Atomic Force Microscopy of Liposomes Bearing Fibrinogen

Bioconjugate Chem. 2003, 14, 593−600

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Atomic Force Microscopy of Liposomes Bearing Fibrinogen Elisenda Casals,† Albert Verdaguer,‡ Rau´l Tonda,§ Ana Galań,§ Gine´s Escolar,§ and Joan Estelrich*,† Departament de Fisicoquı´mica, Facultat de Farma`cia, Universitat de Barcelona, Barcelona, Spain, Unitat de Te`cniques Nanome`triques, Serveis Cientı´fico-Te`cnics, Universitat de Barcelona, Barcelona, Spain, and Servei d’Hemotera`pia i Hemosta`sia, Hospital Clıńic, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain . Received November 18, 2002; Revised Manuscript Received February 4, 2003

Extruded liposomes formed from dipalmitoylphosphatidylcholine and cholesterol, with and without fibrinogen, were examined by atomic force microscopy (AFM). The sequence of events involved in the transition from attached liposomes to bilayer patches on mica supports was viewed by tapping mode in liquid. After adhesion to the mica surface, both liposomes without fibrinogen and liposomes with attached fibrinogen collapsed into patches. The fibrinogen layer attached to the liposomes was 2.6 nm thick. This implied that the protein was spread over the entire liposome and the protein characteristic trinodular structure disappeared. To check the type of bond between fibrinogen and liposome, sequential images were taken after the incubation of fibrinogen with liposomes with and without a chemical group for attaching the protein. The results clearly confirmed that fibrinogen bound covalently to liposomes.

INTRODUCTION

Liposomes are lipid structures used as a model of biological membranes. They are also part of a drugdelivery system of well-defined physicochemical characteristics (1). The promotion by synthetic phospholipids of procoagulant activity in damaged vessels (2) and the fact that acidic phospholipids contribute to prothrombin activation (3, 4) have opened up new possibilities for the therapeutic use of liposomes. Liposomes can also be used as platelet substitutes in coagulant therapy. Along these lines, some authors have prepared liposomes to carry the domain of the platelet glycoprotein IbR that binds the von Willebrand factor (5, 6). Fibrinogen has a central function in the regulation of hemostasis and thrombosis. It coagulates blood and facilitates adhesion and aggregation of platelets (7). Fibrinogen is a 340 kDa dimeric molecule consisting of two sets of three intertwined polypeptide chains held together by 29 disulfide bonds, three of which connect the N-termini of the dimer subunits in an antiparallel arrangement (7-9). In thrombus formation on thromobogenic surfaces, platelet adhesion is successfully achieved by the surface-bound fibrinogen, while the dimeric structure of fibrinogen enables platelet-platelet bridging, which leads to macroscopic platelet aggregation (10). Thus, the binding of fibrinogen to liposomes favors the further adsorption of fibrinogen on a thrombogenic surface. When mixed with normal platelets, liposomes enhance platelet function. Therefore, when transfused in vivo, these liposomes are expected to adhere to suben* Corresponding author: Dr. Joan Estelrich, Departament de Fisicoquı´mica, Facultat de Farma`cia, Universitat de Barcelona, Avda. Joan XXIII s/n; 08028 Barcelona (Catalonia, Spain). Tel: + 34-93-4024559; fax: + 34-93-4035987; e-mail estelric@ farmacia.far.ub.es. † Departament de Fisicoquı´mica, Facultat de Farma ` cia. ‡ Unitat de Te ` cniques Nanome`triques, Serveis Cientı´ficoTe`cnics. § Servei d’Hemotera ` pia i Hemosta`sia, Hospital Clıńic, Facultat de Medicina.

dothelial matrix and to participate in the formation of platelet thrombi at the site of vascular injury, supporting hemostasis in thrombocytopenic patients. To check this hypothesis, we bound fibrinogen to neutral liposomes covalently. To investigate the location and structure of fibrinogen on the liposomes, we employed atomic force microscopy (AFM1). This technique has been successfully employed to study the dynamic change in surface topography during the adhesion of proteins (11) and polymers (12) to a solid surface. The development of AFM has allowed direct in situ examination of many biological materials, including phospholipid membranes (13). AFM images surfaces at very high resolution in aqueous environments. Liposomes are a great challenge for current AFM technology, since they are fluid and dynamic systems (14-20). MATERIALS AND METHODS

