(Received for publication, September 19, 1996, and in revised form, November 29, 1996)
From the Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996
For many years, the concept that the heparin-binding sequence is sequestered within vitronectin and exposed upon denaturation of the protein has guided experimental design and interpretation of related structure-function studies on the protein. To evaluate binding of heparin to both native and denatured/renatured vitronectin, methods for monitoring binding in solution have been developed. A fluorescence method based on changes in an extrinsic probe attached to heparin has been used to evaluate heparin binding to native and denatured/renatured vitronectin. This approach indicates that there are not major differences in intrinsic heparin-binding affinities between native and renatured protein and invalidate the currently accepted model for a cryptic heparin-binding sequence in the protein. Denaturation and renaturation of vitronectin under near physiological solution conditions is accompanied invariably by self-association of the protein into a multimeric form (Zhuang, P., Blackburn, M. N., and Peterson, C. B. (1996) J. Biol. Chem. 271, 14323-14332), resulting in exposure of multiple heparin-binding sites on the surface of the oligomer. On the basis of the binding data from solution studies and interaction of the native monomer and the denatured multimeric form of vitronectin with a heparin column, along with evaluation of the ionic strength dependence of heparin binding to these vitronectin forms in solution, an alternative model is favored to account for the altered heparin binding properties of vitronectin associated with denaturation of the protein. This model proposes that multivalent interactions between heparin and multimeric vitronectin are responsible for differences in heparin affinity chromatography and ionic strength dependence compared with the native protein.
Within the circulation, complicated networks of interactions between proteins and other macromolecules are important for maintaining stasis. An example of the complex interplay that must exist among these biomolecules is provided by the human glycoprotein, vitronectin. Vitronectin interacts with a wide variety of ligands that are involved in control of diverse physiological processes including coagulation, fibrinolysis, tumor metastasis, the humoral immune response, and cellular migration (reviewed in Refs. 1-6). A partial list of target macromolecules that interact with vitronectin includes heparin, collagen, plasminogen, plasminogen activator inhibitor-1, serine protease inhibitor-protease complexes, and a subclass of integrin receptors on the surface of cells.
Indeed, one of the first molecules shown to interact with vitronectin was heparin (7-11), and this interaction has since been widely investigated (12-16). Much of the early work on vitronectin was devoted to evaluating structural requirements for heparin binding, including tests of the effects of denaturation or proteolysis of vitronectin on heparin binding activity. Binding activity was evaluated from vitronectin interactions with a heparin column, estimated in terms of the salt concentrations required to elute bound vitronectin. From these studies came a most striking observation: vitronectin binding to heparin was enhanced upon treatment of the protein with urea, heat, or acid (9). This phenomenon was exploited by Yatohgo et al. (17) to develop an affinity-based purification scheme for vitronectin that invoked chemical denaturation of bulk plasma prior to chromatography on heparin-agarose. As early as 1984, a model emerged in which the heparin-binding region is buried within the native molecule, so that heparin binding is induced when this cryptic binding site is exposed upon denaturation of vitronectin (11, 16). This model has been generally accepted, and more recent work has included attempts to correlate the heparin binding properties of native and denatured/renatured vitronectin with other functions of the protein (12, 18-27). The two forms of the protein, which are sometimes denoted the "non-heparin-binding" (native) and "heparin-binding" (denatured/renatured) species, are characterized in much of the ongoing work in the field.
Many years after the initial work examining effects of denaturation on vitronectin, it was recognized that denaturation is accompanied by self-association of the protein into a multimeric form (28, 29). Multimerization of vitronectin ensues following unfolding of the protein to a structurally altered form that can be detected with a variety of conformationally sensitive monoclonal antibodies (29-31). This partially folded intermediate form has a high propensity to self-associate into multimers that do not readily interconvert with native monomers. The net effect is that multimer formation is essentially irreversible (32). Recently it has been shown that the process of self-association into multimers versus refolding to a monomeric form is highly sensitive to ionic strength (31), with multimer formation almost exclusively favored at physiological salt concentrations. From these analyses of denaturation and self-association, it is now clear that the vitronectin species that are commonly classified as heparin-binding are, in fact, multimeric forms of the protein.
