Biofunctional Coatings via Targeted Covalent Cross-Linking of

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Biomacromolecules 2009, 10, 2408–2417

Biofunctional Coatings via Targeted Covalent Cross-Linking of Associating Triblock Proteins Stephen E. Fischer,† Lixin Mi,‡,# Hai-Quan Mao,†,§ and James L. Harden*,| Department of Materials Science and Engineering, Department of Chemical and Biomolecular Engineering, and Whitaker Biomedical Engineering Institute, Johns Hopkins University, Maryland Hall, 3400 North Charles Street, Baltimore, Maryland 21218, and Department of Physics and Institute for Systems Biology, University of Ottawa, MacDonald Hall, 150 Louis Pasteur, Ottawa, ON K1N-6N5, Canada Received February 16, 2009; Revised Manuscript Received June 8, 2009

A method for creating tailorable bioactive surface coatings by targeted cross-linking of network-forming CRC protein polymers is presented. The proteins are triblock constructs composed of two self-associating leucine zipper end domains (C) separated by a soluble, disordered central block (R) containing a cell or molecular binding sequence. The end domains preferentially form trimeric bundles, leading to the formation of a regular, reversible hydrogel network in a wide range of solution conditions. These hydrogel-forming proteins are useful for creating bioactive surface coatings because they self-assemble into networks, physically adsorb to a variety of substrate materials, and can be tailored to display numerous extracellular matrix (ECM)-derived peptides that interact with cells and biological macromolecules. Moreover, due to the close proximity of complementary Glu and Lys residues in the trimeric C bundles, these protein coatings can be stabilized in a targeted manner by covalent cross-linking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Here, we demonstrate that such EDCcross-linked protein coatings are stable in cell culture media and maintain a significant level of biofunctionality when various ECM-derived peptides are embedded in the central soluble block of the proteins. First, we show that EDC cross-linking enables bioinert CRC protein coatings (those without embedded cell binding domains) to resist the adhesion of human foreskin fibroblasts in normal serum medium, but does not impair the ability of cross-linked coatings of CRC-RGDS (proteins with an embedded RGDS integrin binding domain) to promote cellular attachment, focal adhesion formation, and proliferation of these cells. Next, we show that the ability of cross-linked coatings of several new CRC-based proteins containing embedded heparin-binding sequences to bind biotinylated heparin is not significantly impacted over a range of EDC concentrations. The ability to target specific functional groups for covalent cross-linking is made possible by the specificity of protein-protein interactions and represents an important advantage of protein-based materials.

1. Introduction Cell-substrate interactions play an important role in promoting cell survival, controlling cell proliferation and migration, and directing cell fate decisions.1,2 The substrate surface, in particular, dictates cellular behavior via the presentation of various biophysical and biochemical signals. How a cell ultimately reacts to a substrate is determined by the cell’s orchestrated response to the molecular interactions occurring with these surface cues. Therefore, techniques capable of finely controlling substrate surface properties are of great value. One strategy for inducing specific cell-substrate interactions is to functionalize substrate surfaces with bioactive peptides capable of inducing receptor-mediated cellular responses.3-5 Several methods have been developed to immobilize bioactive peptides to surfaces, including those that aim to covalently link the bioactive peptide directly to the surface6-9 and those that seek to link the peptide to surface-active molecules.10-12 In the * To whom correspondence should be addressed. E-mail: jharden@ uottawa.ca. † Department of Materials Science and Engineering, Johns Hopkins University. ‡ Department of Chemical and Biomolecular Engineering, Johns Hopkins University. § Whitaker Biomedical Engineering Institute. | University of Ottawa. # Current address: Dept. of Oncology, Lombardi Comprehensive Cancer Center, Georgetown Univ., Washington, DC 20057.

latter approach, self-assembled monolayer (SAM) technologies have proven especially useful for creating highly organized interfaces decorated with bioactive peptides.13-15 A limitation of SAM technologies, and virtually every other existing surface functionalization strategy, is the need for tailoring to particular peptide and surface properties. An alternative approach that may enable controlled peptide modification of a wider range of substrate materials is to incorporate the peptides into multifunctional artificial proteins. Using genetic engineering, one can create novel proteins possessing independent molecular recognition, bioactive, and mechanical domains. The molecular recognition domains are particularly important because they enable individual proteins to assemble into complex supramolecular structures, such as hydrogel networks and fibrils,16-22 through precise intermolecular interactions. A key design feature of such proteins is their modularity, enabling them to be embedded with various bioactive domains without disrupting their self-assembly behavior. Naturally, the ability of these proteins to form biofunctional surface coatings is dependent on their possessing a certain degree of surface activity. However, their capacity to selfassemble into networks expands their surface functionalization capabilities over simple amphiphilic molecules since surface affinity limitations are mitigated through multiple surface contact points with the network.

10.1021/bm900202z CCC: $40.75  2009 American Chemical Society Published on Web 08/05/2009

Biofunctional Coatings via Triblock Proteins

Biomacromolecules, Vol. 10, No. 9, 2009

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Figure 1. Amino acid sequences for the CRC and CRC-X triblock proteins, along with the CR diblock protein. The amino acid sequences for the major domains are given. The major domains are joined by short sequences introduced during construction of the expression vectors.

We have developed a series of network-forming CRC-X protein polymers that form biofunctional surface coatings on a variety of substrate materials. The proteins are triblock constructs composed of two self-associating leucine zipper end domains (C) separated by a soluble, disordered central block (R) containing a cell or molecular binding sequence (denoted by X). Figure 1 shows the sequences of the CRC-X and CRC triblock proteins, along with that of a diblock CR version used for characterization studies of intermolecular association and cross-linking. The C domain is a slightly modified version of a de novo sequence designed by Lombardi et al. as a member of a putative metal-binding heterotrimer.23 Although the metalbinding functionality of the original design was not realized, it was found that the original C sequence formed helical domains with a propensity to associate into trimeric bundles. The modifications of the current C sequence are limited to two substitutions: a single GlufLys in one e position and a single MetfLeu near the NH2-terminus. Except for single occurrences of His and Trp residues in a positions, all other a and d heptad positions in the modified C helix are occupied by leucine (L) residues, which favors the formation of trimer bundles.24 The His and Trp substitutions in a positions favor parallel association of the C helices in a bundle to sequester these residues in common regions of the hydrophobic pocket. The C sequences contain primarily glutamic acid residues (E) in the e and basic lysine residues (K) in the g positions, giving them a polyampholytic character. Favorable electrostatic interactions in moderate pH conditions between the acidic residues in the e and the basic residues of the g positions also favors parallel trimeric bundles of C helices, as sketched in the helix wheel diagrams shown in Figure 2A. The unstructured R block, which nominally consists of 10 repeats of the sequence (AlaGly)3ProGluGly, is based on the design by Tirrell and colleagues16 and serves as a water-soluble bridge between the leucine zipper cross-links. Trimeric association of C domains for the CR construct was also demonstrated in analytical ultracentrifugation studies,25

