plasma with levels almost equivalent to that of vitronectin. High local concentrations of fibrin-bound platelet PAI-1 were found to be 10 µg/mL14 or more,15 indicating that both high-affinity and low-affinity binding sites can be involved in anchoring PAI-1 within the fibrin network. Mechanisms alternative to that using vitronectin were described and proposed to accumulate and concentrate PAI-1 at the cell surface or extracellular matrix.6,16 Components of the fibrinolytic system are implicated not only in removing fibrin deposits from the vasculature but also in extracellular matrix remodeling.17,18 Unexpectedly, these new functions are not dependent on the presence of plasminogen or the generation of plasmin.16 Thus, via formation of multimolecular complexes on the cell membranes or in extravascular areas, PAI-1 can have profound effects on cells, including their attachment, detachment, and migration in the extracellular matrix. Hence, knowledge about medium or low affinity binding sites for PAI-1 and their cross-talk with high-affinity sites can be critical in understanding these mechanisms and explaining the complex biology of PAI-1. Therefore, in the present work we characterize binding sites for PAI-1 present in fibrinogen and describe their interaction with different molecular forms of PAI-1.
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Materials and Methods |
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Proteins and Reagents
Human PAI-1 and vitronectin were purchased from Calbiochem (La Jolla, Calif). Different molecular forms of PAI-1 such as active, latent, and substrate as well as PAI-1 mutant Q123K, which does not interact with vitronectin obtained from Dr P. Declerck (Katholieke Universiteit Leuven, Belgium). Affinity-purified sheep anti-human vitronectin IgG (SAHVn) was obtained from Affinity Biologicals (Hamilton, Ontario, Canada). The BIACORE® biosensor system, reagents, and CM5 sensor chips (research grade), were from Biacore AB (Uppsala, Sweden). Vitronectin enzyme-linked immunosorbent assay kit HVNKT was purchased from Innovative Research Inc. (Southfield, Mich). Fragments D and E were kindly donated by Dr L. Medved (American Red Cross, Rockville, Md).
Fibrinogen
Fibrinogen was depleted from vitronectin by immunoaffinity chromatography using affinity-purified sheep anti-human vitronectin IgG immobilized on Sepharose 4B. Contamination of fibrinogen with vitronectin was analyzed by a sandwich-type immunoassay using the enzyme-linked immunosorbent assay kit HVNKT. Such a purification procedure reduced the vitronectin levels to approximately 100 pg per ml of 1 µmol/L fibrinogen solution.
Fibrinogen Recombinant Fragments
To obtain fibrinogen (16-120), a 256-bp cDNA fragment corresponding to 64 to 320 bp was generated by digestion with EcoRI of pGAD-10 containing this fragment and selected after library screening.6 This fragment was subcloned into the EcoRI site of the pRsetA expression vector. Then, a 204-bp cDNA fragment encoding (20-88) was generated from pRset A-Fg(16-120) and subcloned into the BamHI and XhoI sites of pRset A expression vector.
2D SDS PAGE and Microsequencing
Samples of fibrinogen and vitronectin-free fibrinogen were separated by 2D gel electrophoresis using ready-made gels with immobilized pH gradients (Amersham Biosciences) and spots expected to contain vitronectin were excised from the gel and subjected to in-gel digestion with trypsin and microsequenced using an electrospray (ISI-Q-TOF-Micromass) spectrometer as we described previously.19
Enzyme Immunoassay
To monitor exposure of fibrinogen (16-120) epitopes induced by plasmin cleavage of fibrinogen, and the presence of vitronectin in fibrinogen in both samples of fibrinogen separated by 2D SDSPAGE, a competitive inhibition enzyme-linked immunosorbent assay was used as we described previously.20
Solid Phase Binding Assays
Direct binding assays were performed according to the procedure described in our previous work6 by adding increasing concentrations of PAI-1 (the active, latent, or substrate form, the stable PAI-1 mutant 14-1B and PAI-1 Q123K) to the wells of 96-well microtiter plates coated with fibrinogen, fibrin, and fibrinogen recombinant fragments (16-120) and (20-88) or fragments D and E, respectively. The same system was used to evaluate competitive inhibition of PAI-1 binding to fibrinogen produced by plasmin derived fragments of fibrinogen. Independently, binding of PAI-1 (the active, latent, and substrate forms as well as PAI-1 mutant Q123K) was performed in Leuven according to the procedure described by Gils et al.21
Surface Plasmon Resonance
The kinetic parameters (association and dissociation rate constants, kon and koff, respectively) and the affinity constant (KD) for binding of the stable PAI-1 mutant 14-1B to fibrin and fibrinogen fragments were measured by surface plasmon resonance using a BIAcore X (Biacore AB) as we described previously.6 The overall affinity constant, KD, was derived from kon/koff.
