Measurement of Time-Dependent Functional ... - ACS Publications

Downloaded by NORTH CAROLINA STATE UNIV on December 14, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch017

Chapter 17

Measurement of Time-Dependent Functional Activity of Adsorbed Fibrinogen and Platelet Adhesion on Material Surfaces Li-Chong Xu,1 Pranav Soman, Bryan M. Scheetz, and Christopher A. Siedlecki*,1,2 1Department

of Surgery and Biomedical Engineering Institute, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 2Department of Bioengineering Biomedical Engineering Institute, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 *E-mail: [email protected]. Phone: (717) 531-5716. Fax: (717) 531-4464

Fibrinogen adsorbed on material surfaces undergoes conformational changes and subsequently mediates platelet adhesion. Recent evidence has shown that the platelet binding epitope located in the γ-chain dodecapeptide plays an important role in platelet adhesion to biomaterials. In this chapter, we describe a series of studies using an immuno-AFM technique employing an antibody-modified probe for measuring the probability of this γ-chain dodecapeptide epitope (γ392-411) being available (a measure of protein functional activity) following fibrinogen adsorption to model material surfaces. AFM measurements show that the functional activity of fibrinogen appears to be both time and substrate dependent, with the surface characteristics and compositions of protein solution affecting the time dependence. The probability of antibody binding correlates well with temporal changes in platelet adhesion to these material surfaces, suggesting that the availability of the γ-chain in fibrinogen is a useful predictor of platelet adhesion. Results demonstrated that the utility of

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

this approach for measuring protein function at or near the molecular scale and offers new opportunities for improved insights into the molecular basis for the biological response to biomaterials.


1. Introduction With the widespread and remarkable successes of medical devices, biomaterials have had an enormous impact on health care (1). Much of this success has been achieved through judicious selection of existing materials, with little understanding of the fundamental molecular scale structure-function relationships in these materials. In the case of blood-contacting devices, blood–material interactions trigger a wide range of adverse biological responses including complement activation, blood coagulation, and thrombosis (2). Surface-induced thrombosis has particularly remained a main problem for the biocompatibility and eventual success of both implanted and peripheral medical devices. One important mechanism of surface-induced thrombosis involves the interaction of circulating blood platelets with plasma proteins adsorbed on a biomaterial surface, as protein adsorption is viewed as the initial event occurring when a biomaterial comes into contact with blood. While blood contains a multitude of proteins having a variety of biological functions and activities, fibrinogen has been identified as one of the most important proteins involved in platelet adhesion/activation and thrombus formation, particularly in areas of low shear stress (3). An improved understanding of adsorption and functional activity of fibrinogen on biomaterial surfaces is critically important for the development and application of new blood contacting biomaterials. Fibrinogen is the third most abundant blood plasma protein, and plays the dual roles of serving as a ligand for platelet adhesion to a surface and/or as a linker for platelet aggregation (4), in addition to its roles in coagulation. Circulating inactive platelets do not bind soluble fibrinogen in its native conformation, but readily adhere to adsorbed/activated fibrinogen through the platelet integrin receptor αIIbβ3 (GPIIb/IIIa) (5). There is increasing evidence that the conformational state and the availability of platelet-binding sites in adsorbed fibrinogen may actually be a more important indicator of platelet adhesion than simply the amount of adsorbed fibrinogen present at the surface (6–9). Fibrinogen is a symmetric molecule with a 2-fold axis of symmetry and each side of the molecule is composed of three pairs of intertwined polypeptide chains designated as Aα, Bβ, and γ. Each fibrinogen molecule possesses three pairs of potential platelet binding peptide sequences, two RGD-containing sequences in each of the Aα chains (RGDF and RGDS) and an epitope (HHLGGAKQAGDV) located at the C-terminus in each of the γ chains. The γ-chain dodecapeptide sequence, the structure of which has already been reported elsewhere (10, 11), is generally viewed as the primary ligand for platelet adhesion to adsorbed fibrinogen as mutations in this region have dramatic effects on platelet adhesion to surfaces (12, 13). Thus, the availability of dodecapeptide in γ-chain of fibrinogen molecule is defined as the functional activity of fibrinogen in platelet adhesion within this 374 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