Figure 2. (A) Helical wheel representation of a parallel homotrimer of C helices. EDC primarily targets the Glu and Lys residues in the e and g positions, respectively, of neighboring helices. The targeted Glu and Lys residues of putative cross-links are indicated by red connection lines. (B) Sketch of a surface-adsorbed CRC-based protein coating showing the homotrimeric C helix bundle cross-links and the embedded biofunctional domains (in blue).

described below. We hypothesize that the end domains also preferentially form reversible trimeric bundles in the CRC triblock constructs, leading to the formation of a reversible hydrogel network with predominantly trifunctional cross-links in a wide range of solution conditions. These proteins may prove useful for creating bioactive surface coatings because they selfassemble into networks, physically adsorb to many surfaces, and can be tailored to display numerous extracellular matrixderived peptides that interact with cells and biological macromolecules (see Figure 2B).26,27 We recently illustrated this approach using CRC-RGDS, a triblock protein possessing an embedded RGDS cell adhesion sequence.27 It was demonstrated that surfaces could be coated with CRC-RGDS and that such coatings could facilitate the attachment and spreading of human foreskin fibroblasts (HFFs), human umbilical vein endothelial cells (HUVECs), and rat neural stem cells (rNSCs). Furthermore, cell response could be precisely modulated with this protein coating (through control of RGDS surface density) by preparing mixed surfaces of CRC-RGDS and its bioinert homologue, CRC. A major limitation of such protein-based materials, in general, is that their self-assembly is driven by molecular interactions that are physical in nature and, thus, inherently reversible. While this can lead to desirable features like facile and controlled network formation, it can also cause the material to have reduced stability under physiological conditions. In the case of our CRCbased proteins, self-assembly into a coating occurs via the combined effect of the proteins’ adsorption to surfaces and intermolecular association of their terminal leucine zipper blocks into trimeric bundles.27 Under physiological salt conditions, these leucine zipper cross-links are somewhat weakened because

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they are partially dependent on stabilizing electrostatic interactions. This screening of electrostatic interactions by buffer electrolytes results in a reduced coating layer. Moreover, when incubated in the presence of molecules with a higher surface activity (e.g., the proteins in cell culture medium containing 10% serum), the layer may be further reduced by competitive displacement of the triblock proteins from surfaces.27 Here, we demonstrate that our CRC-based surface coatings can be effectively stabilized against the effects of electrolytes and serum proteins through targeted chemical cross-linking of the associated leucine zipper domains with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). While the distribution of chemical cross-links in a polymer hydrogel would normally be somewhat random and difficult to control, our protein-based system enables the cross-links to be templated through its network architecture. In particular, the EDC crosslinking scheme targets Glu and Lys residues that are brought in close proximity upon parallel association of the terminal leucine zipper domains, as sketched in Figure 2A. By predominantly cross-linking the end blocks of our proteins, not only can the network structure be stabilized, a significant level of functionality can also be maintained for various bioactive peptides embedded in the unstructured middle block. This point is demonstrated in two ways. First, cell studies are carried out to show that EDC cross-linking enables bioinert CRC to resist cell adhesion in normal serum medium (due to its enhanced surface stability), but does not impair the ability of CRC-RGDS to promote cellular attachment, focal adhesion formation, and proliferation. Next, ELISA experiments are carried out with a series of new CRC-based proteins containing embedded heparinbinding sequences to show that the ability of several of these proteins to bind biotinylated heparin (b-heparin) is not significantly impacted over a range of EDC concentrations.

2. Materials and Methods 2.1. Reagents. Restriction enzymes were purchased from New England Biolabs (Beverly, MA). The XL-1 Blue E. coli cloning strain and QuikChange II Site-Directed Mutagenesis Kit were purchased from Stratagene Inc. (La Jolla, CA). The pQE vectors, SG13009 E. coli expression strain, DNA purification kits, and Ni-NTA resin were purchased from Qiagen (Valencia, CA). Na125I was obtained from MP Biomedicals (Irvine, CA). EDC, the mouse antihuman vinculin IgG antibody, and biotinylated heparin (b-heparin) were purchased from Sigma-Aldrich (St. Louis, MO). All mammalian cell culture reagents, reagents for fluorescent labeling of cells, and the CyQuant cell proliferation assay kit were purchased from Invitrogen Inc. (Carlsbad, CA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). 2.2. Cloning and Protein Expression/Purification. Cloning of the CRC-RGDS gene is described elsewhere.27 Here we prepared a set of new triblock constructs by inserting the DNA sequences for four different heparin-binding peptides into the central R block of the CRC gene. These heparin-binding sequences are WQPPRARI from fibronectin,28 LQVQLSIR29 and KNRLTIELEVRT30 from laminin, and KAFAKLAARLYRKA from antithrombin III.31 As before, DNA sequences for the peptides were embedded into the R block via site-directed insertion mutagenesis using Stratagene’s QuikChange II Kit.27 The standard QuikChange protocol was utilized. For each of the two larger peptides, the DNA sequences were split up and inserted consecutively using two different pairs of mutagenic primers. DNA sequences were verified by the Johns Hopkins Genetic Resources Core Facility. These four new proteins are abbreviated CRC-WQPP, CRC-LQVQ, CRCKNRL, and CRC-KAFAK. The sequences of these proteins are given in Figure 1. Protein expression and purification were carried out as previously reported.27 Protein molecular weights were confirmed by matrix-assisted

