Journal of Biomaterials and Nanobiotechnology, 2012, 3, 487-498
http://dx.doi.org/10.4236/jbnb.2012.324050 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)
487
Effects of Plasma Proteins on Staphylococcus epidermidis
RP62A Adhesion and Interaction with Platelets on
Polyurethane Biomaterial Surfaces
Li-Chong Xu1*, Christopher A. Siedlecki1,2
1Department of Surgery, Biomedical Engineering Institute, College of Medicine, The Pennsylvania State University, Hershey, USA;
2Department of Bioengineering, Biomedical Engineering Institute, College of Medicine, The Pennsylvania State University, Hershey, USA.
Received August 11th, 2012; revised September 13th, 2012; accepted September 24th, 2012
ABSTRACT
Plasma proteins influence the initial adhesion of bacteria to biomaterials as well as interactions between bacteria and
blood platelets on blood-contacting medical devices. In this paper, we study the effects of three human plasma proteins,
albumin, fibrinogen (Fg), and fibronectin (Fn), on the adhesion of Staphylococcus epidemidis RP62A to polyurethane
biomaterial surfaces, and also address how these three proteins affect bacterial interactions with human platelets on ma-
terials. Measurements of bacterial adhesion on polymer surfaces pre-adsorbed with a variety of proteins demonstrate
that Fn leads to increased bacterial adhesion, with the order of effectiveness being Fn Fg > albumin. Immuno-AFM
(atomic force microscopy) was used to assess the Fn adsorption/activity on surfaces and bacterial cell membranes by
looking at molecular scale events. A correlation between molecular scale Fn adsorption and macroscale bacterial adhe-
sion was observed, with an increased numbers of Fn-receptor recognition events measured on cell surfaces as compared
to Fg-receptor recognition events, suggesting Fn is an important protein in bacterial adhesion. Monoclonal antibodies
recognizing either the carboxyl-terminus or amino-terminus of Fn were coupled to AFM probes and used to assess the
orientation of Fn adsorbed on a surface, with an increased amount of Fn carboxyl-terminus availability corresponding to
higher bacterial adhesion. Interactions between bacteria and platelets were demonstrated with fluorescence and AFM
imaging on the polyurethane surfaces, with albumin inhibiting bacteria-platelet interaction and platelet activation, and
both Fg and Fn promoting adhesion of bacteria to platelets and apparent platelet activation, resulting in bacteria/platelet
aggregation.

Keywords: Bacterial Adhesion; Staphylococcus epidermidis; Fibronectin; Bacteria-Platelet Interactions
1. Introduction
Bacterial adhesion to biomaterials causing microbial in-
fection and poor tissue integration is one of the main
problems associated with the use of blood-contacting
devices. Bacteria adhere to a material surface, where they
proliferate and colonize to form biofilms on implanted
devices, eventually leading to a biomaterial associated
infection. These infections are extremely difficult to treat
by use of antibiotics alone due to the formation of
biofilm, which consists of a microbial community en-
trapped within a polymer matrix secreted by the adherent
microbes, and serves to protect the community from an-
timicrobial agents [1,2]. The increase in antibiotic resis-
tance of bacterium has also contributed to the increase in
infections that are refractory to treatment [3,4]. Thus,
surgical removal and replacement of the implanted de-
vices is often the only treatment, causing significant mor-
bidity and mortality [5,6].
Coagulase-negative staphylococci, particularly Staphy-
lococcus epidermidis, rank first among the causative
agent of nosocomial infections and represent the most
common source of infections associated with the use of
implanted medical devices such as intravascular and
peritoneal dialysis catheters, prosthetic heart valves or
orthopedic prostheses [7]. The defined virulence associ-
ated with S. epidermidis is its ability to colonize and
form biofilm on biomaterials [8]. Pathogenic bacteria
associated with biomaterial-centered infections have the
potential to enter the human circulatory system where
they can interact with platelets, resulting in platelet acti-
vation/aggregation. For example, S. aureus, another im-
portant staphylococcal bacterium found in nosocomial
infection, leads to platelet activation/aggregation [9-12]
and subsequently leads to abnormal blood function such
*Corresponding author.