chapter. Many investigators have reported that the functional activity is related to the conformation and/or the orientation of adsorbed fibrinogen (6, 14–16). Fibrinogen undergoes conformational changes upon adsorption to material surfaces and the transient exposure of functional epitopes appears to be both time and surface dependent (15, 17–19). Agnihotri and Siedlecki (19) measured the time-dependent changes in the structure of individual fibrinogen molecules under aqueous conditions following adsorption on hydrophobic graphite and hydrophilic mica surfaces, respectively. On the basis of differences in the relative height of the D and E domains, four orientation states were observed for fibrinogen adsorbed on these surfaces, and the conformational changes could be explained by a two-step spreading model where in the first step the protein spreads very rapidly after adsorption, and in the second step the spreading can be modeled as exponential decay of height resulting from interactions with the substrate. Material surface properties, including surface charge, roughness, wettability, and the specific chemical groups on the surface all appear to affect the conformational changes of fibrinogen upon adsorption on surfaces (20–24). However, these studies were often carried out on model substrates such as mica and gold, and the conformational change of fibrinogen response to biomedical related polymeric surfaces is less well understood, in part due to complicated surface chemistry and topography. The development of immunological atomic force microscopy (AFM) techniques makes it possible to measure the availability of functional epitopes in proteins adsorbed on surfaces regardless of material surface property. For example, we measured the adsorption and functional activity of fibrinogen adsorbed on segmented polyurethane biomaterial surfaces and found that fibrinogen activity was dependent on soft and hard segments chemistries, and hard segment content (25). These polyurethane biomaterials undergo reorientation and rearrangement during hydration, resulting in net hard segment enrichment at the surface. This dynamic phase restructuring of hard domains leads to a decrease in fibrinogen adsorption and functional activity, as well as subsequent platelet adhesion (26). The success in application of immuno-AFM technique makes it practical to measure the functional activity of proteins adsorbed on surface by looking at the molecular scale, and is helpful for understanding the biological responses at material surface.

2. Measurement of Functional Acticity of Fibrinogen by Immuno-Atomic Force Microscopy 2.1. Immuno-Atomic Force Microscopy AFM has been used extensively for studying biological molecules including proteins, lipid membranes, DNA and cells under physiologically relevant aqueous conditions with nanometer scale resolution, although generally on ultrasmooth model surfaces such as mica, high oriented pyrolytic graphite or self-assembled monolayers (23, 27–29). The low surface roughness of these model surfaces is ideal to characterize protein features including specific domain and conformational changes in the proteins upon adsorption. However, it is very difficult to distinguish 375 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


the proteins of interest from the complicated multi-proteins adsorption based on topography alone. More importantly, clinically used biomaterials have rough topographies and make detection/identification of specific proteins on these surfaces even more difficult. Although it has been reported that the fibrinogen on two clinically relevant biomaterials, poly(dimethylsiloxane) PDMS and low density polyethylene (LDPE) can be detected by phase imaging (30), the surface is often too rough to clearly distinguish the adsorbed proteins (31). There remains a need to develop new techniques that do not rely on topography to recognize specific proteins. An alternative non-conventional AFM technique, force spectroscopy, provides a unique method for detection of specific proteins without dependence on topography. Hinterdorfer et al. (32) used the methodology for the study of molecular recognition of single binding events and for the localization of sites on biosurfaces by combining force microscopy with molecular recognition by specific ligands. This method indicated the potential application for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes. We previously (33) used an AFM probe functionalized by covalently linking polyclonal antibodies against fibrinogen to detect fibrinogen in a dual-protein film. The patterned dual component protein films were formed by microcontact printing bovine serum albumin on a mica surface and then backfilling with fibrinogen. The adhesion mode was used to generate binary recognition images where the specific and non-specific interactions were differentiated based on a statistically derived cut-off value with a recognition efficiency >80%. On the basis of our experience with AFM protein recognition by polyclonal antibodies, we extended the force spectroscopy technique to study the protein activity and indirectly the putative conformational changes in protein upon adsorption. This approach was also similar to measurements of functional activity and conformational changes in proteins that were previously detected by monoclonal antibodies (mAb) against specific peptides of the protein after adsorption by enzyme-linked immunosorption assay or radiolabeling (34, 35). Balasubramanian et al. (15) investigated the time-dependent functional changes in fibrinogen adsorbed to polytetrafluoroethylene, polyethylene, and silicone surfaces using a mAb against the C-terminal dodecapeptide sequence 400-411 of the γ chain and confirmed that fibrinogen undergoes biologically significant conformational changes upon adsorption to polymeric biomaterials. Alternatively, the force measurement mode of AFM provides another approach to study the functional activity of protein at molecular scale by measuring antigen-antibody debonding forces with a mAb functionalized probe. Furthermore, probability of recognition of a specific epitope can be recorded as a function of time so that the time-dependent functional activity of adsorbed protein is revealed (16). 2.2. Functionalization of AFM Probe and Operation of AFM To recognize the specific epitopes in proteins adsorbed on surfaces, the AFM probe is functionalized with the appropriate monoclonal antibodies. The following protocol decribed is one of the methods for attachment of antibodies. Although it 376 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