Analytical Procedures
The protein content of fibrinogen fragments was determined by the bicinchoninic acid method.22 The purity of proteins was analyzed by SDS-PAGE.23
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PAI-1 Binding Sites in Fibrinogen
(16-120) Sequence Identified by the 2-Hybrid SystemIn our preliminary studies, several proteins were identified to interact with PAI-1 after using the yeast 2-hybrid system and screening human liver cDNA libraries cloned into the GAL4 activation domain, with PAI-1 fused to the GAL4 DNA binding domain used as the bait protein. Among His+LacZ+ colonies isolated, there was also a colony giving a strong positive reaction, identified by DNA sequencing to encode the
(16-120) fragment of fibrinogen (Figure I, available online at http://atvb.ahajournals.org). To verify this interaction, 2 recombinant fragments of the fibrinogen chain, (16-120), and its shorter version (20-88), were expressed in Escherichia coli as His-tag fusion proteins, purified on nickel chelating columns in 6 mol/L urea, and refolded by sequential dialysis to remove the denaturant. The final products were soluble in aqueous buffers and were homogenous as assessed by SDS-PAGE and reacted with polyclonal antibodies to human fibrinogen (Figure I).
Characterization of Vitronectin-Free Fibrinogen
Because PAI-1 binds tightly to vitronectin,3,13,24 it was important to establish that the fibrinogen used in these studies was devoid of vitronectin. For this purpose, fibrinogen was depleted of vitronectin using immunoaffinity chromatography with immobilized sheep anti human vitronectin antibodies. The fibrinogen-vitronectin mixture (1 µg of vitronectin added to 100 µg fibrinogen) (Figure 1A, upper gel) and vitronectin-free fibrinogen (Figure 1A, lower gel) were reduced and separated by 2D gel electrophoresis. After silver staining, the 2D protein patterns of fibrinogen enriched in vitronectin clearly shows the presence of this protein migrating as a double spot. As described before,25 the A chain was heterogeneous both in molecular mass and charge, whereas the Bß chain appeared as 4 distinct spots of equivalent size. Identification of protein spots migrating with size and charge similar to vitronectin was performed by peptide sequencing and peptide mass fingerprinting using an electrospray (ISI-Q-TOF-Micromass) spectrometer (Table I, available online at http://atvb.ahajournals.org). There was no vitronectin detectable in the fibrinogen purified by affinity chromatography (Figure 1A). To further determine whether vitronectin was present in the fibrinogen, a competitive inhibition enzyme-linked immunosorbent assay specific for vitronectin was used (please see supplement III). Figure 1B shows that fibrinogen, when used at 100 000-fold higher concentration than vitronectin, did not produce any competitive inhibition indicating that vitronectin, if present at all, is at very low levels in the fibrinogen preparation. This was further evidenced by using a commercial sandwich-type immunoassay kit for vitronectin, which showed that 1 mg of fibrinogen used in these experiments contained <300 pg of vitronectin.
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Interaction of Different Molecular Forms of PAI-1 With Fibrin and Its Fragments
In the next set of analyses, the 2 -chain fragments were immobilized onto microtiter wells, and their binding of the stable PAI-1 mutant (14-1B) was compared with that of fibrinogen, fibrin and its 2 major cleavage products, fragments D and E. Surprisingly, PAI-1 bound to the same extent to both fibrinogen and fibrin. Both recombinant fragments, (16-120) and (20-88), displayed the capacity to bind PAI-1 at levels only slightly lower than those observed with fibrinogen. Both fragments D and E showed only residual ability to interact with PAI-1 (Figure 2A). Furthermore, the binding of PAI-1 to fibrinogen and fibrin was inhibited by 80% in the presence of 100-fold molar excess of the (16-120) fragment, indicating that the latter contains a major interaction site of fibrinogen for the inhibitor (Figure 2B). To evaluate which molecular forms of PAI-1 interact with these sites we analyzed binding of active, latent, and substrate PAI-1. Figure 2C shows that these binding sites preferentially interact with active form, to a lesser extent with latent PAI-1, and only weakly with substrate PAI-1. Furthermore, PAI-1 Q123K, which has dramatically reduced binding affinity to vitronectin,26 showed unchanged binding properties toward fibrinogen when compared with the wild-type active PAI-1(Figure 2C). Essentially the same binding characteristics were obtained when binding to fibrin was analyzed (not shown). Because PAI-1 14-1B showed the same binding characteristics as the active form of wild PAI-1, it was used in all remaining experiments to maintain PAI-1 functional activity during the extended incubations required for different assays.