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
488
as blood coagulation and thrombosis [13,14]. While in-
teractions of S. aureus and platelets have been inten-
sively studied, less information is available on S. epider-
mids. Further knowledge of the interactions between S.
epidermidis and blood contact devices as well as the in-
teractions between bacteria and platelets is crucial in
developing effective strategies for preventing biomate-
rial-centered infection and its subsequent complications.
Bacterial adhesion is the critical step in the pathogene-
sis of biomaterial associated infection. Biomaterial sur-
face chemistry characteristics have been shown to influ-
ence the initial adhesion and aggregation of S. epider-
midis on biomaterials [15]. However, when a biomaterial
is implanted in contact with blood, plasma proteins can
rapidly adsorb onto the material surface to form a “con-
ditioning film”. This adsorbed protein layer may mini-
mize the effect of biomaterial surface properties on bac-
terial adhesion [16,17] so that interactions between the
bacteria and the proteins mediate bacterial adhesion. In
vitro studies have shown that the presence of serum pro-
teins generally suppresses initial bacterial adhesion due
to the lack of a specific interaction between albumin and
bacteria [18,19] while the effect of plasma proteins fi-
brinogen (Fg) and fibronectin (Fn) on bacterial adhesion
is inconclusive. It has been reported that Fg or Fn coated
substrata enhanced the adhesion of S. epidermidis [20-22]
while there were reports also reporting that both proteins
inhibited or had no effect on bacterial adhesion [23-25].
The increase in bacterial adhesion related to adsorbed Fg
or Fn is regarded to be due to specific ligand/receptor
events between plasma proteins and bacterial cell surface
proteins known as the microbial surface components re-
cognizing adhesive matrix molecules (MSCRAMM)
[26-29]. Multiple MSCRAMM have been found on S.
epidermidis surface to promote adhesion of bacteria.
These molecules include proteins such as SdrG [20,30],
SdrF [29,31], and Embp [28,32], which were identified
to bind Fg, collagen, and Fn, respectively. It has also
been demonstrated that SdrG promotes platelet adhe-
sion/activation and aggregation [33].
Microscopy analysis and quantification of adherent
bacteria are generally used to evaluate bacterial adhesion
on material surfaces. This approach reveals cellular and
macroscopic scale phenomena of bacterial adhesion un-
der a variety of conditions and provides useful informa-
tion on the relationships between bacterial adhesion and
various experimental parameters. However, the direct
measurement of interactions between material surfaces,
plasma proteins, and the bacterial cell surface at the mo-
lecular scale is particularly important for understanding
the mechanisms of bacterial adhesion and pathogenic
infection. Atomic force microscopy (AFM) is a powerful
tool in studying bacterial adhesion, not only for imaging
of bacterial cells under physiological conditions, but also
for probing the nano-Newton (or less) interaction forces
between bacteria and various substratum surfaces or bio-
logical molecules [34]. Méndez-Vilas et al. [35,36] char-
acterized the surfaces of slime covered S. epidermidis
and the nano-mechanical properties of cell walls, show-
ing the importance of cell surface properties to adhesion.
Successful coating of bacteria on the AFM probe made it
possible to directly measure the molecular interaction
forces between bacterium and surfaces, indicating the
probability of adhesion. Liu et al. [37] measured the ad-
hesion forces between S. epidermidis and self-assembled
monolayers surfaces in the presence of proteins and
found that molecular adhesion forces between bacteria
and Fn were much greater than the forces between bacte-
ria and fetal bovine serum. Other investigators [38]
measured the time-dependent bacterial adhesion forces of
S. epidermidis to hydrophilic and hydrophobic surfaces
using a similar approach and found different bond-
strengths for staphylococcal adhesion to surfaces with
different wettability.
In this paper we studied the effects of plasma proteins
on adhesion of S. epidermidis as well as bacteria interact-
tions with platelets on microphase-separated polyure-
thane (PU) biomaterial surfaces, an important material
used for blood-contacting devices for over 30 years.
Three plasma proteins (albumin, Fg, and Fn) were pre-
adsorbed on PU surfaces and bacterial adhesion was
measured. AFM was used to detect the molecular-scale
Fn adsorption/orientation and to measure the interaction
forces between proteins and bacterial cell surfaces to
reveal the role of protein in bacterial adhesion. The cor-
relation between molecular scale results and macroscale
bacterial adhesion yields important information for un-
derstanding the mechanisms of bacterial adhesion and
biological responses to materials.