The native and multimeric forms of the protein are thought to represent the two forms that exist in vivo, with most of the circulating pool of vitronectin in a monomeric form, whereas matrix- and other tissue-associated forms appear to be predominantly multimeric. The hypothesis that guided this work is that the two forms of vitronectin exhibit ligand binding properties that differ according to their function and distribution within the body. The goal of this study was to enumerate the energetics of heparin binding to both the native monomer and multimeric vitronectin and to distinguish differences in heparin affinity and differences in numbers of heparin-binding sites on the two forms of vitronectin. In addition to the affinity chromatography approach mentioned above, other methods that have been used previously to evaluate heparin binding to vitronectin have either relied on indirect kinetic methods (14-16, 33) or assays that immobilize one or the other reactant on a plastic surface (16, 28, 29). For a rigorous quantitative analysis of the binding of heparin to both forms of vitronectin, it was essential to develop a solution-based method to monitor the bimolecular interaction. This study provides the first measurements of the affinity and stoichiometry of heparin binding to both forms of the protein using a fluorescently labeled form of heparin to quantify the interactions.
Vitronectin was purified by a modification of the
original procedure of Dahlback and Podack (34), essentially as
described by Bittorf et al. (28). The molecular weight of
the protein is 72,000, and the extinction coefficient is 1.02 ml·mg1·cm
1 (32). Multimeric vitronectin
was prepared by treatment of protein in 8 M urea at room
temperature for 2 h, with subsequent removal of denaturant by
dialysis into 0.1 M sodium phosphate, pH 7.4, containing
0.15 M NaCl and 1 mM EDTA (32).
Unbleached, unfractionated heparin was the kind gift of Dr. Michael N. Blackburn (Department of Macromolecular Sciences, SmithKline
Beecham, King of Prussia, PA). Human thrombin was provided by Dr. Frank
C. Church (Department of Hematology, University of North Carolina,
Chapel Hill, NC). The succinimidyl ester of
7-diethylaminocoumarin-3-carboxylic acid was acquired from Molecular
Probes. Urea was a product of U. S. Biochemicals. Chromozym-TH was
purchased from Boehringer Mannheim. Sephadex G-100 Super Fine was
obtained from Pharmacia Biotech Inc. All other chemicals were of
reagent grade purity.
Unbleached heparin
was fractionated by molecular weight on two columns in series of
Sephadex G-100 Super Fine (2.5 × 100 cm each) linked in series,
essentially as described (35). Fractions from the broad elution peak of
heparin were combined into separate pools, dialyzed against deionized
water, and lyophilized. For labeling with the fluorescent probe, 10 mg
of fractionated heparin were dissolved in 1 ml of 0.1 M
NaHCO3, pH 8.3. 7-Diethylamino-coumarin-3-carboxylic acid,
succinimidyl ester (1 mg dissolved in 100 µl of dimethylformamide) was added dropwise to the heparin solution and incubated overnight in
the dark at room temperature. Unbound coumarin was removed by
exhaustive dialysis into 0.1 M NaHCO2, pH 8.3. Labeling stoichiometries were determined spectrophotometrically using
an extinction coefficient for coumarin at 432 nm of 57,000 M1·cm
1. Heparin
concentrations were determined by an azure A dye binding method (36).
Molecular weights for size-fractionated, coumarin-labeled heparin
samples were determined by sedimentation equilibrium in a Beckman
Optima XL-A analytical ultracentrifuge as described previously (35).
For ultracentrifugation experiments, heparin samples were dissolved in
2 M NaCl, 20 mM Tris-HCl, pH 7.4, and a partial
specific volume of 0.47 ml·g
1 was used for heparin
(37). Absorbance in the ultracentrifugation experiments was monitored
at 430 nm, and interference patterns were also recorded at equilibrium.
Molecular weights calculated from the absorbance and interference
optical patterns were the same within experimental error; this result
indicates that the small molecular weight probe was quantitatively
bound to the macromolecule, heparin.
Binding of heparin to vitronectin was monitored by
alterations in fluorescence of the extrinsic coumarin probe that was
incorporated into heparin. The change in the fluorescent properties of
the coumarin probe upon vitronectin binding was measured by changes either in quantum yield or polarization. Changes in quantum yield were
measured on a Perkin-Elmer LS-50B spectrofluorimeter, using a
thermostatted cell holder connected to an external circulating water
bath. Small volume aliquots of native vitronectin (at least 20 mg·ml1) were added to a microvolume cuvette with a 1-cm
path length, containing 150 µl of the labeled heparin sample
(2.0-30.0 µM) in 0.02 M Tris-HCl, 20 mM NaCl, pH 7.4. Measurements of the fluorescence emission
at 480 nm using an excitation wavelength of 430 nm were recorded in
triplicate. The data were mathematically corrected for dilution upon
addition of vitronectin. To evaluate binding by changes in polarization
of the coumarin probe on heparin, fluorescence anisotropy was measured
with an SLM 8000 fluorimeter with film polarizers. Excitation was at
400 nm, and emission was measured at 480 nm utilizing a cut-off filter
at 440 nm. Small volume aliquots of vitronectin (10 mg·ml
1 stock) were titrated into 2 µM
labeled heparin in 20 mM Tris buffer, pH 8.0, containing 20 mM NaCl. The change in fluorescence anisotropy of the
coumarin-labeled heparin was monitored as a function of the total
native vitronectin concentration.
