Stepwise Assembly of Fibrin Bilayers on Self-Assembled

Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, and Hope Heart Program, Benaroya Research Institute at Virgin...
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J. Phys. Chem. C 2007, 111, 8504-8508

Stepwise Assembly of Fibrin Bilayers on Self-Assembled Monolayers of Alkanethiolates: Influence of Surface Chemistry Hua Wang,† Buddy D. Ratner,*,† E. Helene Sage,*,‡ and Shaoyi Jiang*,† Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195, and Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 ReceiVed: October 24, 2006; In Final Form: March 25, 2007

Fibrin bilayers are formed in a stepwise manner on four model surfaces: hydrophobic, neutral hydrophilic, positively charged, and negatively charged. These surfaces are self-assembled monolayers (SAMs) of alkanethiolates on gold with different terminal groups. A surface plasmon resonance (SPR) sensor is used to monitor the formation of fibrin bilayers in real time. The structures of the fibrin bilayers formed on SAMs of different surface chemistries are proposed, based on the amount of fibrinogen in each layer. The efficiency of the physically adsorbed fibrinogen to promote fibrin formation is assessed by the ratio of the amount of fibrinogen in the second layer to that in the first layer. It is shown for the first time that the orientation/ conformation of an adsorbed fibrinogen layer determined by underlying surface chemistry affects its reactivity with soluble fibrinogen, resulting in fibrin bilayers with different molecular arrangements. Bovine aortic endothelial cells cultured on fibrin bilayers formed on CH3- and OH- SAMs exhibited different morphologies and growth characteristics. Results show that fibrin bilayers with different molecular arrangements and thus different mechanochemical properties lead to different cellular responses.

Introduction The interactions of proteins with surfaces govern many biological processes. Upon contact of a foreign surface with the biological environment, the first major events that occur are the adsorption and activation of proteins.1,2 Particularly, the adsorption of fibrinogen plays a critical role, because of its high concentration in plasma and its regulation of hemostasis and thrombosis.3 Fibrinogen is also the precursor of fibrin, a participant in many physiological and pathological processes, including wound healing.4 Both the amount and conformation of adsorbed fibrinogen depend on surface chemistry.5-7 Although many studies have shown that the conformation of surface-bound fibrinogen plays an important role in platelet aggregation and thrombus formation,8,9 little is known about the influence of surface chemistry on fibrin assembly. Fibrinogen is a 340 kDa glycoprotein consisting of two identical subunits; each composed of three polypeptide chains (AR, Bβ, and γ) joined by 29 disulfide bonds. The native structure of fibrinogen can be described as trinodular, consisting of a central E and two outer D globular domains connected by R-helical coiled coil domains.10 The molecular length of fibrinogen is 47.5 nm, with the diameter of the roughly spherical D and E domains to be 6.5 and 5 nm, respectively11 (Figure 1). Fibrin monomer is formed via thrombin cleavage of two pairs of fibrinopeptides from the N-terminal ends of AR and Bβ polypeptides located in the E domain. Since the thrombincleaved E domain carries a net positive charge, whereas the D domain has a net negative charge, the electrostatic interactions * To whom correspondence should be addressed. (S.J.) E-mail: [email protected]. Phone: (206) 543-4548. Fax: (206) 685-3451. (B.D.R.) E-mail: [email protected]. Phone: (206) 6851005. Fax: (206) 616-9763. (H.S.) E-mail: [email protected]. Phone: (206) 903-2026. Fax: (206) 903-2144. † University of Washington. ‡ Benaroya Research Institute at Virginia Mason.