Materials. DL-R-Dipalmitoylphosphatidylcholine(DPPC), cholesterol (CHOL), and type-I fibrinogen (from human plasma) came from Sigma (St. Louis, MO); N-succinimidyl-S-acetylthioacetate (SATA) and Excellulose GF-5 desalting column were from Pierce (Rockford, IL); egg phosphatidylethanolamine (PE) and 1,1-dipalmitoyl-snglycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (N-MPB-PE) were from Avanti Polar Lipids (Alabaster, AL). The buffer used was 50 mM Hepes, pH 7.5 (with and without 1 mM EDTA for liposomes with and without fibrinogen). Other reagents used were of analytical grade. Water was distilled twice and deionized (Millipore, Molsheim, France). Preparation of Thiolated Fibrinogen. The procedure for thiolating proteins with SATA is similar to that described by Heeremans et al. (21). Fibrinogen was 1 Abbreviations: AFM, atomic force microscopy; CHOL, cholesterol; DPPC, DL-R-dipalmitoylphosphatidylcholine; EDTA, ethylenediaminetetraacetic acid; N-MPB-PE, 1,1-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]; PE, phosphatidylethanolamine; SATA, N-succinimidylS-acetylthioacetate.

10.1021/bc025641t CCC: $25.00 © 2003 American Chemical Society Published on Web 04/18/2003

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dissolved in saline solution (145 mM NaCl) at 60 µM. Immediately before reaction, about 14 mg of SATA was dissolved in 1.0 mL of dimethyl sulfoxide. One milliliter of the fibrinogen solution was combined with 10 µL of the SATA solution in a test tube. This corresponded to a SATA:fibrinogen molar ratio of 10. After mixing, the resulting solution was left to react at room temperature for 30 min. Fibrinogen was purified by size-exclusion chromatography by means of Excellulose GF-5 equilibrated with Hepes/EDTA buffer. The presence of fibrinogen in the eluates was checked by monitoring their absorbance at 278 nm. Fractions containing the protein were pooled. Acetylthioacetyl-protein (1 mL) was deacetylated by adding 100 µL of a freshly prepared 0.5 M hydroxylamine‚HCl solution containing 50 mM Hepes and 25 mM EDTA, pH 7.5. The incubation lasted for 2 h at room temperature. Then, thiolated protein was purified again by size-exclusion chromatography. The number of introduced thiol groups was assayed according to Ellman, using cysteine for the calibration curve (22). In this assay, the blank consisted of all components of the sample itself, the only difference being that, instead of thiolated fibrinogen, the same concentration of nonthiolated protein was used. According to this assay, the number of introduced thiol groups (expressed as mol SH per mol fibrinogen) was 1.6 ( 0.1. Liposome Preparation. Phospholipids (DPPC:CHOL: N-MPB-PE and DPPC:CHOL:PE at the molar ratio 50: 20:30) were dissolved in chloroform in a round-bottomed flask and dried in a rotary evaporator under reduced pressure at 50 °C to form a thin film on the flask. The film was hydrated with Hepes buffer to give a concentration of 20 mM. Multilamellar liposomes were formed by constant vortexing for 4 min on a vortex mixer and sonication in a bath for 4 min. Multilamellar liposomes were downsized to form uni- or oligolamellar vesicles by extrusion through polycarbonate membranes of a nominal size of 200 nm (Poretics, Livermore, CA) at 50 °C in an extruder device (LiposoFast, Avestin, Ottawa, Canada). Coupling of Thiolated Fibrinogen to Liposomes. A volume of liposome suspension (DPPC:CHOL:N-MPBPE) was combined with an equal volume of fibrinogen solution. The resulting suspension was left to react overnight at room temperature, while being shaken at 1000 rpm. The coupling reaction was stopped by adding 50 µL of N-ethylmaleimide (8 mM in Hepes buffer), and the liposomes were separated from free protein by two centrifugation steps in a Centrikon T-1170 (Kontron Instruments, Milan, Italy) for 40 min at 100 000 × g and at 7 °C. Liposome Characterization. Phospholipid content was based on the phosphorus content (23), the fibrinogen concentration was determined by the Bradford’s method (24) after liposome disruption in sodium dodecyl sulfate (10%), the vesicle size distribution was determined by photon correlation spectroscopy with an Autosizer IIc spectrometer (Malvern Instruments, Malvern, UK), and surface charge was determined as ζ potential by Doppler microelectrophoresis in a Zetasizer 4 (Malvern Instruments, Malvern, UK) using the Henry correction of Smoluchowski’s equation (25). Substrate Preparation and AFM Observation. A Digital Instruments Nanoscope IIIa AFM (Santa Barbara, CA) with a 12 µm scanner (E-scanner) and Tapping Mode liquid cell was employed. Before use, the liquid cell was cleaned with ultrapure water, then with ethanol, and was rinsed again with ultrapure water. A small square (≈ 5 × 5 mm) of muscovite mica was glued with waterinsoluble glue to a Teflon disk with a supporting steel