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PAI-1 Binding Sites and (16-120) Sequence Are Exposed on Fibrinogen Binding to Solid Surfaces
Because PAI-1 binding to fibrinogen could be detected by a solid phase binding assay, we further tested the mechanism by which the binding sites are exposed in fibrinogen molecule. Therefore, to further characterize this reaction, the real-time bimolecular interactions in vitro during complex formation between PAI-1 and fibrinogen, fibrin or its chain fragments, were next monitored by surface plasmon resonance. For this purpose, limiting amounts of fibrin monomers, fibrinogen, (16-120) or (20-88) were immobilized on separate sensor chips through amine coupling. Injection of active PAI-1 as the analyte to the sensor chip containing fibrinogen produced a typical surface plasmon resonance binding signal (Figure 3A). Similar response was produced when PAI-1 binding to fibrin was analyzed. The binding of PAI-1 was dose-dependent and the maximum response monitored at the end of the protein injection phase was 325 and 330 for 5000 RU of initially immobilized fibrinogen and fibrin monomers, respectively. The specific signal produced by binding of PAI-1 to (16-120) or (20-88) was equal to 300 and 290 RU for 3200 and 3100 RU, respectively, of the immobilized peptides. There was no binding of PAI-1 to the immobilized fragment D or fragment E thus supporting data produced by enzyme-linked immunosorbent assay. When PAI-1 was immobilized on a sensor chip and (16-120) or (20-88) were used as analytes, they showed almost the same values of analytic parameters, including KD, as those estimated in the reversed system (Table). In contrast, under such conditions, soluble fibrinogen showed only residual binding to PAI-1, characterized by a KD 2 orders of magnitude higher than that calculated for binding of PAI-1 to the immobilized fibrinogen (Figure 3B). As is shown in the Table, PAI-1 interacts with fibrin and (16-120) with the same binding affinity. Furthermore, binding of vitronectin or PAI-1 to fibrin is described by the same values of KD, indicating the same binding affinity of fibrin for both proteins. To analyze whether binding of fibrinogen to solid surfaces results in exposure of the (16-120) sequence, we next analyzed expression of (16-120) epitopes in both fibrin and fibrinogen by a solid phase enzyme immunoassay. For this purpose, polyclonal antibodies to (16-120) were isolated from anti-fibrinogen A antiserum by affinity chromatography using the recombinant (16-120) fragment immobilized on Sepharose 4B. Figure 3C shows binding of affinity-purified polyclonal antibodies to (16-120) epitopes on fibrinogen and fibrin but lack of binding to FgD. This experiment indicates that adsorption of fibrinogen to a solid plastic surface results in exposure of the (16-120) sequence to the same extent as that observed in fibrin.
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PAI-1 Binding Sites and (16-120) Sequence Are Exposed by Plasmin Cleavage of Fibrinogen
Next, experiments were designed to characterize the interaction of fibrinogen with PAI-1 in a liquid phase. For this purpose, fibrinogen was first preincubated with PAI-1 in 10-fold molar excess and then aliquots of the mixture were added to the wells of 96-well microtiter plates coated with fibrinogen. Figure 4A shows that preincubation of PAI-1 with soluble fibrinogen did not influence its interaction with the immobilized fibrinogen. However, cleavage of fibrinogen with plasmin almost completely exposed PAI-1 binding sites in degradation products. Thus, 2 hours of fibrinogen digest, when preincubated with PAI-1 at a 10-fold molar excess, abolished the interaction of PAI-1 with the immobilized fibrinogen. SDS PAGE of such digests showed exclusively the presence of final degradation products of fibrinogen, namely FgD1, FgD2, FgD3, and FgE3 (Figure 4A, inset). This ability to form a complex with PAI-1 in a liquid phase and thus inhibit its interaction with the immobilized fibrinogen was directly associated with a progressive exposure of (16-120) epitopes, as evidenced by a competitive inhibition enzyme immunoassay (Figure 4B). In this experiment, aliquots of the affinity-purified anti (16-120) antibodies were preincubated with increasing concentrations of a fibrinogen digest, withdrawn and quenched at different time points, and then introduced to the wells of 96-well microtiter plates coated with recombinant (16-120) fragment. The recombinant (16-120) fragment used in parallel was a control. These data indicate that after 2 hours of digestion with plasmin, (16-120) epitopes are almost fully exposed, thus supporting their role in binding of PAI-1. To further support location of the PAI-1 binding site in fibrin/fibrinogen, we next attempted to purify peptide fragments from the digestion mixture by an affinity chromatography on immobilized PAI-1. For this purpose, the 120 minutes of plasmic digest of fibrinogen was subjected into the recombinant PAI-1 immobilized on the Ni2+-HiTrap column attached to fast protein liquid, then the column was washed extensively with 0.14 mol/L NaCl buffered with 0.1 sodium phosphate, pH 7.3, and peptides bound were eluted with 0.1 mol/L acetic acid. The bound fragments were identified by sequencing using an electrospray (ISI-Q-TOF-Micromass) spectrometer (please see supplement IV). There were several short fragments of fibrinogen identified in peptide material eluted from the column. Among them there were N71SLFEYQK78, G96DFSSANNR104, as well as D172YEDQQKQLEQVIAK191. Two first peptides derived from the region of chain which was originally identified by the yeast 2-hybrid system to bind PAI-1.