Figure 1. Trinodular model of the structure of a fibrinogen molecule.

between the D and E domains give rise to the characteristic half-staggered structure of a fibrin strand.12 The noncovalent assembly of fibrin could occur between two fibrin monomers or between a fibrin monomer and a native fibrinogen molecule. Indeed, dimeric fibrinogen-fibrin monomer complexes have been found in patients with venous thrombosis.13 The objective of the present study is to build fibrin bilayers in a stepwise manner through the interactions between surface-adsorbed fibrin monomer and soluble fibrinogen. Since the fibrin monomer is generated by the enzymatic conversion of surface-adsorbed fibrinogen, fibrin can only be formed directly on the surface and not via polymerization in solution. Successive fibrin layers can therefore be added. Unlike previous studies, in which the parameters such as pH, ionic strength, and fibrinogen/thrombin concentration were adjusted to modulate the formation of bulk fibrin gels,14,15 in our study, a layer-bylayer fibrin structure is modulated by the control of surface chemistry. Self-assembled monolayers (SAMs) of alkanethiolates terminated with amine (NH2-), carboxylic acid (COOH), methyl (CH3-), and hydroxyl (OH-) groups, are used to functionalize SPR chips for fibrin assembly. They represent positively and negatively charged, hydrophobic, and neutral hydrophilic surfaces, respectively. It is expected that the orientation/conformation of an adsorbed fibrinogen layer determined by underlying surface chemistry will affect the

10.1021/jp066960a CCC: $37.00 © 2007 American Chemical Society Published on Web 05/27/2007

Stepwise Assembly of Fibrin Bilayers accessibility of the thrombin-cleavage sites on the central E domains and, thus, its reactivity with soluble fibrinogen. Therefore, the molecular arrangements of fibrin bilayers can be controlled by modulating surface chemistry. Since the fibrin assembly process can be monitored in real-time by surface plasmon resonance (SPR), the compositions and structures of the fibrin bilayers can be deduced from SPR results. As a result of vascular injury, blood coagulation and platelet aggregation generate a fibrin-rich clot to facilitate hemostasis, promote inflammatory responses, and recruit tissue cells to the injured site.16 In particular, neovascularization (the formation of new blood vessels) frequently occurs in a fibrin-rich extracellular matrix (ECM), where fibrin serves as a provisional matrix for endothelial cell migration and adhesion.17 Endothelial cells attach to and spread on fibrinogen through the cell surface receptor RVβ3 integrin, which binds to an Arg-Gly-Asp (RGD) sequence near the C-terminus of the R chain of fibrinogen.18 Fibrin has also been reported to induce angiogenesis in vitro, with the formation of capillary-like structures critically dependent on the configuration of the fibrin gels.19,20 It has been found that malleable gels stimulate the formation of capillary-like structures better than rigid gels.21 In this study, bovine aortic endothelial (BAE) cells are cultured on fibrin bilayers formed on different surface chemistries. We report here on the effects of the molecular arrangement of a fibrin bilayer on the behavior of endothelial cells. Materials and Methods Materials. Tris-buffered saline (25 mM, with 125 mM NaCl and 1 mM CaCl2, pH 7.2) was used for all experiments except cell culture. Purified human plasma fibrinogen was acquired from Enzyme Research Laboratories (South Bend, IN). Thrombin was purchased from Calbiochem (San Diego, CA). Bovine serum albumin (BSA), phenylmethylsulphonyl fluoride (PMSF), Triton X-100, and both thiols of 1-dodecanethiol [HS(CH2)11CH3] and 11-mercapto-1-undecanol [HS(CH2)11OH] were obtained from Sigma-Aldrich (St. Louis, MO). 11-Mercaptoundecanoic acid [HS(CH2)11COOH] was purchased from ProChimia (Sopot, Poland), whereas 11-amino-1-undecanethiol [HS(CH2)11NH2] was from Dojindo Molecular Technologies (Gaithersburg, MD). SPR Measurements. A custom-built dual-channel SPR sensor was used in this work, which is based on the Kretschmann configuration exploiting attenuated total reflection (ATR).22 In this configuration, light from a broadband source passes through the optical prism and excites a surface plasmon wave at the outer boundary of the metal layer of the sensing chip. The amount of adsorbed protein is monitored by measuring the resonant wavelength, which is determined by locating a minimum in the spectrum of the reflected optical wave using an optical spectrometer. SPR chips were functionalized by immersing them into thiol solutions as described in previous papers from our group. In particular, an improved method for the preparation of COOH- and NH2- SAMs was used.23 Figure 2 shows the assembly of a fibrin bilayer monitored by SPR. Following the initial adsorption of the first layer of fibrinogen (2 mg/mL), 6 U/mL of thrombin was injected, and the reaction was allowed to proceed for 30 min to convert the adsorbed fibrinogen to fibrin monomer. After the enzyme was quenched by PMSF (1 M) for 10 min, the surface was blocked with 1 mg/mL BSA in 1 M PMSF. Then, another solution of fibrinogen (2 mg/mL) in 1 M PMSF was applied to react with the surface-bound fibrin monomer to form the second layer of fibrin. The sensor chip was washed with Tris-buffered saline