Fischer et al. laser desorption ionization-time-of-flight mass spectroscopy (MALDITOF) on a Voyager DE-STR (Applied Biosystems, Foster City, CA) at the Johns Hopkins University AB Mass Spectrometry/Proteomics Facility. 2.3. Cross-Linking Diblock Protein Solutions with EDC or Glutaraldehyde. CR, a diblock version of CRC (see Figure 1) prepared previously,25 was reacted with various concentrations of EDC or glutaraldehyde to investigate the feasibility of introducing specific covalent cross-links into the protein assembly. A solution of CR was prepared in pure water (final concentration 0.25 mg/mL) and added to several tubes. For the EDC reactions, the cross-linker was dissolved in anhydrous DMSO at 100 mg/mL and serially diluted in the same solvent. Cross-linking was carried out by mixing a small volume of the EDC solutions with CR and incubating overnight at room temperature. A constant DMSO concentration (2%) was maintained for all samples while varying cross-linker concentration in the range of 50 µM to 12.8 mM. DMSO was used as the solvent for EDC to prevent hydrolysis prior to addition to CR. For the glutaraldehyde reactions, various concentrations of the cross-linker were prepared by serially diluting from a 25% (v/v) stock solution with water. After mixing a small volume of glutaraldehyde with CR, the reactions were incubated at room temperature for a period of 2 h. The range of concentrations tested for glutaraldehyde was 44 µM to 11.3 mM. All samples were analyzed with SDS-PAGE/Coomassie Blue staining. 2.4. Sedimentation Equilibrium Studies of Uncross-Linked CR Diblock Protein. Sedimentation equilibrium studies were performed in a Beckman Optima XL-1 analytical ultracentrifuge with Rayleigh interference optics using an 8-hole rotor, 12 mm carbon-filled epoxy double-sector centerpieces, and sapphire windows. Lyophilyzed HPLC-purified CR diblock proteins were dissolved in 10 mM MOPS and 100 mM NaCl buffer with pH 7.0 at a concentration of 2 mg/mL. This stock solution was centrifuged at 14000g to remove any large aggregate impurities prior to dialyzing against the same buffer overnight to equilibrate the solutions. Measurements of the interference fringe J (proportional to the local protein concentration) as a function of radius r were performed on each sample at 28000 and 32000 rpm for 24 h at 20 °C. Two loading concentrations of 0.32 and 0.96 mg/mL (corresponding to absorbances of 0.17 and 0.48 at 280 nm) were measured and analyzed. The point-averaged molecular weight Mw was extracted from the local slope of the logarithm of fringe ln J versus r2 using standard methods.32 2.5. Cross-Linking Surface-Adsorbed Triblock Protein with EDC. Solutions of CRC or CRC-X triblock proteins were prepared in 10 mM phosphate buffer pH 7.5, filtered through 0.22 µm syringe filters, and then coated at a concentration of 10 µM onto various substrates (glass coverslips and tissue cultured treated polystyrene (TCPS)) by passive adsorption overnight at 4 °C. After rinsing the substrates excessively with pure water to remove unbound protein, cross-linking with EDC was performed. In preparation for cross-linking, an EDC stock solution (25 mg/mL) was prepared in anhydrous DMSO, sterilized by filtration, and diluted in sterile DMSO to various concentrations. Cross-linking was initiated by mixing predetermined volumes of the EDC solutions with water, vortexing, and immediately adding to the protein-functionalized substrates. A constant DMSO concentration (2%) was maintained for all samples while varying cross-linker concentration in the range of 25-1600 µM. Substrates were incubated with EDC overnight at room temperature and then rinsed excessively prior to use. 2.6. 125I-Labeling of CRC-RGDS and Surface Stability Studies. CRC-RGDS was labeled with 125I via its tyrosine residues to characterize the stability of protein surface coatings cross-linked with EDC. 125I-labeling of CRC-RGDS was carried out as previously reported27 using IODO-GEN Pre-Coated Iodination Tubes (Pierce, Rockford, IL). Labeled protein was separated from free 125I by passing through a D-Salt Polyacrylamide Desalting Column (Pierce) through 1 mL additions of tris iodination buffer (25 mM Tris-Cl, 0.4 M NaCl, pH 7.5) containing 5 mM EDTA and 0.05% sodium azide. A specific activity of approximately 1 × 108 cpm/mL was obtained.