Casals et al.

disk, and 120-µm-long V-shaped cantilevers from Olympus Ltd. (Tokyo, Japan) with Si3N4 tips and a nominal force constant k ) 0.1 N‚m-1, which minimized force during scans, were used. The scanning line speed was optimized to 2 Hz. Height and amplitude of the images of 256 × 256 pixels were taken simultaneously. Height images were flattened and plane adjusted. All measurements were taken on the height images, but breadth images were also shown for easier viewing of the processes. Before the adsorption to freshly cleaved mica, the samples were diluted to a lipid concentration of 0.1 mM in buffer solution. To achieve a better interaction between mica and liposomes, 50 µL of 0.5 M NaCl was deposited on a mica surface. After 10 min, the surface was rinsed gently with Hepes buffer. Then, 40-50 µL of liposome sample was added. After 10-15 min of incubation, the surface was rinsed gently with Hepes buffer at room temperature, and the samples in buffer solution were examined directly. Heights of the images were determined by cross-sectional analysis of a software zoom of the image, and the results are expressed as mean ( standard deviation, n being the number of liposomes measured. RESULTS AND DISCUSSION

Liposome Characterization. The incubation ratio of SATA/fibrinogen of 10 (molar ratio) resulted in the introduction of 1.5-1.7 mol of thiol groups per mole of fibrinogen. For the different fibrinogen-liposome batches tested in this study, 33 to 53 µg of fibrinogen per µmole of phospholipid were coupled, which corresponded with about 34-55 protein molecules per liposome, assuming unilamellarity, uniform particle size, spherical particle shape, and a phospholipid molecular area of 0.70 nm2. Photon correlation spectroscopy found that liposomes were monodisperse and monomodal in their distribution. Their average diameter was 200 ( 5 nm for liposomes lacking protein, and 250 ( 20 nm after the coupling procedure. As the average end-to-end length of fibrinogen is about 46 nm (26-28), the increase of vesicle diameter is coherent with the presence of the protein molecule on the liposomal surface. This effect has been described by other authors (29). The polydispersity index, which ranges from 0.0 for an entirely monodisperse sample to 1.0 for a highly polydisperse sample, was always less than 0.2. In this way, despite the presence of more than one thiol group per protein molecule, and that theoretically a multivalent interaction could exist, the results of vesicle size and polydispersity seem to rule out the possibility that fibrinogen had cross-linked more than one liposome. When the electrophoretic mobility of liposomes was determined by Doppler microelectrophoresis, an average value of -18 mV was obtained for the composition DPPC: CHOL:PE. This figure corroborates previous values (30) for liposomes bearing the same percentage of the zwitterionic PE. However, in liposomes with the PE conjugated to the maleimide group (DPPC:CHOL:N-MPB-PE), the average ζ potential is close to -1 mV. In fibrinogenbearing liposomes, the ζ potential rose to -60 mV. This was due to negative net charge on the protein at pH 7.5, since the isoelectric point of fibrinogen is 4.9. The ζ potential of the mica surface was determined by means of the plane interface technique (31), in which the velocity profile of a standard particle is extrapolated. On the basis of the electro-osmotic flow to the cell wall of the mica plate, this technique gave highly negative values. As the electrostatic interaction between liposomes