J. Phys. Chem. C, Vol. 111, No. 24, 2007 8505

Figure 2. (a) SPR sensorgram and (b) model for the stepwise assembly of a fibrin bilayer.

between additions of reagents to remove loosely bound proteins. The whole system was sequentially washed with acid and base between runs to ensure complete protein removal. Freshly prepared chips were used for each experiment. Using the software ClampXP Biosensor Data Analysis V. 3.50 developed by T. Morton and D. Myszka (Center for Bimolecular Interaction Analysis, University of Utah), data were fitted to the model for simple 1:1 interactions for the binding of soluble fibrinogen to surface-bound fibrin monomer (1 nm from the SPR response is equivalent to 214.6 BIACORE RU @ 760 nm). The forward (ka) and reverse (kd) rate constants of the formation of the second layer of fibrin were obtained from the fitting. The equilibrium dissociation constant was calculated as Kd ) kd/ka. Cell Culture and Characterization. BAE cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), passaged once a week, and discarded after 15 passages. CH3and OH- SAMs with fibrin bilayers were transferred into 24well culture plates and blocked with 1 mg/mL sterile BSA for 30 min. 2 mL of a suspension of BAE cells were plated in DMEM at a density of 150 000 cells/mL. Overall cell morphology was photographed using a phase-contrast microscope (Nikon TE200) with a ×10 objective. Results and Discussion Formation of Fibrin Bilayers on Various Surfaces. The SPR sensorgram of the stepwise formation of a fibrin bilayer on the hydrophobic CH3- SAM is shown in Figure 2.a, with a model of the fibrin assembly shown in Figure 2.b. It can be seen from the sensorgram that fibrinogen adsorption on the CH3SAM functionalized SPR chip increases rapidly in the initial stage and reaches a plateau within 5 min, suggesting that a protein monolayer is formed. The first layer of adsorbed fibrinogen was then activated by thrombin. Before the reintroduction of fibrinogen solution into the system, a buffer containing PMSF (an inhibitor of thrombin) was applied to deactivate any thrombin bound to the surface and to ensure that no polymerization occurred in the solution. A second layer of fibrinogen was formed at a much lower rate than that of the

8506 J. Phys. Chem. C, Vol. 111, No. 24, 2007

Wang et al.

TABLE 1: Amounts (ng/cm2) of Fibrinogen in the First and Second Layers of the Fibrin Bilayers, Their Respective Ratios, and Equilibrium Dissociation Constants of the Binding of Soluble Fibrinogen to Surface-Bound Fibrin Monomer on Various SAMsa first layer second layer ratio (second/first layer) Kd (µM)

CH3-

NH2-

COOH-

OH-

445 (41.2) 183 (11.6) 0.41 (0.04)

710 (41.6) 245 (25.2) 0.35 (0.03)

305 (10.0) 77.5 (5.51) 0.25 (0.01)

171 (8.41) 88.0 (9.80) 0.52 (0.06)

0.29 (0.04)

0.33 (0.06)

0.35 (0.08)