Binding constants were estimated from heparin titration data using a nonlinear least squares computer fit to the equation:
![]() |
(Eq. 1) |
To determine whether the incorporation of an
extrinsic probe into heparin affects binding to vitronectin,
competitive binding studies using a fixed concentration 3 µM coumarin-labeled heparin in the absence or the
presence of 15, 45, or 75 µM unlabeled heparin were
performed. Aliquots from a concentrated stock of native vitronectin (at
least 20 mg·ml1) were added to a microvolume cuvette
containing a sample of 150-µl volume. Measurements of the
fluorescence emission at 480 nm with an excitation wavelength of 430 nm
were recorded. The fluorescence intensity data were mathematically
corrected for dilution.
An equation analogous to that described by Bock et al. (39, 40) was derived to analyze the competitive vitronectin binding to coumarin-labeled and unlabeled heparin, using the labeled heparin as the probe. The equation can be written as:
![]() |
(Eq. 2) |
Unfolding of vitronectin was induced by urea and monitored by changes in intrinsic protein fluorescence as described (32). Protein fluorescence measurements were made on a Perkin-Elmer LS-50B spectrofluorimeter. Emission spectra were recorded between 300 and 450 nm using an excitation wavelength of 290 nm and a path length of 1 cm. Buffer for the unfolding experiments was 0.1 M sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl, 1.0 mM EDTA and 0.04% (v/v) Tween 20, and the 10 M urea stock solution was made in the same buffer. Unfolding curves were generated from fluorescence measurements on vitronectin incubated overnight in mixtures of the urea stock and buffer to give solutions of intermediate urea concentrations.
Differential Scanning CalorimetryFor differential scanning
calorimetry experiments, vitronectin samples were dialyzed overnight
against 0.1 M sodium phosphate buffer, pH 7.4, containing
0.15 M NaCl, 1 mM EDTA. Vitronectin solutions
(0.9 mg/ml) in the absence and the presence of added heparin
(Sigma, Mr ~6000) at a 100 µM concentration were scanned at
1 °C·min1 using a high precision differential
scanning calorimeter (model DS-93). Samples and reference solutions
were properly degassed and carefully loaded into the calorimeter to
eliminate artifacts that might arise from spurious bubbles in the
solution. Reproducibility of base lines was verified by multiple
overnight scans. The analysis of the differential scanning calorimetry
data was performed with software developed by Biocalorimetry Center
(Johns Hopkins University, Baltimore, MD) (41). Absolute and excess
heat capacities were determined.
Inactivation of thrombin was measured
by the ability of antithrombin to inhibit the amidolytic activity of
the enzyme essentially as described (38). The rate of inactivation of
thrombin by antithrombin plus heparin was monitored continuously by
hydrolysis of the thrombin substrate, Chromozym-TH, to product that
absorbs at 405 nm. Heparin with an average molecular weight of 6000 (product of Sigma) was used in the kinetic analyses at
a final concentration of 1 µg·ml1. The final
concentration of chromogenic substrate was 0.19 mM, and
thrombin concentration was fixed at 2 nM in the assay.
Rates of reaction using a fixed antithrombin concentration of 60 nM were measured at 30 °C in the presence of various
amounts of added vitronectin. The standard assay buffer was
phosphate-buffered saline (40 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl) containing 0.1% (w/v) polyethylene
glycol-8000. Absorbance at 405 nm was recorded on a Perkin-Elmer model
Lambda 3B spectrophotometer interfaced with an IBM computer for
automated data acquisition. Pseudo-first order rates of reaction were
determined by nonlinear least squares analysis using the IGOR software
package (Wavemetrics, Oswego, OR).