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Discussion |
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In this work we provide evidence that PAI-1 interacts with the fibrinogen
(16-120) sequence (see supplement V), which is cryptic in the intact fibrinogen molecule but can be exposed either on its adsorption to solid surfaces or proteolytic cleavage by thrombin and plasmin. This conclusion is supported by the following observations. This fibrinogen segment first identified by the yeast 2-hybrid system to bind PAI-1, when obtained as the recombinant peptide, interacted predominantly with active PAI-1. The affinity-purified anti-(16-120) polyclonal antibodies bound to fibrinogen immobilized on the plastic wells, indicating that their (16-120) epitopes are well exposed (see supplement VI). Based on the recently published crystal structure, this fragment is located entirely in the -helical coiled-coil domain region of fibrinogen.27 Tryptic digest of the peptide material eluted from the immobilized PAI-1 showed a presence of several short peptides including N71SLFEYQK78, G96DFSSANNR104, which are from the same region as (16-120). Interestingly, these tryptic peptides are derived from the soluble fibrin degradation products released from a fibrin clot perfused with plasmin.28 Fibrinogen and fibrin, when immobilized on the plastic wells or covalently attached to BIAcore CM5 chips, bound PAI-1 with the same binding capacity and affinity. They interacted the most efficiently with active PAI-1, to lower extent with its latent form, and weakly with cleaved PAI-1 (see supplement VII). Soluble fibrinogen did not bind to BIAcore CM5 chips with immobilized PAI-1 and did not compete for PAI-1 with the immobilized fibrinogen even when used in a large molar excess. It proves that PAI-1 binds to fibrinogen only when the latter is adsorbed to solid surfaces but does not interact with soluble fibrinogen (see supplement VIII). Degradation of fibrinogen with plasmin progressively exposes cryptic (16-120) sequences, and plasmic fragments obtained after 2 hours showed almost complete expression of both (16-120) epitopes and PAI-1 binding sites. Vitronectin and PAI-1 individually bind to different regions of fibrin but show the same binding affinity for fibrin, with measured KDs of 2.58x10–7 and 1.88x10–7 M, respectively. PAI-1 directly binds to the N-terminal region of the chain, which is in close vicinity to fibrinopeptide A being released by thrombin during conversion of fibrinogen to fibrin. Thus, this region is exposed in fibrin monomer, explaining why PAI-1 preferentially recognizes fibrin over soluble fibrinogen. Binding sites for vitronectin were suggested to be localized in the C-terminal part of the fibrinogen 
chain, namely the A/’ variant.13 The apparent binding affinity estimated by SPR (KD of 0.26 µmol/L) is higher but consistent with that previously determined by solid-phase binding assay (KD of 
0.60 µmol/L) in which radiolabeled vitronectin was bound to pre-formed fibrin matrices.11
PAI-1 release can be induced from a number of cells (platelets, monocytes, macrophages, endothelial cells, and smooth vessel cells) by several cytokines during different processes including thrombosis, wound healing, and inflammation. Thus, it is conceivable to expect accumulation of PAI-1 at sites of injury and inflammation and fibrin seems to be the best candidate to concentrate this inhibitor. This effect may be particularly important in the case of platelets, which release large amounts of PAI-1. Although, there is a continuous production of active PAI-1 in platelets,29 only a small proportion of PAI-1 was reported to be functionally active.30 Thus, PAI-1 released from platelet -granules is mostly in the latent state and it cannot interact with vitronectin, which is known to exclusively bind the active inhibitor.3 It suggests that large amounts of PAI-1, which are found in fibrin clots and that originated from activated platelets are directly bound to the (20-88) region of fibrin. Previous studies described 2 classes of binding sites in fibrin that interact with the activated PAI-1. The first class consisted of a small number of high-affinity sites with a KD < 1 nM, whereas the second class, recognizing both active and latent PAI-1, contained a large number of low-affinity sites with an approximate KD of 3.8 µmol/L.9 Our present studies identified the nature of the second class and verified its binding affinity by measuring the apparent dissociation constant to be >10-fold lower than that published previously.9 Interestingly, the PAI-1/vitronectin complex can potentially interact with the same binding affinity with 2 sites of fibrin, the first primarily recognizing PAI-1 ((52–88)) and the second one interacting with vitronectin (A/'). In the thrombus, all these sites are likely to be saturated because of the high concentration of fibrin-bound platelet PAI-18,9 (see supplement IX).
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Acknowledgments |
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This work was supported by projects KBN 3/PO4A 068 23 and PBZ-KBN-039/P04/2001.
Received May 24, 2005; accepted September 8, 2005.
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