Biofunctional Coatings via Triblock Proteins The stability of EDC-cross-linked coatings of 125I-labeled CRCRGDS was evaluated on two different substrates (glass coverslips and TCPS disks). The TCPS disks were prepared in-house by plasma treating polystyrene films for a period of 30 s in air. The polystyrene films were obtained from Goodfellow Corp. (Oakdale, PA) and were punched into 0.77 cm2 disks using a small diameter hole punch. Standard glass coverslips were cut to the desired size using a diamondtipped scribe. A 5 µM solution of CRC-RGDS in low salt buffer (10 mM phosphate, 10 mM NaCl, pH 7.5) containing approximately 0.5 × 106 cpm/mL of radioactivity was adsorbed onto the substrates and then cross-linked with different concentrations of EDC (in the range of 25-1600 µM) as described above. Three samples were prepared for each condition. After allowing the EDC reactions to proceed overnight, the substrates were rinsed several times with PBS and then challenged with HFF culture medium containing 10% fetal bovine serum (FBS) at 37 °C. At each time point, the HFF culture medium was removed and the substrates were rinsed twice with PBS prior to quantifying radioactivity. For quantification, the substrates were transferred to a glass test tube and counted for a period of 3 min in a Packard Cobra Quantum gamma counter (Perkin-Elmer, Inc.). The mass of protein detected on the substrates was obtained by comparing the radioactivity of the samples with that of the initial CRC-RGDS solutions. 2.7. Cell Response to EDC-Cross-Linked Protein Coatings. Human foreskin fibroblasts (HFFs) were obtained from ATCC and maintained in a complete growth medium consisting of RPMI 1640 supplemented with 300 mg/L L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were cultured in a humidified incubator at 37 °C and 5% CO2 and when nearly confluent, harvested with a solution of 0.05% trypsin-EDTA. Relative proliferation of HFFs on EDC-cross-linked protein coatings was quantified by measuring total cellular DNA using the CyQuant Cell Proliferation Assay Kit. Triplicate wells of 96-well TCPS plates were coated with either CRC or CRC-RGDS and cross-linked with various concentrations of EDC (in the range of 25-1600 µM) as described above. A cell suspension was then prepared at a density of 7000 cells/mL in complete growth medium without phenol red and 150 µL was seeded into each well. The cells were cultured for a period of 4 days without changing the medium and then frozen directly in the wells at -80 °C. After performing three freeze/thaw cycles, 50 µL of CyQuant GR reagent diluted 400-fold in cell lysis buffer was added to each well, and the plate was put on a shaker for a period of 30 min. Sample fluorescence was then measured in a Gemini XPS microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA) set to 485 nm excitation and 538 nm emission. Fluorescent staining of cytoskeletal components was performed for HFFs cultured on 12 mm diameter glass coverslips coated with CRCRGDS and cross-linked at various concentrations of EDC (in the range of 25-1600 µM). Approximately 20000 cells were seeded onto each coverslip in 500 µL of complete growth medium and then cultured for a period of 6 h. After fixation in 3% paraformaldehyde, cellular vinculin was immunostained using a mouse antihuman primary antibody, and Alexa Fluor 546-conjugated goat antimouse secondary antibody, while actin filaments were stained with Alexa Fluor 488-conjugated phalloidin. A more detailed account of the staining procedures has been described previously.27 2.8. Biotinylated-Heparin ELISAs. Biotinylated-heparin (b-heparin) ELISAs were carried out to assess the heparin-binding capacity of various triblock proteins (CRC, CRC-WQPP, CRC-LQVQ, CRCKNRL, and CRC-KAFAK) and to determine the impact of EDC crosslinking on b-heparin-binding. Triplicate wells of 96-well TCPS plates were coated with the proteins overnight at 4 °C. When appropriate, the proteins were cross-linked with various concentrations of EDC (in the range of 25-1600 µM) as described above. After coating, the wells were blocked with 3% bovine serum albumin (BSA)/0.05% Tween20 in PBS (3% BT) and then incubated with 100 µL of b-heparin for a period of 3 h at room temperature. For the uncross-linked samples, b-heparin solutions were prepared at concentrations ranging from 1 to


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5000 ng/mL in 0.1% BSA/0.05% Tween20 in PBS (0.1% BT). For the cross-linked samples, only the high concentration b-heparin solution was utilized (5000 ng/mL b-heparin in 0.1% BT). Following this incubation, the wells were rinsed four times with 0.1% BT (5 min for each rinse). Subsequently, 100 µL of streptavidin-HRP at 500 ng/mL in 0.1% BT was added to the wells and incubated at room temperature for 30 min. After an additional four rinses with 0.1% BT, 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) was added to the wells, followed a few minutes later by 50 µL of 2 N H2SO4. The amount of b-heparinbinding to each sample was determined by measuring the absorbance at 450 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA). As a negative control for b-heparin-binding, a set of wells that had not been coated with the triblock proteins was also blocked with 3% BT. This is referred to simply as “BSA” in the figures.

3. Results and Discussion Modular artificial proteins are useful for creating tailored bioactive surface coatings because they can easily be embedded with various biofunctional moieties and their self-assembly is driven by very precise molecular interactions. Unfortunately, proteins with engineered self-assembly behavior may suffer from limited stability under physiological conditions because these molecular interactions are usually physical rather than covalent33 and, thus, are reversible. For our CRC-based proteins, selfassembly is driven by both hydrophobic and electrostatic interactions occurring between the terminal leucine zipper domains within the helix bundles (see Figure 2).27 Although the precise contribution of interhelical salt bridges to the stability of leucine zipper bundles is somewhat controversial in the literature,34,35 it is clear that the salt bridges in self-assembled CRC hydrogels play a stabilizing role because these hydrogels dissolve in open systems under physiological salt concentrations but are quite stable in pure water. The goal for this work was to demonstrate that our CRC-based protein coatings can be stabilized for use under physiological conditions without significantly impacting the functionality of embedded bioactive peptides. 3.1. Feasibility of Cross-Linking Proteins with EDC. EDC is a cross-linker commonly used for peptide conjugation and other applications. It reacts with carboxyl groups to form an amine-reactive O-acylisourea intermediate. In aqueous solution this intermediate is short-lived; if it does not encounter an amine group it will quickly hydrolyze and regenerate the carboxyl. We took advantage of this characteristic of EDC chemistry to introduce targeted covalent cross-links into our CRC-based protein coatings. Our hypothesis was that the short half-life of the O-acylisourea intermediate would allow us to selectively couple those carboxyl and amine groups brought into close proximity to each other upon assembly of the protein network. In our system, the most likely location for EDC cross-linking to occur is in the salt bridges that are formed between the Glu and Lys residues of neighboring helices within the helix bundles (see Figure 2A). Glu also occurs every ninth residue in the central R block of the proteins; however, the R block is unstructured and EDC-activated carboxyls have a much lower chance of encountering an amine before becoming hydrolyzed. To further increase the probability that EDC would selectively couple the salt bridge residues, we carried out the cross-linking reactions in pure water (rather than in buffers) since such electrostatically driven interactions are more stable in the absence of salt. We utilized CR, a diblock version of CRC (see Figure 1) prepared previously,25 to test the feasibility of cross-linking with

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Fischer et al.

Figure 3. Apparent point-averaged molecular weight of CR oligomers as a function of fringe displacement obtained from analytical centrifugation studies of samples with 0.32 and 0.96 mg/mL at two different rotation speeds. The upper and lower horizontal dashed lines indicate the molecular weight of potential trimer and dimer species, respectively.