Because previously published methods for evaluating interactions between vitronectin and heparin have relied either on measurements with one of the species bound to a solid phase or on complicated kinetic experiments with multiple interactions, a primary goal of this work was to develop a method to monitor the binary interaction in solution. Evaluation of changes in fluorescence of vitronectin upon interaction with heparin was an appealing possibility, because the protein contains nine tryptophans at positions 181, 209, 261, 294, 303, 320, 382, 405, and 450 within the 459-amino acid chain. Note that tryptophans 320 and 382 are located in the primary sequence within the general vicinity of the heparin-binding region, which has been localized to amino acids 341-375 (12). However, binding of heparin to vitronectin could not be reliably measured by changes in fluorescence that were attributable to the interaction.1
Binding of Vitronectin to Heparin Is Detected by Monitoring Changes in the Fluorescence of an Extrinsic Probe on HeparinBecause one of the goals of this research was to compare binding energetics for heparin with both monomeric and multimeric forms of vitronectin, the strategy adopted was to introduce an extrinsic fluorescent probe into heparin that would be sensitive to changes in environment upon binding to either form of the protein. Similar approaches have been previously used successfully to monitor heparin interactions with a variety of proteins, including histidine-proline-rich glycoprotein (38), thrombin (35, 42), antithrombin (35, 43, 44), and fibronectin (45, 46).
Fig. 1 shows binding isotherms in which coumarin-labeled
heparin was titrated with native vitronectin. The two panels of the figure illustrate that binding of vitronectin can be measured either by
changes in fluorescence intensity (Fig. 1A) or polarization (Fig. 1B) of the coumarin probe. The quantum yield of the
coumarin probe is increased upon interaction with vitronectin, and the steady-state anisotropy of the fluor increases as would be expected upon formation of a molecular complex of larger hydrodynamic volume. The dissociation constants derived from the two equilibrium binding isotherms shown in Fig. 1 are in excellent agreement with that calculated from changes in intensity (Fig. 1A) equal to
5.4 × 106 M and that measured by
polarization methods (Fig. 1B) equal to 4.2 × 10
6 M.
Care was taken to establish that the coumarin probe on heparin did not
perturb the binding equilibrium with vitronectin by performing rigorous
competition studies in which vitronectin-binding isotherms with
coumarin-labeled heparin were measured in the presence and the absence
of several fixed concentrations of unlabeled heparin (Fig.
2A). These data were used to calculate
Ki, the binding constant for unlabeled heparin, and
compare it with the binding constant measured for the labeled heparin
sample (Fig. 2B). From this analysis, the ratio between the
binding affinities of labeled and unlabeled heparin samples was 1.08, indicating that the coumarin probe itself does not affect the measured
binding equilibrium. The competition studies were also important to
determine the stoichiometry of interaction between the heparin sample
and native vitronectin, which is given from the y intercept
of the plot of Fig. 2B. The stoichiometry determined from
simultaneous analysis of the three competition curves in Fig. 2 was 1.1 mol of vitronectin bound/mol heparin of molecular weight 9600. It is
important to note that size-fractionated heparins were used for all of
the experiments described in this paper, because the number of protein
binding sites will vary proportionately to the size of the heparin
chain.
Binding of Multimeric Vitronectin to Heparin Is Measured by the Fluorescence Assays
Because previous results had indicated that
structural changes induced upon denaturation of vitronectin result in
apparent higher affinity interactions with heparin, a major objective
of this study was to compare binding of heparin with the native protein and vitronectin that had been treated with denaturant. Recent work from
this and other laboratories (28, 29, 31, 32) has shown that alteration
of vitronectin by chemical or thermal denaturation at physiological
ionic strengths invariably produces a multimeric form of the protein.
The fluorescence methods developed in this work for evaluating binding
of heparin to native protein were also useful for evaluating the
energetics of interaction of heparin with this multimeric form of
vitronectin. Fig. 3 shows results from a binding
experiment in which increasing quantities of vitronectin multimer were
added to fixed amounts of coumarin-labeled heparin of molecular weight
9600 (same sample used in Figs. 1A and 2 with native
vitronectin). Note that the concentration of vitronectin indicated on
the x axis corresponds to the concentration of monomeric
vitronectin subunits contained within the multimer, i.e. the
molar concentration of vitronectin monomers (Mr
72,000) rather than the molar concentration of multimeric vitronectin with an average molecular weight equal to 420,000. The dissociation constant for heparin binding to denatured/renatured vitronectin is
calculated from the isotherm in Fig. 3A to be 5.1 × 106 M, virtually identical to that for the
native protein. Table I summarizes heparin-binding data,
including dissociation constants and stoichiometries of binding, for
both the native and multimeric forms of the protein gathered under a
variety of experimental conditions.
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The distinguishing feature comparing the binding of native and multimeric vitronectin with heparin is the stoichiometry for the protein-heparin interaction. Fig. 3B shows a titration of coumarin-labeled heparin with multimeric vitronectin that was performed using a fixed heparin concentration of 30 µM, which greatly exceeds the measured Kd. Under these conditions, the titration reaches a clear end point that is a direct measurement of the stoichiometry of the interaction. From this analysis, the stoichiometry of binding equals 1.8 mol of vitronectin protomer/heparin chain. The inference from this analysis is that the heparin-binding site within the multimeric form of the protein spans more than one vitronectin monomer. This interpretation of the data indicates that the binding of heparin to multimeric vitronectin involves more intermolecular interactions than the binding to native vitronectin at a single site on the protein.