Atomic Force Microscopy of Liposomes Bearing Fibrinogen

Bioconjugate Chem., Vol. 14, No. 3, 2003 595

Figure 1. Amplitude tapping mode images in liquid of a mica surface after exposure to liposomes formed by DPPC:CHOL:PE (50:30:20, molar ratio) at 0.1 mM lipid concentration and bearing fibrinogen (30% of attachment sites, molar ratio). The images show flattened liposomes and liposomes with a second layer of rough surface. The cross-section of the areas identified by the line (measured from the height images) shows that the height of the flattened liposome is 5.9 nm, and of the liposome for the rough surface, 8.4 nm. The difference in height is due to the network that fibrinogen forms on the liposome surface.

Figure 2. Images of height (left) and width (right) of liposomes formed by DPPC:CHOL:PE (50:30:20, molar ratio). The top images are 5 × 5 µm, and the bottom ones of a more detailed image of 1 × 1 µm. After incubation with 5 µL of 1.5 µM fibrinogen and further rinsing with Hepes buffer, the liposome surface is clean, whereas the mica surface is covered by fibrinogen. The stronger interaction between the mica and the fibrinogen avoids the sweeping of the protein.

and the mica surface is an important factor in determining the adhesion rate of liposomes to a mica surface, the interaction between two negative entities did not lead to adhesion. The incubation of the mica surface with NaCl reduces or even overcompensates the negative ζ potential.

Therefore, in electrostatic terms, it can be assumed that mica treated with 0.5 M NaCl will adhere well to liposomes. AFM Imaging of Liposomes. When liposomes not bearing fibrinogen were deposited on mica, the AFM


Casals et al.

Figure 3. Images of height (left) and width (right) of liposomes formed by DPPC:CHOL:MPB-PE (50:30:20, molar ratio). The top images are 5 × 5 µm, and the bottom ones of a more detailed image of 1 × 1 µm. After incubation with 5 µL of 1.5 µM fibrinogen and further rinsing with Hepes buffer, the liposome surface is covered by fibrinogen in a way similar to the mica surface. The attachment of fibrinogen to the liposome is now strong enough to keep the protein bound to the vesicles.

imaging showed that larger areas of the surface were covered by liposomes (image not shown). However, only a few of these liposomes were attached intact to the surface or partly flattened. Most of them were completely flattened, forming layers of two different heights. The value of the higher layer was 12.0 ( 1.6 nm (n ) 5). This may correspond to a double-bilayered structure. The lower layer, at 5.9 ( 0.7 nm (n ) 5), is characteristic of a single bilayer. Sequential imaging (not shown) had the following steps: (1) adsorption to surface, (2) flattening and spreading; and (3) fusing and spreading of bilayers. More explicitly, the initially adsorbed liposomes seemed to collapse from the outer periphery toward the center of the liposome. The process continued until the whole liposome had produced two bilayers stacked one on top of the other. At this point the double-bilayer disk abruptly fused with already formed bilayer patches, and its thickness changed to that of a single bilayer. The absence of any elastic or restorative forces in this process indicates two main features. First, the fused bilayer fragments slide over each other in the aqueous layer that separates them, and, second, the relatively high energetic cost of exposing the hydrophobic inner membrane precludes the sliding of the tails over each other. This is the same pattern of liposome collapse as is described in the literature (18). When the different stages of collapse of liposomes bearing fibrinogen were observed under the same conditions as those described above, the same smooth layer,