EDC. CR consists of the same disordered R block as CRC, but it lacks the second leucine zipper domain. By virtue of its single associating C domain, CR can aggregate into small assemblies (via helical bundle formation), but it cannot form networks. We first performed sedimentation equilibrium studies on solutions of CR to characterize self-association behavior. Figure 3 shows semilog plots of the point-averaged molecular weight of putative oligomers of CR as a function of interference fringe displacement, J, for two concentrations (0.32 and 0.96 mg/mL) and two rotations speeds (28000 and 32000 rpm). The point-averaged molecular weight of all four data sets increases with increasing J, indicating the coexistence of monomeric CR with higher order oligomers. With increasing fringe displacement, the pointaveraged molecular weights approach that of a pure trimer (40416 Da, upper dashed line). Additional analysis indicates that this CR sedimentation equilibrium data are consistent with a monomer-trimer equilibrium model,25 although it is difficult to rule out an intermediate dimer state. There is no direct evidence that helix bundles in CRC solutions behave in the manner demonstrated for CR diblocks. However, because bundle formation is qualitatively similar for solutions of C and CR, it is our hypothesis that bundle association behavior is governed primarily by the interactions between C helices and is only weakly dependent on the connectivity the of C and R blocks. Next, we covalently coupled these physically assembled bundles using similar concentrations of either EDC or glutaraldehyde and analyzed the results via SDS-PAGE/Coomassie Blue staining to gain insight into the selectivity of the cross-linking reactions. Glutaraldehyde is well-known for its ability to crosslink proteins and was chosen strictly for comparison purposes. As shown in Figure 4, electrophoretic separation of uncrosslinked CR (lane 2 in both Figure 4A and B) consistently results in monomer, dimer, and trimer bands. Evidently, the SDS cannot completely break up these physical assemblies. Covalent linkage of CR multimers with EDC becomes apparent at a cross-linker concentration of 200 µM, as seen in Figure 4A. Densitometric analysis of this gel (data not shown) showed that the trimeric species becomes the most abundant for the two highest concentrations of EDC tested. Figure 4A also appears to indicate that EDC is selective for cross-linking physically assembled CR bundles, because the dimer and trimer bands remain relatively distinct with increasing cross-linker concentration and only

Figure 4. Coomassie Blue stained SDS-PAGE gels of CR (0.25 mg/ mL) cross-linked with different concentrations of (A) EDC or (B) glutaraldehyde. For (A) the lanes are (1) BenchMark His-Tagged Protein Standard (Invitrogen), (2) 0 µM EDC, (3) 50 µM EDC, (4) 200 µM EDC, (5) 800 µM EDC, (6) 3.2 mM EDC, (7) 12.8 mM EDC. For (B) the lanes are (1) SeeBlue Plus2 Pre-Stained Standard (Invitrogen), (2) 0% Glut, (3) 0.0004% Glut, (4) 0.00156% Glut, (5) 0.00625% Glut, (6) 0.025% Glut, and (7) 0.1% Glut. Abbreviations: Glut, glutaraldehyde; M, monomer; D, dimer; T, trimer.

minor amounts of additional bands are generated. The band smearing and increase in electrophoretic mobility with increasing cross-linker concentrations indicated in Figure 4A suggests that various conformations for each species of CR (monomer, dimer, trimer) are formed upon cross-linking with EDC. This may be due to the ability of the unstructured R blocks to fold over and become chemically coupled in various configurations to the C helices (recall that the R block contains a Glu every ninth residue; see Figure 1). However, if EDC was not selective for cross-linking the bundles, bands with a higher molecular weight than the trimer would appear in greater abundance. This is precisely what occurs when CR is cross-linked with glutaraldehyde, as several higher molecular weight bands (tetramer and higher order) become apparent when increasing concentrations of glutaraldehyde are used (see Figure 4B). Such a result indicates the ability of glutaraldehyde to polymerize CR to some extent, which is not surprising given that this homobifunctional cross-linker is known to react with multiple amino acids36 and its reactive groups have a long half-life in solution.37 Clearly, glutaraldehyde is less selective than EDC in cross-linking physically assembled bundles of CR. 3.2. Effect of EDC Cross-Linking on Stability of Protein Coatings. Next, we applied this EDC cross-linking scheme to our CRC-based triblock protein coatings to investigate whether it could help stabilize the coatings under physiological conditions. Our approach was to allow the proteins to self-assemble into surface coatings before adding the cross-linker. For the diblock protein, the results described above suggest that the



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Figure 5. Effect of EDC cross-linking on the amount of 125I-labeled CRC-RGDS remaining on (A) glass coverslips or (B) TCPS disks over time after incubation in HFF culture medium containing 10% serum. Protein amounts were determined by directly quantifying the radioactivity of the substrates using a gamma counter. The data for each group was normalized by dividing the protein amount at each time point by the amount at 0 h.

disordered R block can become covalently coupled to a helix via intraprotein cross-linking in the presence of EDC. Such a scenario is much less likely to occur when cross-linking surface adsorbed triblock proteins since the mobility of their R blocks becomes more constrained with the addition of the second helix. This second helix could become adsorbed to the surface or involved in a network junction, either of which would restrict the mobility of the R block. Additionally, the C domains were inserted with same sequence orientation within each triblock design to favor intermolecular association over intramolecular association.27 With this sequence arrangement, the formation of a parallel intramolecular bundle with the opposite helical domain would require the R block to adopt a rather tight, entropically disfavored loop conformation. To characterize the efficacy of cross-linking surface-adsorbed triblock proteins with EDC, the stability of these coatings in HFF culture medium containing 10% FBS was analyzed using 125 I-labeled CRC-RGDS. Figure 5A and B demonstrate the stabilizing effect of EDC at various concentrations on the amount of protein remaining over time on glass and TCPS substrates, respectively. For both substrates, the amount of CRCRGDS that initially adsorbed was approximately 200-300 ng/ cm2. However, it is clear that cross-linking impacted the stability of the protein more significantly on glass. Without cross-linking, CRC-RGDS desorbed rapidly from glass in the presence of serum. Use of only 25 µM EDC for cross-linking enabled nearly 90% of the protein to be retained on glass after 4 days of incubation with 10% serum; further increasing the concentration of EDC did not result in a considerably higher stability for CRCRGDS on glass. Although approximately the same mass of CRCRGDS adsorbed to the TCPS films used for these experiments, the protein demonstrated a stronger adsorption to the TCPS than to the glass coverslips. As seen in Figure 5B, approximately 75% of the protein coating was retained on the TCPS films after 4 days, even in the absence of cross-linking. Cross-linking with an EDC concentration as low as 100 µM significantly enhanced the stability of the protein coating on these films. For both the glass and the TCPS substrates, the efficiency of cross-linking surface-adsorbed triblock protein coatings appears to be higher than that observed for coupling diblock protein since the required EDC concentration is significantly less (as shown in Figure 4). The likely reason for this is that the surface concentration of adsorbed triblock proteins is much higher than the solution concentration of the diblock protein. Additionally, the mobility of the helical domains of surface-adsorbed proteins is more constrained than it is in solution, where at any point in time a greater fraction of monomeric helices likely exists due