It is also instructive to consider the results from the stoichiometry
measurement reported in Fig. 3B if protein concentration is
expressed in terms of the molar concentration of multimeric vitronectin. This treatment of the data yields a binding stoichiometry of 0.4-0.5 mol of multimeric vitronectin/mol heparin. A stoichiometry of less than 1 is observed in many cases for proteins binding to
shorter heparin chains. This result indicates that a binding site large
enough to accommodate multimeric vitronectin is not present on all
heparins of this size. In contrast, the binding stoichiometry for this
heparin sample with native vitronectin is nearly 1:1. This result is as
expected, because fewer molecules of larger molecular weight multimer
can be bound to heparin chains that are equal in size. If the data in
Fig. 3A are expressed in terms of moles of multimeric
vitronectin (Mr ~420,000) with the stoichiometry of approximately 0.5, the best fit of the data yields a
dissociation constant of 0.2 × 106 M.
The tighter apparent binding affinity results from the multiplicity of
heparin-binding sites within multimeric vitronectin.
To further evaluate the contribution of ionic and other types of interactions to the binding of heparin to the two forms of vitronectin, a van't Hoff analysis was pursued in which the temperature dependence of the binding equilibrium was evaluated for native and multimeric protein (Table II). A linear van't Hoff relationship was observed for both forms of vitronectin over the experimentally tested temperature range from 25 to 40 °C. For both the native and multimeric proteins, the binding constant differs 2-fold over the given temperature range. Small differences in the corresponding Kd values at a given temperature that are observed comparing the native and multimeric protein are within experimental error. Because it has been established that different types of intermolecular forces contribute differently to the enthalpic and entropic terms for a given binding equilibrium and because these different types of interactions exhibit different temperature dependences (47), this analysis was pursued to evaluate the type of interactions that account for the free energy of binding heparin to vitronectin. Enthalpic and entropic contributions to the heparin/vitronectin equilibrium, derived from the van't Hoff analysis, are summarized in Table II. These values are compatible with a predominance of ionic forces in the binding interaction, as would be expected from the highly basic nature of the heparin-binding sequence in vitronectin and the polyelectrolytic features of heparin. This analysis demonstrates that the energetics of interaction between heparin and either form of vitronectin are indistinguishable and that the binding interaction is dominated by enthalpic contributions with a smaller contribution from entropic forces. The temperature dependence of the vitronectin-heparin interaction is similar to that for the Kd of the antithrombin-heparin interaction, which varies 1.8-fold in the range from 15 to 37 °C. However, the situation may be contrasted with that for thrombin interactions with heparin, in which there is less of a temperature dependence for the binding equilibrium.2
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The dependence of binding on heparin chain length was
examined to determine what effects binding at multiple sites along the chain has on the equilibrium. The data obtained from measurements of
native vitronectin binding to three different molecular weight heparin
species are shown in Fig. 4A and summarized
in Table I. It is apparent from this analysis that the number of sites
at which native vitronectin binds on the heparin chain increases with
molecular weight, as expected. However, there is not a significant difference in the binding affinities measured with the three different sizes of heparins. This result indicates that vitronectin monomers bind
independently at separate sites along the heparin chain.
The question of heparin size effects is especially pertinent for the multimeric form of the protein, in which the heparin-binding site appears to be "shared" between a minimum of two vitronectin protomers within the multimeric protein. One would predict that an interaction that involves multiple binding sites on both the target ligand and the protein would exhibit tighter binding as the number of sites on the ligand increases. Binding isotherms that describe the interaction of multimeric vitronectin with heparins of varying size are shown in Fig. 4B and listed in Table I. The three heparin species are the same that were used in the measurements with native vitronectin in Fig. 4A. In a similar fashion to the native protein, the number of vitronectin protomers bound per heparin chain increases with the molecular size of the heparin. Interestingly, the molar ratio of protomers bound in multimeric vitronectin to the ratio of native vitronectin monomers bound for a given heparin sample is relatively constant (approximately 1.6-2.0) for all of the heparins tested. The consistency of the stoichiometries comparing multimeric and native vitronectin among the heparin series is striking, and it substantiates the argument that the heparin-binding site within multimeric vitronectin encompasses more than one vitronectin polypeptide chain. A further contrast to the independent binding behavior observed with the native protein is the observation that multimeric vitronectin exhibits progressively tighter binding to heparin as the chain length of the polysaccharide increases.