0.26 (0.05)

Data shown are the average results (n ) 3) with standard deviations (in parentheses). a

physically adsorbed first layer when another solution of fibrinogen was injected. The second solution of fibrinogen was of the same concentration (2 mg/mL) as the initial one, and BSA was used to block unoccupied sites. Control experiments showed that no response from the application of the second solution of fibrinogen was detected in the absence of the thrombin activation step (data not shown), indicating that the second layer of fibrin was immobilized via interactions with the activated adsorbed fibrinogen (i.e., fibrin monomer). The same cycle of thrombin and fibrinogen can be repeated to add successive layers of fibrinogen in a stepwise manner. From the SPR sensorgrams of the fibrin bilayers assembled on surfaces of various chemistries, the amounts of fibrinogen in the first and second layers of the fibrin bilayers and their ratios (second/first layer) were calculated (Table 1). It can be seen that the physical adsorption of fibrinogen (first layer) was dependent on surface chemistry. The positively charged surface adsorbed most fibrinogen, followed by the hydrophobic and negatively charged surfaces. As expected, the neutral hydrophilic surface adsorbed the least amount of fibrinogen. The abilities of different surface chemistries to facilitate fibrin proliferation were assessed by the ratios of the amounts of fibrinogen in the second layer to those in the first layer. Student t-test showed that the means of the ratios on various SAMs were statistically different from each other at the 0.05 confidence level. The largest ratio of the amount of fibrinogen in the second layer to that in the first layer was observed on the neutral hydrophilic surface, indicating that, despite a relatively low level of fibrinogen adsorption, the adsorbed fibrinogen on the neutral hydrophilic surface upon activation by thrombin had relatively high reactivity with soluble fibrinogen. The capacity of the physically adsorbed fibrinogen to form a fibrin bilayer was greatly impaired on the negatively charged surface, as evidenced by the lowest ratio (second/first layer) on the COOH- SAM. The overall fibrin bilayer coverage on various SAMs decreased in the following order: NH2- > CH3- > COOH- > OH-. Furthermore, the equilibrium dissociation constants Kd (calculated as Kd ) kd/ka) for the formation of the second layer of fibrin bilayers on various SAMs were determined by fitting the binding data of soluble fibrinogen to surface-bound fibrin monomer to Langmuir binding isotherms. Student t-test showed that the means of the dissociation constants on various SAMs were not statistically different from each other at the 0.05 confidence level, indicating that the mechanism of fibrin bilayer formation was similar on different surface chemistries. We showed that the effectiveness of the adsorbed fibrinogen in promoting fibrin formation was dependent on surface chemistry, a result consistent with previous work. For example, a previous study using quartz crystal microbalance (QCM) and scanning electron microscopy (SEM) by Evans-Nguyen et al.24 showed that the conversion of fibrinogen to fibrin on negatively charged surfaces was significantly reduced as compared with

Figure 3. Schematic representation of the structures of fibrin bilayers formed on (a) CH3-, (b) NH2-, (c) COOH-, and (d) OH- SAMs. Fibrinogen molecules in the first and second layers are represented by solid and open ovals, respectively.