5.9 ( 0.8 nm thick (n ) 5), that was observed in liposomes lacking fibrinogen was also appreciated (Figure 1). Sometimes an additional layer, with a rougher aspect, 8.5 ( 0.4 nm thick (n ) 5), was found. The fibrinogen molecules may form a network over the whole surface, in which case the characteristic trinodular structure disappears. According to the model of the transformation of the liposomes in a planar bilayer proposed by Jass et al. (18) the transition from a double bilayer to a single bilayer involves the movement of the upper bilayer from the lower one to unoccupied areas of the surface. This movement can occur by either sliding or rolling, leading in both cases to a single bilayered structure. As a consequence of such processes, much of the face viewed by AFM is the internal layer of the liposomes. Moreover, in the presence of fibrinogen, the strong interaction between the mica and the protein increases the proportion of the inner layer that can be seen. Therefore, the smooth bilayers displayed in Figures 1 and 2 correspond to the internal layer of the liposome, as the external bilayer is in contact with the mica. Occasionally, some fibrinogen fibers could be observed just at the edges of the lipid layers (not shown in the figures). As stated above, the height of the bilayer observed is, as a general rule, on average the same (5.9 nm), irrespective of the presence of fibrinogen. This implies that when fibrinogen is present, it is in contact with the mica and must be extremely compressed. In a few such cases, a rough layer



Figure 4. Series of height (left) and width (right) of 2 × 2 µm images in liquid taken to perform a kinetic experiment. After flattening DPPC:CHOL:MPB-PE liposomes at 0.1 mM lipid concentration on the mica surface, 1.5 µM fibrinogen was injected directly until its presence on the mica surface was clear. This occurred at various times after fibrinogen incubation. (A) Image showing liposomes before the injection of fibrinogen, (db): double bilayer, (m): mica. (B) First stages of the deposition of fibrinogen on the mica surface (m). (C) In this image some changes can be appreciated on the lipid bilayers: apparition of a new smooth layer (sl) spread principally on double bilayers. (D) Most of the single bilayer is still unaltered, but the new smooth layer (sl) can be appreciated on some parts of the single bilayer. Mica (m) and double bilayers are now completely covered.

of 8.5 nm was observed, which should correspond to the external face of the liposome covered by fibrinogen. Thus, the layer of fibrinogen attached to the liposome was 2.6 nm thick. The value of the additional thickness found in the indicated liposomes confirms that the height of fibrinogen alone deposited on mica varies from 2.3 to 2.5 nm (31). As indicated above, the molecular length of fibrinogen is about 46 nm, and when the fibrinogen is in solution (aqueous buffers are good solvents for proteins),

the protein chains extend into the solution and gain conformational entropy. Under such conditions, the high degree of freedom of the protein molecule is favored by the covalent bond between liposome and protein. This can explain the difference of 50 nm between liposomes bearing fibrinogen and liposomes without it found by photon correlation spectroscopy. Macroscopic and microscopic studies have shown that fibrinogen binds strongly and in high amount to most


Casals et al.

Figure 5. The same kind of images as those in Figure 4, but now using liposomes without a specific binding-point for fibrinogen. (A) image showing some flattened liposomes (db) at the first stages of deposition of fibrinogen on mica surface (m). (B) After 15 min, the percentage of protein on mica has increased. (C) The mica surface is steadily covered by fibrinogen. No changes on the liposomal surface (b) can be appreciated.

surfaces (32). Ta et al. (33) have proposed a model for fibrinogen adsorption both on hydrophobic and hydrophilic surfaces. At the mica-buffer interface, fibrinogen forms a monolayer, and it lies flatter than on a hydrophobic surface. The external side of a liposome bilayer has hydrophilic properties, and, therefore, one can expect a similar liposome-fibrinogen interaction. Moreover, the flattening effect of the tip must be also considered. At the moment, findings have shown only that fibrinogen is present on liposomes, but this does not ensure that the protein binds covalently to the liposome, since fibrinogen could be attached to the liposome by adsorption or by a hydrogen bridge. To determine how the protein is bound, two kinds of liposomes were prepared: liposomes composed of DPPC:CHOL:N-MPB-PE (50:20: 30, molar ratio), i.e., liposomes with the point of attachment free, and liposomes composed of DPPC:CHOL:PE (50:20:30, molar ratio), which had no specific place for attaching the fibrinogen. Liposomes were deposited on