Interaction of Native or Multimeric Vitronectin with Heparin Is Disrupted by Increases in Ionic StrengthThe observation that binding constants for heparin with native and denatured/renatured vitronectin are essentially the same was surprising in light of the previous work that had indicated tighter binding of the denatured form of vitronectin to a heparin column. To test whether the vitronectin preparations used in this study behaved similarly, interaction of the native and multimeric forms of vitronectin with heparin-Sepharose was evaluated by using a salt gradient elution. The protein is quantitatively bound to the heparin matrix under low ionic strength conditions. Native protein was eluted in a single symmetrical peak with a midpoint near 0.2 M NaCl. However, higher salt concentrations were required to elute multimeric vitronectin from the heparin column, with a midpoint in the elution near 0.4 M NaCl. Thus, similarly to previous findings, the denatured form of vitronectin used in this study bound to a heparin column so that a higher ionic strength was necessary for elution than that required for the native protein.
Ionic strengh effects on the binding affinity were measured directly
using the coumarin-labeled heparin. Fig. 5A
shows titrations of coumarin-heparin with native vitronectin, and Fig.
5B shows titrations with multimeric vitronectin at various
salt concentrations. These analyses are summarized in Table I. From
these binding isotherms, it is obvious that the native and multimeric
forms of vitronectin exhibit different sensitivities to ionic strength variations. Differences are pronounced at near physiological NaCl concentrations, with the native form of vitronectin exhibiting significantly weaker binding (Kd = ~26
µM) than the multimeric form of the protein
(Kd = ~5 µM) at 100 mM
NaCl. It should be noted that the fluorescence of the coumarin probe on
free heparin (i.e. that which is not bound to vitronectin)
is not altered over the experimental ionic strength range. The direct
titrations at various ionic strengths shown in Fig. 5 demonstrate clear
differences in binding affinities in the range from 100 to 400 mM NaCl. Higher concentrations of salt are required to
disrupt binding of multimeric vitronectin to heparin because of the
more extensive binding site that appears to be shared between at least
two adjacent vitronectin protomers. More ionic interactions
contribute to the binding of heparin to the multimeric form compared
with native vitronectin.
The Anticoagulant Activity of Heparin Is Neutralized by Either Native or Multimeric Vitronectin
It was important to determine
the effects on heparin anticoagulant activity that resulted from the
observed differences in heparin binding properties of the two forms of
vitronectin. There has not been a previous comparison of native and
multimeric vitronectin in neutralizing heparin using the kinetic assay
system. To compare the consequences of heparin binding by native and
multimeric vitronectin on the protease inhibition, reaction kinetics
were measured using fixed concentrations of enzyme, inhibitor, and
heparin and by varying the concentration of vitronectin. The behavior
of native and multimeric vitronectin in this type of assay is compared
in Fig. 6. Native vitronectin binds and prevents
antithrombin and/or thrombin from binding to heparin in a
concentration-dependent fashion. Similar inhibition
profiles have been reported previously (15, 16, 48) with a
Ki of 0.6 µM for the
heparin-vitronectin interaction estimated from Dixon analysis of
kinetic data (15). The results from the present study agree with this
estimate, because the concentration of native vitronectin that gives
50% inhibition is almost exactly equal to 0.6 µM.
However, another study by Preissner and colleagues using Factor Xa
rather than thrombin as the target protease concluded that the
Kd for heparin interaction with native vitronectin
was lower, equal to 1 × 108 M (16).
This calculation was based on attempts to determine concentrations of
free and bound heparin from these assay mixtures with multiple
components, three of which bind to heparin. The validity of this
approach is questionable, and the calculated Kd is 2 orders of magnitude lower than the near micromolar values generated in
the current work. Furthermore, the stated Kd is
incongruent with the kinetic data from which it is derived, in which
effects in the assay are observed using vitronectin concentrations in
the micromolar range (15, 16).
For the first time, inhibition of heparin activity by multimeric vitronectin has been measured and compared with the inhibition characteristic of native vitronectin (Fig. 6). The midpoint for inhibition for the multimer is shifted to lower vitronectin concentrations, near 0.2 µM, compared with the 0.6 µM value observed for native vitronectin. The rather modest difference in concentrations of the two forms of vitronectin for inhibition of heparin activity reflects the difference in binding affinity of native and multimeric vitronectin at physiological ionic strength. Heparin activity is appreciably inhibited by both forms of vitronectin, an observation that indicates that heparin binding to either form of the protein may be consequential for the biological activity of heparin.