to helix exchange (inherent in this dynamic system). It is also possible, however, that the number of covalent cross-links required to stabilize the surface coating against 10% serum is sufficiently low so as to be undetectable in the SDS-PAGE analysis. 3.3. Effect of EDC Cross-Linking on Mediation of Cell Adhesion and Proliferation. Under serum-free conditions, CRC-RGDS surface coatings were shown previously to induce attachment and focal adhesion formation for both HFFs and HUVECs, while CRC surface coatings were shown to resist cell attachment.27 In 10% serum, these protein coatings behave much differently. As shown above, non-EDC-cross-linked protein coatings desorb from glass and TCPS substrates to varying degrees in serum containing medium. Cross-linking with EDC helps to stabilize these coatings and prevent desorption. Here, the effect of cross-linking the protein coatings with EDC on HFF culture was analyzed in medium containing 10% FBS. Figures 6 and 7 demonstrate the stabilizing effect of EDC cross-linking on surface-coated triblock proteins on TCPS. This can be seen most clearly with the CRC-coated surfaces both in the phase contrast images of cells (Figure 6) and cell proliferation data (Figure 7). When TCPS was coated with CRC and not cross-linked, HFFs seeded onto the surface appeared to attach, spread, and proliferate well by day 4. For these noncrosslinked surfaces, the cells did not attach to CRC itself (which is intrinsically resistant to cell adhesion), but presumably to cell adhesive serum proteins that had replaced CRC on the surface. When the CRC coatings were cross-linked, however, fewer cells attached and more cellular aggregates were observed (Figure 6). Because HFFs are an adhesion-dependent cell line, this reduction in cell adhesion led to a reduction in cellular proliferation (as measured by quantifying cellular DNA, Figure 7). HFF proliferation on CRC-coated surfaces generally decreased with increasing EDC concentration, reaching a minimum by approximately 400 µM EDC, at which point the cross-linked CRC appears to have completely resisted the competitive adsorption of serum proteins. Figures 6 and 7 also show that the HFFs attached, spread, and proliferated well on CRC-RGDS at all EDC concentrations examined in this study. The sharp contrast between the CRC and CRC-RGDS groups at higher EDC concentrations confirms that not only did EDC increase the stability of the triblock protein coatings in serum, it appeared to do so in a manner that did not prevent the RGDS ligand from inducing cell adhesion and proliferation. We further investigated the initial adhesive interactions of HFFs with EDC-cross-linked CRC-RGDS to determine whether

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Figure 6. Phase contrast images of HFFs after 4 days on TCPS substrates coated with CRC or CRC-RGDS and cross-linked with different concentrations of EDC.

Figure 7. Proliferation of HFFs after 4 days on TCPS substrates coated with CRC or CRC-RGDS and cross-linked with different concentrations of EDC. Proliferation fold was determined by quantifying cellular DNA using the CyQuant kit (Invitrogen) and dividing the amount of DNA on day 4 by that on day 0 for each group.

cross-linking impacted the capacity of this bioactive coating to induce focal adhesion formation and cytoskeletal organization within the cells. As markers for focal adhesions, we stained for cytoskeletal F-actin and vinculin, a focal adhesion associated protein that functions as a linker between actin filaments and integrins.38,39 Figure 8 shows representative images of the cells at both low (25 µM EDC) and high (1600 µM EDC) crosslinking conditions. After 6 h of culture under 10% serum conditions, a well-developed F-actin stress fiber network (indicated in green), terminating at focal adhesions (indicated in red), was observed at all concentrations of EDC utilized for cross-linking. These results confirmed that the EDC-cross-linked CRC-RGDS coating presents the RGDS ligand in a manner appropriate to induce focal adhesion formation and cytoskeletal organization by HFFs. 3.4. Effect of EDC Cross-Linking on Heparin-Binding Capacity of Protein Coatings. An important advantage of recombinant protein-based coatings is that they can be embedded with virtually any bioactive sequence through the use of genetic engineering techniques. Here, we generated four new heparinbinding triblock proteins (denoted CRC-LQVQ, CRC-WQPP,

CRC-KAFAK, and CRC-KNRL) by inserting short peptide sequences known to bind heparin into the R block of CRC (see Figure 1 for the precise sequences). Such proteins could potentially be used to immobilize heparin-binding growth factors to surfaces via a heparin bridge. As heparin is a highly negatively charged molecule, interactions between heparin and proteins are primarily ionic in nature, and heparin-binding sites within proteins are characterized by clusters of positively charged basic amino acids.40 Therefore, it was not known whether the short heparin-binding peptides we selected would maintain their functionality when inserted into the negatively charged R block of CRC. To investigate the heparin-binding capacity of the new triblock proteins, and to determine whether EDC cross-linking impacts this functionality, biotinylated heparin (b-heparin) ELISAs were carried out. We first checked whether the triblock proteins could bind b-heparin in the absence of EDC cross-linking. TCPS substrates were coated with the proteins, incubated in the presence of various concentrations of b-heparin, and then probed with streptavidin-HRP. The ELISA results shown in Figure 9A demonstrate that all of the new proteins bind b-heparin in a dose-dependent manner. CRC-KAFAK and CRC-KNRL demonstrate the highest affinity for b-heparin. CRC-LQVQ and CRCWQPP demonstrate a somewhat lower but still significant b-heparin-binding capacity. Importantly, CRC proved to be bioinert with respect to b-heparin binding. Without a heparin binding sequence inserted, the central R block of CRC possesses only negatively charged residues (one Glu every nine residues) and likely repels b-heparin. We then investigated the effect of EDC cross-linking on the capacity of the triblock protein coatings to bind b-heparin. The proteins were coated onto TCPS substrates, cross-linked in the range of 0-1600 µM EDC, incubated in the presence of b-heparin (500 ng/well), and then probed with streptavidin-HRP. Absorbance values for each EDC concentration were normalized by the value for the same protein at 0 µM EDC. As shown in Figure 9B, three of the heparin-binding proteins (CRC-WQPP, CRC-KNRL, and CRC-KAFAK) demonstrated a significant increase in relative b-heparin-binding for low concentrations



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Figure 8. Confocal microscopy images of HFFs cultured on EDC-cross-linked CRC-RGDS (the concentration of EDC is indicated). Fluorescentlylabeled F-actin is shown in green and fluorescently labeled vinculin is shown in red.