that on hydrophobic surfaces. In their study, fibrin was formed by exposing adsorbed fibrinogen to both thrombin and soluble fibrinogen simultaneously. Thus, multiple-layer fibrin was formed, resulting from fibrin formation both on the surface and in the solution. In our study, the surface-adsorbed fibrinogen was exposed to thrombin and fibrinogen sequentially. Thus, fibrin can only be formed directly on the surface, and successive layers of fibrin can be added layer by layer with precise control. An atomic force microscopy (AFM) study by Sit et al. also found that the sequential addition of fibrinogen, thrombin, and soluble fibrin monomer into the AFM system resulted in the formation of an extensive multilayer fibrin network on the hydrophobic graphite substrate but not on a negatively charged mica substrate.25 The consistency between our results and those reported previously shows that the surface chemistry of the underlying substrate has significant impact on the formation of a surface-bound fibrin network. We therefore asked why adsorbed fibrinogen promoted fibrin formation most efficiently on the neutral hydrophilic surface, whereas least on the negatively charged surface. On the basis of the molecular dimensions (47.5 nm × 6.5 nm × 6.5 nm) and weight (340 kDa) of fibrinogen, the surface coverage of a fibrinogen monolayer with all fibrinogen molecules having “side-on” orientation (lying on the surface) was calculated to be ∼183 ng/cm2 (equivalent to a wavelength shift of ∼9.2 nm in the SPR sensorgram), while the surface coverage of a fibrinogen monolayer with fibrinogen molecules taking “endon” orientation to be ∼1337 ng/cm2 26. The structures of the fibrin bilayers (i.e., the orientations of the fibrinogen molecules) on various SAMs illustrated in Figure 3 can be estimated based on the adsorbed amounts of fibrinogen in each fibrin layer (Table 1) with reference to the theoretical fibrinogen monolayer. It is speculated that most of the adsorbed fibrinogen molecules took

Stepwise Assembly of Fibrin Bilayers an “end-on” orientation on the positively charged and hydrophobic surfaces. The tightly packed central E domains, which carry the two pairs of fibrinopeptides to be cleaved, were less accessible to thrombin and soluble fibrinogen. On the neutral hydrophilic surfaces, the adsorbed fibrinogen had a lower surface coverage. Both of the relatively weak interaction with the surface and the “side-on” orientation of the adsorbed fibrinogen favored fibrin formation. On the negatively charged surface, however, the thrombin-cleaved E domain with a net positive charge could have strong electrostatic interactions with the negatively charged surface. Although the adsorbed fibrinogen had a “side-on” orientation, the strong engagement of the thrombin-cleaved E domains with the surface made them much less accessible to the D domains of soluble fibrinogen, leading to minimal fibrin formation on the negatively charged surface. In summary, the orientation/conformation of the physically adsorbed fibrinogen in the first layer is critical to the subsequent formation of fibrin. BAE Cells on Fibrin Bilayers with Different Molecular Arrangements. The effects of bulk fibrin gel structures on endothelial cell behavior have been actively studied.14,15 Unlike previous studies, in which bulk fibrin gels with different structures were synthesized by adjusting gelling parameters, we examined fibrin bilayers formed on surfaces (i.e., hydrophobic CH3- and hydrophilic OH- SAMs). It can be seen from the cartoons in Figure 3, based on data collected in this study, that the molecular arrangements of these two fibrin bilayers are different. Overall, the fibrin bilayer on the hydrophobic surface is thicker, with a higher fibrinogen density (most molecules in the “end-on” orientation as illustrated in Figure 3a), whereas the fibrin bilayer on the neutral hydrophilic surface is thinner with fewer fibrinogen molecules (most of them in the “sideon” orientation as illustrated in Figure 3d). BAE cells were cultured on both fibrin bilayers under serum-free conditions. Serum was excluded from the culture medium because it contains other cell adhesive proteins, such as fibronectin and vitronectin, as well as growth factors. It can be seen from Figure 4 that cellular responses to the fibrin bilayers formed on different SAMs were different. On day 1 (Figure 4a,b), cells were adherent but appeared more elongated on the CH3- SAM. On days 3-7, cells plated on the CH3- SAM formed cord-like structures, whereas cells cultured on the OH- SAM continued to proliferate until a confluent monolayer of polygonal cells was formed (Figure 4c-f). Finally, when the original culture medium was replaced with fresh serum-free medium on day 7, the cells on the OH- SAM exhibited minimal morphological change (Figure 4h), whereas the cells on the CH3- SAM assembled into cord-like networks (Figure 4g) and subsequently detached from the substrate within a 24-h period. It is likely that the cells cultured on the CH3-SAM secreted their own growth factors that were critical for the maintenance of their phenotype shown in Figure 4, panels a, c, and e. After these factors were removed by the replacement of the original culture medium, the cells assumed a network configuration and detached. These results are consistent with those reported previously from cultures on bulk fibrin gels.27 Adhesion to a rigid substratum appears to promote endothelial cell proliferation and to inhibit differentiation, whereas adhesion to a malleable substratum that permits multicellular retraction appears to stimulate “capillary tube” formation.28 In our study, the fibrin bilayer formed on the hydrophobic surface was thicker and we believe more malleable than that formed on the neutral hydrophilic surface. The different mechanochemical properties of these two fibrin bilayers induced by different surface chemistries may be responsible for varied cellular responses.