uses a chemical fixation technique, the method provides sufficient mobility and flexibility for proteins to rotate and orient themselves for binding (36). Briefly, pyramidal Si3N4 AFM probes are cleaned in 10ml of acetone for 15 minutes and then glow-discharge plasma treated for 30 minutes at 100W power. The tips are modified by placing into 10 ml of 1% (v/v) aminopropyltriethoxysilane (APTES) in ethanol for 1 hr to provide a reactive amine group, and then rinsed 3 times using DI water. Tips are then reacted in 15 ml of 10% glutaraldehyde for 1 hr and washed 3 times using DI water to remove the glutaraldehyde. The reaction of glutaraldehyde with the amino silane on the nitride surface provides a bridging agent between the solid support and the antibodies. The activated probes are incubated for 1 hr with mAb (NYB4-2xl-f, Accurate Chemical, Westbury, NY) (25 μg/ml) that recognize fibrinogen γ392-411, a region that includes the platelet binding dodecapeptide sequence γ400-411. Modified tips are stored in PBS at 4°C and used within 2 days. 2.3. Measurement of Functional Activity of Proteins by Immuno-AFM and Platelet Adhesion Human platelet poor plasma (PPP) and purified proteins in PBS were used for studies. Proteins were adsorbed onto the material surface for 5 min in an external fluid cell, and remaining free protein was washed away 3 times using a syringe pump for 5 min each, while the residence time was recorded. AFM Si3N4 probes modified with mAb were used to detect the fibrinogen activity on surfaces. The techniques for fibrinogen activity measurements by AFM have been described in a previous publication (16). Briefly, the force volume mode of AFM was used to measure interaction forces between fibrinogen and the probes modified with mAbs across the area. Force curves were continuously acquired as 32×32 array of individual force curves cross the each scanning area at a certain scan rate. Thus, the amount of time required for completion of one recognition map could be determined. As controls for these measurements, material samples were incubated with bovine serum albumin (BSA) (1 mg/ml) for 5 min and used for measuring the nonspecific interactions of the antibody with proteins. To correlate the molecular scale protein functional activity to macroscale platelet adhesion, platelet adhesion was carried on material surfaces which were pre-adsorbed with proteins or PPP and then allowed to reside in PBS for the desired time periods. Human platelet rich plasma (PRP) was collected from either human blood donated by healthy volunteers in accordance with institutional policies or from the blood bank at the Hershey Medical Center. Fresh blood was collected in Vacutainer® blood collection tubes with EDTA, and no other agents were added to the blood. The blood was centrifuged at 180g and 25°C for 20 min to collect PRP and to remove the blood cells, followed by careful removal of the upper platelet-rich layer with a polyethylene pipette. PRP was centrifuged at 1500g for 10 min to separate platelets into pellet. The supernatant was collected as PPP. The platelet pellet was washed with PBS and gently re-suspended in PBS, and then diluted with PBS or PPP, recalcified with CaCl2 to yield a final platelet concentration of 2.0×108 platelets/ml and Ca2+ concentration of 2.5mM. Platelet adhesion was performed by incubation of materials in platelet solution for 10 min 377 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

under static condition. The platelet solution was aspirated and then the samples were rinsed with PBS for 5 times. Platelets adhered on surfaces were analyzed by fluorescence microscopy (37) or lactate dehydrogenase (LDH) assay (16), which have been described in previous publications.


2.4. AFM Data Analysis The individual retraction force curves were extracted and analyzed off-line with tools developed in Matlab software. The rupture force and rupture length were calculated from each retraction force curve. The rupture force was calculated from the distance between the zero deflection value to the point of maximum deflection during probe separation from the surface for each force curve using Hook’s law, Frupture = −k × dmax,, where k is the cantilever spring constant. The rupture length was calculated from the distance that the tip moves from the zero interaction force during separation to the position where the probe has separated and returned to zero deflection (Figure 1). Both rupture force and rupture length can be used to distinguish the specific and non-specific interactions.

Figure 1. A representative force curve of specific interactions showing the calculation of rupture force and rupture length.