the mica. The tip imaging was left until most of the liposomes were completely flattened on the surface. At that point, 5 µL of 1.5 µM fibrinogen solution was added to the liposomal sample. After 10 min of incubation, the sample was rinsed with Hepes buffer. For reference images, the same procedure was applied to freshly cleaved mica to which only fibrinogen was added. Figure 2 shows that the flat surface of liposomes without MPB is clean, without fibrinogen, whereas the mica is fully covered by the protein. However, the surface of liposomes bearing MPB was also covered by the fibrinogen, just like the mica surface (Figure 3). In the latter case, multiple rinses of the surface were unable to clean the liposome surfaces. This confirms that fibrinogen is strongly attached to the liposome, since the washing procedure should have removed loosely bound material. To delve further into the process of fibrinogen binding to liposomes, an in situ kinetic experiment was performed. The liposomes bearing the group MPB adhered to the mica


surface. Images were taken periodically, and, after the flattening of liposomes, an area was chosen where the mica surface and the lipid bilayer could be examined in the same image. The tip scan was then left in the same position all the time, and images were taken. Fibrinogen (1.5 µM) in buffer was injected directly into the liquid cell. The process was repeated until the first molecules of fibrinogen became attached to the mica surface. At this moment, the injections were stopped, and the system was left to evolve. In Figure 4, this evolution can be followed in four different images. To show clearly all the changes on the mica surface, images of height and width are shown together. Figure 4A shows the liposome before the injection of fibrinogen. On the mica surface (m) there is a layer of about 6 nm, which may correspond to a lipid bilayer (b); and, in some places, layers with a height of 12 nm, which may logically correspond to a double bilayer (db) structure (see arrows). After 10 min of incubation, the first steps of the deposition can be observed. The existence of roughness on the mica is due to the attachment of the first molecules of fibrinogen (Figure 4B). After 15 min, some changes can be appreciated in the lipid bilayers (Figure 4C). In the places that exhibited a double bilayer, a new smooth layer (sl) spread over the surface. Similar new layers were found in those zones where only a single bilayer appeared before. The rest of the surface did not show any change. In the last image (time elapsed 25 min, Figure 4D) we can see that most of the liposome surface has still not changed. However, mica and the liposomal zones, which had been covered previously by fibrinogen, are now completely covered by a new layer. The liposomes have continued their evolution, and the edges of the single and double lipid bilayers have changed in comparison with the first image. The height of the new layer measured 7.4 ( 0.8 (n ) 5) when it corresponded to the height difference between the mica and the lipid bilayer/fibrinogen layer. Note that this value had to be carefully borne in mind because of the difficulties involved in seeing a zone with clean mica. The direct measurement of the fibrinogen layer from the lipid bilayer gave a height of 2.8 ( 0.4 nm (n ) 4). The difference of height between this second bilayer and the first bilayer was 8.6 ( 0.4 nm. The same in situ experiment was performed with liposomes without a specific site for attaching the fibrinogen. In Figure 5, a surface of mica (m) partially covered by a layer of liposomes can be seen (b). Image 5A was taken 10 min after the injection of the fibrinogen, and it was already possible to see some individual molecules of fibrinogen on the surface. Five minutes later, the number of protein molecules attached to the mica had increased (Figure 5B), and eventually, 20 min after the injection, the entire mica surface was covered by fibrinogen (Figure 5C). However, on the liposome surface no change can be appreciated, even on the small double bilayer that appears at the top of the image. It seems logical to suppose that fibrinogen has been swept off by the tip. CONCLUSIONS

AFM was used for in situ viewing of the dynamic morphological changes in liposome adhesion to a mica surface. The structure observed is strongly dependent upon the interaction between the liposomes and the mica surface. The AFM images display a spontaneous deformation from a spherical liposome to a flat bilayer. Liposomes without fibrinogen mainly formed two layers, of the thickness of one and two bilayers. In some liposomes with fibrinogen, an additional layer appeared. This was due to the fibrinogen spread on the surface.