2. Material and Methods
2.1. General
Phosphate buffered saline (PBS, 0.01 M, pH 7.4, Sigma)
was prepared using purified water (18 M) from a Mil-
lipore Simplicity 185 system. Human fibrinogen (Fg,
100% clottable) from Calbiochem (La Jolla, CA), human
serum albumin (HSA, >99%) and human fibronectin (Fn)
from Sigma Inc were used as received. A polyclonal
anti-Fn antibody was obtained from Abcam (Cambridge,
MA). Monoclonal antibodies (MAb) anti-amino (N-ter-
minus) (MAb1936) and anti-carboxy (C-terminus) (MAb-
1935) of human Fn were purchased from Millipore (Bil-
lerica, MA). MAB1936 specifically recognizes the N-ter-
minal fibrin and heparin binding 29 kDa domain.
MAB1935 recognizes the C-terminal domain containing

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
489
the second fibrin binding site [39].
2.2. Polyurethane Film Preparation
A BioSpan® MS/0.4 segmented polyurethane urea (PUU),
having 22 wt% hard segments, was obtained from the
Polymer Technology Group (Berkeley, CA). PUU films
were prepared by a drop of solution casting onto round
glass coverslips (15 mm dia, Ted Pella Inc., CA) and
dried in a vacuum oven at 65˚C overnight. PUU films on
glass coverslip were soaked in purified water over night
and equilibrated in PBS for 1 hr before use.
2.3. Bacterial Strain Culture and Adhesion
Strain S. epidermidis RP62A (ATCC 35984) was cul-
tured in tryptic soy broth (TSB, BD) at 37˚C for 24 hrs
and collected by centrifuge at 1360 g for 10 min. The
pellet was resuspended in PBS and the concentration of
bacteria was measured by a spectrophotometer at 600 nm.
PUU films on glass coverslip hydrated in H2O for 24 hr
were incubated in protein solutions at the desired con-
centration for 15 min in 12-well tissue culture plate (BD).
After adsorption of proteins, PUU films were rinsed with
PBS three times and incubated in bacterial solution at a
concentration of 1 × 108 cfu/ml for 1 hr with shaking at
250 rpm. The samples were rinsed in PBS 3 times and
fixed in 2.5% glutaraldehyde for 2 hrs, then the bacteria
were stained with Hoechst 33258 (Invitrogen) for 30
mins. Adherent bacteria on PUU films were imaged un-
der a fluorescent optical microscopy with magnification
of 1000× (Nikon, Eclipse 80i) at six random locations.
The DAPI filter was used for the Hoechst 33258 (excita-
tion/emission wavelengths of 352/461 nm). Cell numbers
were quantified using Image J software (NIH, Bethesda,
MD). Adhesion was measured on three replicates of each
protein concentration and presented as average ± stan-
dard deviation.
2.4. Modification of AFM Probes
All AFM experiments were performed using a Multi-
mode AFM equipped with a Nanoscope IIIa controller
system (Veeco Instruments, Santa Barbara, CA). AFM
probes having long-narrow Si3N4 triangular cantilevers
(Veeco Instruments, Santa Barbara, CA, nominal k =
0.06 N/m) were modified with anti-Fn antibodies, puri-
fied Fn or purified Fg. Probes were treated by glow dis-
charge plasma at 100 W power for 30 min and then in-
cubated in a 1% (v/v) solution of aminopropyltriethox-
ysilane (Gelest Inc., PA) in ethanol for 1 hr to provide
reactive amine groups on the tip. After thoroughly rins-
ing with Millipore water, the probes were reacted with
10% glutaraldehyde in aqueous solution for 1 hr. The
probes were again rinsed with Millipore water and incu-
bated in protein solution (~20 µg/ml) for 1 hr. The probes
were rinsed with PBS after removal from protein solution
and were stored in PBS at 4˚C until use within 2 days.
This attachment method has been shown to provide suf-
ficient mobility and flexibility for proteins to rotate and
orient themselves for binding [40,41]. Multiple probes
were prepared together to improve consistency between
experiments. The spring constants of cantilevers (all
taken from the same wafer) were determined using the
thermal tuning method (Nanoscope V6.12r2) using a
multimode AFM with a PicoForce attachment and Nan-
oscope IIIa control system (Veeco Instruments, Santa
Barbara, CA).