Figure 9. (A) Biotinylated-heparin ELISA on protein coated TCPS substrate. Raw absorbance values at 450 nm for the conversion of TMB by streptavdidin-HRP are reported. (B) Biotinylated-heparin ELISA on protein coated TCPS substrates cross-linked with various concentrations of EDC. Raw absorbance values for each EDC concentration were normalized by the value for the same protein at 0 µM EDC.

of EDC, followed by a gradual decrease in b-heparin-binding at higher EDC concentrations. Although the results observed for low EDC concentrations may indicate that cross-linking can improve ligand binding (perhaps through a beneficial conformational change induced in the active peptide), a more likely explanation is that cross-linking improves the surface stability of these protein coatings, allowing them to resist desorption in the ELISA buffers. BSA, a surface-active protein present in all of the buffers, does not bind b-heparin (see Figure 9A), so any BSA that displaced the triblock protein on the surface would have resulted in reduced b-heparin binding. For higher EDC concentrations, cross-linking somewhat reduced b-heparin binding for each of these three proteins. For both CRC-KAFAK and CRC-KNRL, the reduction is minimal. CRC-WQPP showed a more significant reduction but still retained approximately 60% of its maximal b-heparin-binding capacity at the highest concentration of EDC tested here. For the fourth triblock protein, CRC-LQVQ, cross-linking significantly reduced b-heparinbinding, even for low concentrations of EDC. This protein showed virtually no b-heparin-binding for EDC concentrations

of 200 µM and above. We note, however, that because low EDC concentrations (in the 25 µM range; see Figure 5) can have a significant stabilizing effect for the protein coatings, in practice it may still be possible to use CRC-LQVQ as a heparin binding ligand in lightly EDC cross-linked protein coatings. These results indicate that EDC cross-linking can impact the functionality of middle-block embedded heparin-binding peptides at sufficiently high concentrations, but suggests that the degree to which this occurs is peptide specific. This is not surprising because each bioactive peptide has different requirements for binding its ligand. For some carbohydrate binding domains within proteins, ligand binding can be dramatically altered with even a single amino acid substitution.41 The EDC induced changes in ligand binding observed here (especially for higher EDC concentrations) presumably can be caused either by the participation in a cross-link of an important residue within the bioactive peptide itself or by conformational changes induced in the bioactive peptide by cross-links located externally to this sequence. The fact that the two proteins that possess EDCreactive primary amine groups in their active sequences (CRC-

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KAFAK with three Lys residues and CRC-KNRL with one Lys residue) were impacted to a lesser extent by EDC cross-linking than the two proteins without primary amines in their active sequences (CRC-WQPP and CRC-LQVQ) suggests that the latter explanation is more likely. The Lys residues undoubtedly play an important role in the ability of CRC-KAFAK and CRC-KNRL to bind b-heparin so one would expect these proteins to show reduced b-heparin binding if these residues were to become cross-linked.

4. Conclusions We have developed an artificial protein-based system capable of functionalizing surfaces with bioactive peptides in a facile and well-controlled manner. The basis for this system is CRC, a triblock protein that adsorbs to various materials and selfassembles into a surface coating. CRC itself demonstrates very low background levels of binding to both cell adhesion receptors (integrins23) and heparin. Because this protein can be easily modified to contain short bioactive peptides, CRC-based surface coatings offer the possibility of tailoring cell responses through specific cell-ligand interactions. Heretofore, the most significant limitation of our CRC-based coating system was its lack of stability in serum. In cell culture media containing 10% serum, the triblock proteins gradually desorb from surfaces and are replaced by serum proteins possessing a higher surface activity. Here, we have developed a cross-linking scheme that effectively stabilizes surfaceadsorbed CRC-based coatings against serum displacement with limited deleterious effects on bioactivity. The scheme utilizes EDC, a cross-linker that directly couples carboxyl groups to primary amines. Although stabilizing the triblock protein coatings through reaction with an external cross-linking reagent unavoidably introduces some number of unwanted cross-links, our EDC scheme takes advantage of the architecture of selfassembled CRC networks and appears to selectively couple the C helical domains of the triblock proteins (specifically, the complementary Glu and Lys resuides comprising the salt bridges). By targeting the end blocks of the proteins, a significant level of functionality could be maintained for various central R block-embedded bioactive peptides. This was demonstrated both through cellular studies and b-heparin ELISAs. Cellular studies revealed that EDC cross-linking enables CRC to resist cell adhesion in normal serum medium (due to its enhanced surface stability), but does not impair the ability of CRC-RGDS to promote cellular attachment, focal adhesion formation, and proliferation. The ELISAs revealed that the b-heparin-binding capacity of two triblock proteins (CRC-KAFAK and CRC-KNRL) is only minimally affected by EDC cross-linking with EDC, while the b-heparin-binding capacity of a third protein (CRCWQPP) is only moderately impacted. The heparin-binding capacity of a fourth triblock protein (CRC-LQVQ) was shown to be significantly reduced at all but low EDC levels, indicating that this technique does possess some limitations. Clearly, the degree to which this cross-linking scheme impacts the biofunctionality of CRC-based proteins must be determined on a caseby-case basis. Developing a suitable method for stabilizing the network structure of our CRC-based protein coatings has brought us one step closer to realizing our goal of producing biofunctional substrates that can be tailored for specific cell culture applications. Our next step is to further expand this technology from two-dimensional (2-D) surface coatings to more ECM-like threedimensional (3-D) cell culture environments. We have previ-

Fischer et al.

ously demonstrated the feasibility of utilizing our protein coatings to biofunctionalize porous 3-D matrices for short-term, serum-free cell studies.27 Our current efforts are focused on applying our EDC cross-linking scheme to these hybrid substrates such that longer-term cell studies in the presence of serum (if necessary) can be undertaken. Acknowledgment. We thank Dr. Yuan Chuan Lee for use of his laboratory for the 125I-labeled protein studies, Dr. Evangelos Moudrianakis and Dr. Jamie Godfrey for use of AUC facilities, and the JHU Department of Materials Science and Engineering for use of their confocal microscopy facilities. This work was partially supported by NASA through Grant NAG 9-1345 (to J.L.H.) and a GSRP fellowship award (to S.E.F.; Grant No. NGT5-50437), by the National Science Foundation through a Faculty Early Career Award (to H.-Q.M.), and by a Discovery grant from NSERC (to J.L.H.).