Successful imaging of the covalent presence of fibrinogen in liposomes illustrates the potential of AFM for determining the degree to which macromolecules located on the liposome surface are natural or synthetic. ACKNOWLEDGMENT

This research was supported by funds from the FIS 99-0110 (Spain) and FEDER 2FD97-0778. LITERATURE CITED (1) Lasic, D. D. (1993) Liposomes: From Physics to Applications, Elsevier, Amsterdam. (2) Galań, A. M., Hernańdez, M. R., Bozzo, J., Reverter, J. C., Estelrich, J., Roy, M. T., Mazzara, R., Ordinas, A., and Escolar, G.(1998) Preparations of synthetic phospholipids promote procoagulant activity on damaged vessels: studies under flow conditions. Transfusion 38, 1004-1010. (3) Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution. Biochemistry 35, 7482-7491. (4) Koppaka, V., and Lentz, B. R. (1996) Binding of bovine factor Va to phosphatidylcholine membranes. Biophys. J. 70, 29302937. (5) Kitaguchi, T., Murata, M., Iijima, K., Kamide, K., Imagawa, T., and Ikeda, Y. (1999) Characterization of liposomes carrying von Willebrand factor-binding domain of platelet glycoprotein IbR: A potential substitute for platelets transfusion. Biochem. Biophys. Res. Commun. 261, 784-789. (6) Nishiya, T., Murata, M., Handa, M., and Ikeda, Y. (2000) Targeting of liposomes carrying recombinant fragments of platelet membrane glycoprotein IbR to immobilized von Willebrand factor under flow conditions. Biochem. Biophys. Res. Commun. 270, 755-760. (7) Hantgan, R. R., Francis, C. W., and Marder, V. J. (1984) Fibrinogen structure and physiology. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed. (Colman, R. W., Hirsch, J., Marder, V. J., and Salzman, E. W., Eds.) pp 277-300, J. B. Lippincott, Philadelphia. (8) Hoeprich, P. D., and Doolittle, R. F. (1983) Dimeric half molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation. Biochemistry 22, 20492055. (9) Gårlund, B., Hessel, B., and Marguerie, G. (1977) Primary structure of human fibrinogen: characterizations of disulfidecontaining cyanogen-bromide fragments. Eur. J. Biochem. 77, 595-610. (10) Savage, B., Saldivar, E., and Ruggeri, Z. M. (1996) Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 84, 289-297. (11) Drake, B., Prater, C. B., Weisenhorn, A. L., Gould, S. A. C., Albrecht, T. R., Quate, C. F., Cannell, D. S., Hansma, H. G., and Hansma, P. K. (1989) Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243, 1586-1589. (12) Stipp, S. L. S. (1996) In-situ, real-time observations of the adsorption and self-assembly of macromolecules from aqueous-solution onto an untreated, natural surface. Langmuir 12, 1884-1891. (13) Shao, Z., and Yang, J. (1995) Progress in high-resolution atomic force microscopy in biology. Quart. Rev. Biophys. 28, 195-251. (14) Iwamoto, H., and Wakayama, N. (1997) Structural studies on phosphatidylcholine liposomes in various phases using atomic force microscopy. Jpn. J. Appl. Phys. 36, 3913-3916. (15) Egawa, H., and Furusawa, K. (1999) Liposome adhesion on mica surface studied by atomic force microscopy. Langmuir 15, 1660-1666. (16) Thomson, N. H., Collin, I., Davies, M. C., Palin, K., Parkins, D., Roberts, C. J., Tendler, S. J. B., and Williams, P. M. (2000) Atomic force microscopy of cationic liposomes. Langmuir 16, 4813-4818. (17) Sideratou, Z., Tsiourvas, D., Paleos, C., Tsortos, A., Pyrpassopoulos, S., and Nounesis, G. (2002) Interaction of phosphatidyl choline based liposomes functionalized at the

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Atomic Force Microscopy of Liposomes Bearing Fibrinogen

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