The ionic strength dependence of the binding interactions observed for native and multimeric vitronectin account for the differences in neutralization of heparin activity in the antithrombin-thrombin assay comparing the two forms of vitronectin. The net effect of the stronger binary interaction between heparin and multimeric versus monomeric vitronectin at physiological ionic strengths is that lower concentrations of the multimer are required to neutralize the activity of heparin. Heparin-like molecules exist on the lining of the vasculature and are thought to be responsible for maintaining patency of the blood vessels under normal circumstances. Circulating forms of vitronectin are present in micromolar concentrations and are predominantly the monomeric form of the protein, and native vitronectin has the potential to modulate the anticoagulant activity of heparin or heparin-like molecules in much the same way that other proteins that are abundant in the circulation can interfere with heparin activity (38). However, the relative levels of circulating antithrombin (near micromolar), combined with an affinity for heparin that is 2 orders of magnitude tighter than observed with vitronectin, effectively compete for binding of heparin so that its anticoagulant potential in vivo is not substantially impaired by vitronectin under normal conditions. In contrast, multimeric forms of vitronectin that are localized to the extracellular matrix and that are released from platelets will be more effective at binding heparin and modulating its anticoagulant potential in the vicinity of thrombi, which are localized to the vessel wall. Thus, the self-association of vitronectin is required to increase the number of heparin-binding sites and effectively compete with antithrombin in circulation for heparin-like molecules at sites of vascular injury or thrombosis.
Heparin Binding to Vitronectin Does Not Stabilize the Protein in Denaturation ExperimentsThe noteworthy observation that the
heparin-binding sites on native and denatured/renatured vitronectin
exhibit essentially identical binding constants for heparin, in
contradiction to conclusions from most of the earlier work that would
predict large differences in binding affinity, prompted a thorough
effort to compare and contrast interactions with native and multimeric
vitronectin. This laboratory has recently evaluated the denaturation
and renaturation/self-association process for vitronectin in detail
using spectroscopic and hydrodynamic techniques (31, 32). Reproducible
unfolding curves that report changes in protein fluorescence to
characterize denaturation of vitronectin with varying concentrations of
chemical denaturants have been informative. Shown in Fig.
7A are unfolding curves observed when
intrinsic fluorescence of the protein is used to monitor denaturation
of vitronectin in the presence and the absence of added heparin. Note
that the curves do not differ using the two different experimental
conditions tested, although LeChatelier's principle would argue for a
difference between the two curves if heparin binds preferentially to
the unfolded or denatured form of vitronectin. In that case, the
unfolding curve measured in the presence of high heparin concentrations
should be shifted to lower concentrations of denaturant, reflecting the
preference for heparin binding to the denatured protein and shifting
the unfolding equilibrium toward the unfolded form at all
concentrations of urea. The absence of any effect of heparin on the
unfolding curve supports the conclusion from the binding data and
strongly argues that heparin binds with similar affinity to native and denatured vitronectin.
Similar results were obtained in thermal denaturation experiments on native vitronectin. Fig. 7B shows denaturation endotherms for vitronectin measured in the absence and in the presence of saturating concentrations of heparin. The endotherms are indistinguishable, indicating that heparin neither stabilizes nor destabilizes vitronectin, because preferential binding of heparin to either form should perturb the observed endotherm. Effects of heparin binding on stability would be reflected in changes in the tm, the midpoint in thermal denaturation of the protein, and perhaps in the enthalpy of denaturation, measured as the area under the endotherm.
An Alternative Model to Account for Differences in Heparin Binding Properties of Native and Multimeric VitronectinThe model
entailing a heparin-binding sequence, which is encrypted in the native
fold of vitronectin, as represented schematically in Fig.
8A, has garnered wide support because the
initial demonstration that denatured vitronectin binds more efficiently
to a heparin matrix than the starting native material (11). Although
the early work on the protein clearly pointed to differences in heparin binding properties of the two forms of vitronectin, these differences have not been previously evaluated in a quantitative fashion. Therefore, this work represents the first analysis of the heparin binding properties of the two forms of vitronectin using solution-based methods. Previously, distinctions in the concentration of salt required
to elute the native and multimeric forms of vitronectin from a heparin
column have been noted (9, 11, 13), but these data cannot be
interpreted in a straightforward manner in terms of binding affinity.
Furthermore, immobilization of one or the other interacting species on
a solid phase (e.g. matrix or plastic surface) in many cases
perturbs the binding equilibrium so that it is not reflective of the
situation in solution. Although this has not been considered for
heparin-vitronectin interactions, the problem has surfaced in
evaluating vitronectin interactions with certain other target ligands
(49).