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Figure 4. Phase contrast images of BAE cells cultured on fibrin bilayers formed on CH3- (a, c, e, and g) and OH- (b, d, f, and h) SAMs (original magnification ×10). The seeding density of BAE cells was 15 000 cells/mL. Images were taken after culturing of BAE cells in DMEM for 1 day (a and b), 3 days (c and d), and 7 days (e and f). On the seventh day, cells were given fresh DMEM and then photographed on the eighth day (g and h).

The ability to control the layer-by-layer formation of fibrin will allow the systematic analysis of cellular responses to fibrins with different molecular arrangements, as well as to understand novel chemical and mechanical properties of fibrin. Conclusions In this study, fibrin bilayers with different molecular arrangements were formed in a stepwise manner on SAMs of various surface chemistries. The amount of fibrinogen in each layer was measured by SPR. It was shown that adsorbed fibrinogen on the neutral hydrophilic surface upon activation by thrombin had relatively high reactivity with soluble fibrinogen despite a relatively low level of fibrinogen adsorption. The capacity of adsorbed fibrinogen on the negatively charged surface to form a fibrin bilayer was greatly impaired. The ability of adsorbed fibrinogen on different SAMs to promote fibrin formation could be attributed to the orientation/conformation of adsorbed fibrinogen. This is the first report showing that fibrin bilayers can be formed directly on surfaces with molecular arrangements modulated by underlying surface chemistries. Furthermore, results from BAE cells cultured on fibrin bilayers formed on CH3- and OH- SAMs showed that fibrin bilayers with different molecular arrangements or different mechanochemical properties induced by different surface chemistries could lead to varied cellular responses.

8508 J. Phys. Chem. C, Vol. 111, No. 24, 2007 Acknowledgment. This work is supported by NSF EEC9529161 through the University of Washington Engineered Biomaterials (UWEB)-Engineering Research Center and NSF CAREER Award (CTS-0092699). References and Notes (1) Brash, J.; Horbett, T. ACS Symp. Ser. 1987, 343. (2) Andrade, J. In Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985; Vol. 2. (3) Mosesson, M. J. Lab. Clin. Med. 1990, 116, 8. (4) Yamada, K.; Clark, R. Provisional matrix. In Molecular and Cellular Biology of Wound Repair; Clark, R., Ed.; Plenum: New York, 1996; p 51. (5) O’Connor, S.; DeAnglis, A.; Gehrke, S.; Retzinger, G. Biotechnol. Appl. Biochem. 2000, 31, 185. (6) Ostuni, E.; Chapman, R.; Holmlin, E.; Takayama, S.; Whitesides, G. Langmuir 2001, 17, 5605. (7) Welin-Klintstro, S.; Lestelius, M.; Liedberg, B.; Tengvall, P. Colloids Surf. B 1999, 15, 81. (8) Pitt, W.; Park, K.; Cooper, S. J. Colloid Interface Sci. 1986, 111, 343. (9) Balasubramanian, V.; Grusin, N.; Bucher, R.; Turitto, V.; Slack, S. J. Biomed. Mater Res. 1999, 44, 253. (10) Cacciafesta, P.; Humphris, A.; Jandt, K.; Miles, M. Langmuir 2000, 16, 8167.

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