The nonspecific forces were collected from the interactions of albumin and antibodies and pooled to produce a histogram of rupture force (or rupture length). Mean value (μ) and standard deviation (σ) were thus established. A 95% confidence interval limit determined as μ+1.96σ is used as a cut-off value for differentiating the specific and nonspecific interactions of mAb and proteins. Interactions above this limit were considered as specific interactions. Thus, the rupture force (length) map is converted into binary recognition map, where the warm colors indicate the recognition of a specific interaction and cool colors represent the nonspecific interactions (Figure 2). 378 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. (a) A representative rupture length map of interactions of mAb probe and fibrinogen adsorbed surface, and (b) binary recognition map after data were filtered through a cut-off value with 95% confidence. Interactions between protein and the mAb probe were acquired at varied scan rates as a 32×32 array of individual force curves. The time corresponding to each individual force measurement was determined from the scan rate for the particular image. The force data was bundled into groups of 32, corresponding to one scan line across the rupture force or length map. The probability of detecting a positive binding event was calculated as follows: Probability of recognition = number of specific binding events in each scan line /32. The time-dependent probability recognition data from multiple experiments (n≥3) were pooled together and presented as the mean of 5 time points.

3. Fibrinogen Adsorption and Platelet Adhesion on Model Mica Surfaces 3.1. Time-Dependent Functional Activity of Fibrinogen Adsorbed on Mica Surfaces Freshly cleaved muscovite mica was incubated with protein solutions of fibrinogen (100 μg/ml) or bovine serum albumin (BSA, 100μg/ml) for 5 min, and rupture forces were collected by a functionalized AFM probe having a mAb and as a function of residence time (16). Figure 3a shows the distribution of rupture forces between a mAb-modified AFM probe and bare mica substrates for one particular probe. Most of the rupture forces are found in the range of 400 to 700 pN with a maximum occurring at ~520 pN. Figure 3b shows force distributions between adsorbed BSA and the same mAb probe. There is a substantial shift in the force distribution, with most of the rupture forces in the range of 0 – 150 pN. The lack of overlap between force values in Figures 3a and 3b suggests that the probe encounters little bare mica during the measurements. The mean and standard deviation of this distribution were used to establish a cut - off value for specific/nonspecific interactions (150 pN for this particular probe) based on a 95 % confidence interval. Histograms of these rupture forces were in the range of 379 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


0 – 400 pN. Figure 3b and 3c show that there are sufficient differences between the distributions to be confident that the coupled antibody specifically recognizes its antigen. Data obtained spatially was converted to the appropriate time of measurement and probability of antigen recognition at that time was determined as described in section 2.4.

Figure 3. Representative distributions of maximum rupture forces between monoclonal anti-fibrinogen (mAb) modified probe and (a) bare mica (b) BSA adsorbed on mica (100μg/ml) (c) fibrinogen adsorbed on mica (100μg/ml). Scan Rate = 1 Hz. (Reproduced with permission from reference (16). Copyright 2008 American Chemical Society.)

Figure 4. Time-dependent changes in the probability of recognition between an AFM probe coupled with mAb and adsorbed fibrinogen (100μg/ml). (a) Measurement was initialed at 15 min and (b) measurement was initiated at 90 minutes fibrinogen residence time. The black line shows a running 5 point average to guide the eye. (Reproduced with permission from reference (16). Copyright 2008 American Chemical Society.) Figure 4(a) shows an example of a typical probability plot for antigen recognition measured against fibrinogen-adsorbed surfaces. Individual time points are shown by the data points and clearly demonstrate that the activity of fibrinogen is time dependent, with the maximum likelihood of recognition 380 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


occurring in the time range around 45 minutes and then decreasing at longer adsorption times. Due to the time required for setup of AFM experiments, the measurement of AFM is generally initialed at 10-15 min after protein adsorption on mica surface. One concern was that the long scanning time could affect the activity of the mAb on probe. Therefore, measurements were initialed at residence time of 90 min with a fresh mAb probe. The probabilities of recognition were still in the range of 0-0.18, similar to those in Figure 4(a) after 90 minutes. A comprehensive series of control experiments confirmed that the long time scanning of mAb probe has no significant effect on its activity and that changes are due to temporal effects.

Figure 5. The pooled probability data of antigen recognition showing the functional activity of fibrinogen adsorbed on mica surface. (Reproduced with permission from reference (16). Copyright 2008 American Chemical Society.)

Figure 5 shows pooled probability data from multiple experiments (n ≥ 6 for each time point) as a function of fibrinogen residence time on mica substrate. The probability of antibody-antigen recognition, indicating the functional activity of adsorbed fibrinogen, peaks at ~45 minutes post-adsorption and thereafter 381 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

decreases with increasing adsorption time. Although fibrinogen activity at 15, 25 and 35 minutes residence time is not statistically significant when compared to activity at 45 minutes, the recognition probability peak at ~45 minutes was consistently seen in all the experiments. Moreover, statistical analysis indicates that functional activity of adsorbed fibrinogen as measured by AFM force spectroscopy at the 45 minute time point is significantly greater than all time points ≥65 minutes (P

Measurement of Time-Dependent Functional ... - ACS Publications

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