For these reasons, it was imperative that a solution-based method for measuring heparin-vitronectin interactions be developed. Although the solution biochemistry clearly establishes similar binding behavior for native and multimeric vitronectin, there is obviously a difference in effective heparin binding "affinity" for immobilized heparin and ionic strength requirements for the interaction. How can these two results be reconciled? To visualize the differences between the measured equilibria comparing monomeric and multimeric vitronectin, it is helpful to consult the model presented in Fig. 1B. Following denaturation and renaturation, vitronectin associates with itself to form a noncovalently linked multimeric protein. Exposed on the surface of the protein are multiple binding sites for the target ligand, heparin. The ionic strength and molecular weight dependence of the binding interaction appears to be the result of a more extensive binding site within the multimer that incorporates features from individual heparin binding sites on adjacent vitronectin protomers comprising the multimer. Juxtaposition of these multiple binding sites increases their local concentration so that rebinding of the ligand following dissociation is favored. Because it is clear that there is only a single heparin-binding site per vitronectin in its native, monomeric form, the dissociation and reassociation of heparin from native protein is not influenced in this way. This situation described for heparin interactions with multimeric vitronectin is analogous in many respects to the paradigm of avidity effects in binding of antigens to bivalent (or multivalent) antibodies, in which effective binding of antigen to the antibody molecule is much tighter than the measured binding affinity of the separate antigen-combining sites.
Most significantly, the model that is supported by this work (Fig. 1B) does not involve burial of the heparin-binding site within the native structure of the protein. Masking of the heparin site within vitronectin has been widely accepted, despite the fact that the kinetic measurements that were used to initially demonstrate neutralization of heparin by vitronectin were performed using native protein and were used to calculate Ki values representing appreciable affinity for heparin (14, 16, 33). Specific interactions with a relatively acidic region of vitronectin near the N terminus have been proposed previously to mask the heparin binding site in the native protein, with little evidence to support this notion aside from charge considerations (16). As discussed above, the effectiveness of native vitronectin in modulating the anticoagulant activity of heparin certainly discourages application of a model in which the binding site is masked. Furthermore, the heparin-binding region of vitronectin has recently been shown in this laboratory to be susceptible to chemical modification with an arginine-specific fluorescent probe (52), a result that also indicates that this region of the protein is surface-exposed and accessible. It is clear from this work that the heparin binding sites within native and multimeric vitronectin are similarly exposed so that intramolecular interactions involving the acidic region of vitronectin cannot be envisioned to bury the heparin-binding sequence. The two types of vitronectin should no longer be referred to as heparin-binding or non-heparin binding; they are more appropriately identified as native (or monomeric) and multivalent (or multimeric) forms of vitronectin.
The physiological mechanisms that convert vitronectin from a native to a structurally altered multimeric form with an increased local concentration of binding sites for heparin remain elusive. Heparin binding induces conformational changes in native vitronectin that can be detected with monoclonal antibodies (24, 50); although such conformational changes are generally perceived as prerequisites for the self-association of vitronectin leading to multimeric forms, the binding of heparin in itself is not sufficient to induce multimerization of the protein (28, 31). Other studies using conformationally sensitive monoclonal antibodies have indicated that most noncirculating forms of vitronectin exist in the conformationally altered form. From this information, it has been inferred that the form of vitronectin found in the extracellular matrix is multimeric in composition. Self-association to a multimeric form in the extracellular matrix may be advantageous to enhance binding of other macromolecules known to associate with vitronectin, including plasminogen activator inhibitor-1, collagen, and heparin-like molecules. For other macromolecular interactions altered upon denaturation of vitronectin, it will be interesting to consider whether perturbations are due to alterations in intrinsic affinity or clustering of binding sites on the multimer and thus to clarify the importance of the observed conformational lability of vitronectin in regulation.
We thank Steve Olson and Paul Bock for helpful discussions and critical reading of the manuscript. We are indebted to Michael Blackburn from the Department of Macromolecular Sciences at SmithKline Beecham for the use of the Beckman Optima XL-A ultracentrifuge and SLM fluorimeter and for providing the fitting routine for kinetics to use with the IGOR software package. The microcalorimetry work was performed at the Biocalorimetry Center, a Biomedical Research Technology Resource Center sponsored by the National Institutes of Health (RR04328) and Johns Hopkins University (Department of Biology, Baltimore, MD). We are grateful to Ernesto Freire and Craig Johnson for granting instrument time and providing expertise with the instrumentation and analyses at the Biocalorimetry Center.