2.5. Protein Adsorption and Orientation
Detection
An immuno-AFM technique [41] was used to detect the
adsorption/orientation of Fn from mixtures with Fg or
HSA on PUU film surfaces using a probe modified with
anti-Fn antibodies. The orientation of Fn adsorbed on
PUU surface was detected by the monocolonal antibodies
(MAb) coupled AFM probes. PUU films hydrated in
H2O for 24 hr were incubated in protein solutions at the
desired concentrations for 15 min, and rinsed with PBS
to remove the free proteins. The PUU film was mounted
on the AFM stage in an AFM fluid cell filled with PBS.
An array of 32 × 32 force curves were collected by force
volume image mode with a scanning area of 1 × 1 μm2 at
a scan rate of 1Hz and ramp size of 1 μm. As nonspecific
controls for these measurements, polymer films were
incubated with HSA for 10 min and used for measuring
the nonspecific interactions of the antibody with protein.
The individual retraction force curves were extracted and
analyzed off-line with tools developed with Matlab soft-
ware. The maximum debonding forces, defined as rup-
ture force, calculated from the distance between the zero
deflection value to the point of maximum deflection dur-
ing probe separation from the surface for each force
curve, was used as the strength of the interactions. The
rupture length was calculated from the distance that the
tip moves from the zero interaction force during separa-
tion to the position where the probe has separated and
returned to zero deflection. Both rupture force and rup-
ture length can be used to distinguish the specific and
non-specific interactions.
2.6. Measurement of Binding Strengths between
Proteins and Cell Surface
To directly measure the binding strengths between pro-
teins and bacterial cell surface, bacteria were nonspecifi-
cally attached onto the glass coverslips coated with poly-

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
490
L-lysine for 5 min and rinsed with PBS 3 times to re-
move un-attached cells [37]. The cells were kept wet in
PBS buffer for AFM measurement. AFM probes modi-
fied with Fg or Fn were used to measure the interaction
forces between protein-probe and bacterial cell surfaces
in PBS. A probe coated with HSA was used as control
for non-specific interactions between protein and cell
surface. An array of 32 × 32 force curves were collected
by AFM force volume mode. Force curve data were ex-
tracted from AFM files and analyzed off-line with tools
developed using Matlab software, and the maximum de-
flection of the cantilever from each retracting curve was
used to calculate binding strength. The slope of ap-
proaching force curve was used to distinguish the forces
curves measured on cell or substrate surfaces.
2.7. Interactions of Bacteria and Platelets
Salvaged human platelets with citrate phosphate dextrose
anticoagulant were obtained from the Blood Bank at
Hershey Medical Center. A previous study showed that
salvaged human platelets retain functional activity [42].
The platelets were centrifuged at 200 g for 20 min to
remove any remaining red blood cells. The supernatant
was centrifuged at 600 g for 20 min to separate platelets
into a pellet. The platelet pellet was gently resuspended
in 10 ml of PBS, and the platelet count was measured by
a hematology analyzer (Sysmex KX-21N, Japan). The
PUU films were incubated in 2 ml of PBS solution con-
taining bacteria (1 × 108 cfu/ml) and platelets (2.5 ×
108/ml) for 1 hr with shaking at 250 rpm on a shaker
plate. Plasma proteins (HSA (4 mg/ml), Fg (0.3 mg/ml),
and Fn (0.03 mg/ml)) were added into solutions respect-
tively in order to study the influences of proteins on bac-
teria-platelet interactions. After 1 hr, the adherent plate-
lets and bacteria were fixed in 1% paraformaldehyde and
2.5% glutaraldehyde for 1 hr. After washing with PBS,
platelets and bacteria on the PUU surface were stained
and examined with a fluorescence microscopy. For pla-
telet staining, samples were incubated in a primary anti-
body solution containing Ab662 anti-human 
IIb

3 (1.5
µg/ml) in 6% normal donkey serum (Jackson Immu-
noResearch) overnight at 4˚C. Following this labeling
step, samples were washed with PBS and labeled with a
secondary antibody by adding 10 μl/ml of AlexaFluor555
goat anti-mouse antibody IgG (Invitrogen) in 6% normal
goat serum in the dark and at room temperature for 1 hr.