References and Notes (1) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47–55. (2) Sands, R. W.; Mooney, D. J. Curr. Opin. Biotechnol. 2007, 18, 448– 53. (3) Brandley, B. K.; Schnaar, R. L. Anal. Biochem. 1988, 172, 270–8. (4) Kao, W. J.; Lee, D.; Schense, J. C.; Hubbell, J. A. J. Biomed. Mater. Res. 2001, 55, 79–88. (5) Heilshorn, S. C.; DiZio, K. A.; Welsh, E. R.; Tirrell, D. A. Biomaterials 2003, 24, 4245–52. (6) Massia, S. P.; Hubbell, J. A. Anal. Biochem. 1990, 187, 292–301. (7) Sugawara, T.; Matsuda, T. J. Biomed. Mater. Res. 1995, 29, 1047– 52. (8) Sofia, S.; McCarthy, M. B.; Gronowicz, G.; Kaplan, D. L. J. Biomed. Mater. Res. 2001, 54, 139–48. (9) Gabriel, M.; van Nieuw Amerongen, G. P.; Van Hinsbergh, V. W.; Amerongen, A. V.; Zentner, A. J. Biomater. Sci., Polym. Ed. 2006, 17, 567–77. (10) Danilov, Y. N.; Juliano, R. L. Exp. Cell Res. 1989, 182, 186–96. (11) Neff, J. A.; Caldwell, K. D.; Tresco, P. A. J. Biomed. Mater. Res. 1998, 40, 511–9. (12) Janssen, M. I.; van Leeuwen, M. B.; Scholtmeijer, K.; van Kooten, T. G.; Dijkhuizen, L.; Wosten, H. A. Biomaterials 2002, 23, 4847– 54. (13) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55–78. (14) Houseman, B. T.; Mrksich, M. Biomaterials 2001, 22, 943–55. (15) Ochsenhirt, S. E.; Kokkoli, E.; McCarthy, J. B.; Tirrell, M. Biomaterials 2006, 27, 3863–74. (16) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389–92. (17) Lee, J.; Macosko, C. W.; Urry, D. W. Biomacromolecules 2001, 2, 170–9. (18) Haider, M.; Cappello, J.; Ghandehari, H.; Leong, K. W. Pharm. Res. 2007, 25, 692–9. (19) Woolfson, D. N. AdV. Protein Chem. 2005, 70, 79–112. (20) Rajagopal, K.; Schneider, J. P. Curr. Opin. Struct. Biol. 2004, 14, 480–486. (21) Fairman, R.; Åkerfeldt, K. S. Curr. Opin. Struct. Biol. 2005, 15, 453– 463. (22) Bhardwaj, A.; Walker-Kopp, N.; Wilkens, S.; Cingolani, G. Protein Sci. 2008, 17, 1475–85. (23) Lombardi, A.; Bryson, J. W.; DeGrado, W. F. Biopolymers 1996, 40 (5), 495–504. (24) Kohn, W. D.; Hodges, R. S. Trends Biotechnol. 1998, 16, 379–389. (25) Mi, L. Protein Hydrogels with Engineered Biomolecular Recognition; Johns Hopkins University: Baltimore, 2003. (26) Mi, L.; Fischer, S.; Chang, B.; Sundelacruz, S.; Harden, J. L. Biomacromolecules 2006, 7, 38-47. (27) Fischer, S. E.; Liu, X. Y.; Mao, H. Q.; Harden, J. L. Biomaterials 2007, 28, 3325–37. (28) Mooradian, D. L.; McCarthy, J. B.; Skubitz, A. P.; Cameron, J. D.; Furcht, L. T. InVest. Ophthalmol. Vis. Sci. 1993, 34, 153–64. (29) Nomizu, M.; Kim, W. H.; Yamamura, K.; Utani, A.; Song, S. Y.; Otaka, A.; Roller, P. P.; Kleinman, H. K.; Yamada, Y. J. Biol. Chem. 1995, 270, 20583–90. (30) Richard, B. L.; Nomizu, M.; Yamada, Y.; Kleinman, H. K. Exp. Cell Res. 1996, 228, 98–105.

Biofunctional Coatings via Triblock Proteins (31) Tyler-Cross, R.; Sobel, M.; Marques, D.; Harris, R. B. Protein Sci. 1994, 3, 620–7. (32) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry, Part II; W.H. Freeman: San Francisco, 1980. (33) Shen, W.; Lammertink, R. G. H.; Sakata, J. K.; Kornfield, J. A.; Tirrell, D. A. Macromolecules 2005, 38, 3909–16. (34) Burkhard, P.; Stetefeld, J.; Strelkov, S. V. Trends Cell Biol. 2001, 11, 82–88. (35) Phelan, P.; Gorfe, A. A.; Jelesarov, I.; Marti, D. N.; Warwicker, J.; Bosshard, H. R. Biochemistry 2002, 41, 2998–3008. (36) Habeeb, A. J.; Hiramoto, R. Arch. Biochem. Biophys. 1968, 126 (1), 16–26.


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(37) Avrameas, S.; Ternynck, T. Immunochemistry 1971, 8 (12), 1175–9. (38) Ziegler, W. H.; Liddington, R. C.; Critchley, D. R. Trends Cell Biol. 2006, 16, 453–60. (39) Zaidel-Bar, R.; Cohen, M.; Addadi, L.; Geiger, B. Biochem. Soc. Trans. 2004, 32, 416–20. (40) Munoz, E. M.; Linhardt, R. J. Arterioscler., Thromb., Vasc. Biol. 2004, 24, 1549–57. (41) Revelle, B. M.; Scott, D.; Beck, P. J. J. Biol. Chem. 1996, 271, 16160–70.

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