Samples were rinsed with PBS and incubated in Hoechst
33258 solution for 30 min to stain bacteria. After rinsing
in PBS, the sample was mounted under a coverslip with
antifade gel (Biomeda) and stored at 4˚C overnight. The
adherent platelets and bacteria were examined under
fluorescence microscopy with appropriate fluorescence
filters. In another experiment, the fixed platelets and
bacteria samples on PUU surfaces were washed by pure
water and dried in air for AFM imaging.
2.8. Image and Data Analysis
The fluorescence images were analyzed by Image J
software. Statistical analysis of bacterial adhesion data
was performed by ANOVA utilizing the commercial
software program GraftPad Instat (version 3.06). p < 0.05
was considered statistically significant. Significant dif-
ferences are denoted by symbols (* or #) with one sym-
bol denoting p < 0.05, two symbols denoting p < 0.01,
and three symbols denoting p < 0.001.
3. Results
3.1. S. epidermidis RP62A Adhesion on PUU
Surfaces with Pre-Adsorption of Proteins
Bacterial adhesion values for S. epidermidis RP62A on
PUU surfaces pre-adsorbed with different proteins and
different concentrations are illustrated in Figure 1. For
surfaces adsorbed with single proteins, the bacterial ad-
hesion was significantly increased when surfaces were
pre-adsorbed with Fn (either concentration) compared to
either Fg or albumin. HSA appears to impart a small in-
hibition of bacterial adhesion, evidenced by the lower
adhesion against albumin compared to the control surface
without any protein adsorption, although the result is not
statistically significant. Fg produced a slight increase in
adhesion compared to HSA, but adhesion to Fg was still
much lower than Fn. Results showed the general trend of
effect of single protein adsorption on bacterial adhesion
to be in the order of Fn Fg > HSA. When surfaces
were pre-adsorbed with dual component proteins solu-
tions (either Fn + Fg or Fn + HSA), the Fg + Fn combi-
nation showed greater bacterial adhesion than either the
control or Fn + HSA samples, as expected. It is interest-
ing to note that increasing the amount of Fg with respect
to Fn in solution had no significant effect on bacterial
adhesion (perhaps even a small drop in adhesion, though
not significant) while increasing Fn concentration sig-
nificantly increased the bacterial adhesion (Fn 0.01 + Fg
0.3 mg/ml and Fn 0.03 + Fg 0.3 mg/ml).

3.2. Fn Adsorption on PUU Surfaces
The Fn adsorption on polyurethane surfaces was meas-
ured by an immuno-AFM technique. Utilizing an AFM
probe modified with polyclonal anti-Fn antibody (pAb),
Fn on the polymer surface can be recognized by differen-
tiating specific and non-specific interactions between
antibody and adsorbed proteins [41]. Either the rupture
force or rupture length (stretching of the interaction)

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
491
Figure 1. S. epidermidis RP62A adhesion on PUU surfaces after pre-adsorption of plasma proteins at varying concentrations.
(n = 3; ***: p < 0.001; Fn = fibronectin; Fg = fibrinogen; HSA = human serum albumin).
from each force curve can be used to characterize the
interactions between antibodies and proteins. It should be
noted that non-specific interactions measured between
pAb anti-Fn and HSA in this case show a wide range of
rupture forces, similar to the interaction forces between
pAb and Fn, however, the rupture length of non-specific
interactions varied over a small range, compared to the
distribution of rupture lengths of pAb and Fn (Figure 2).
Results here suggest that rupture length is more suitable
than rupture force for distinguishing non-specific and
specific interactions in this system. Therefore, the distri-
bution of rupture length of non-specific interactions be-
tween pAb and HSA was used to build a 95% confidence
interval limit, similar to what we have done previously
[41]. This resulted in a value of 93.6 nm as the cut-off
value. Interactions with a rupture length above this limit
were considered as specific interactions, and the per-
centage of curves across an array of force curves show-
ing specific interactions was used to indicate the recogni-
tion of Fn on the material surfaces.
Figure 3 illustrates the molecular Fn recognition data
from the dual component protein adsorption on PUU
surfaces. A lower Fn fraction was recognized on the sur-
faces after adsorption of Fn and HSA, while higher Fn
recognition was observed on surfaces with adsorption of
Fn or Fn + Fg. A good correlation between molecular
scale measurements of Fn adsorption and macroscale
bacterial adhesion was observed, suggesting Fn plays an
important role in bacterial adhesion.
To assess the orientation of adsorbed Fn on PUU sur-
faces, the amino (N)- or carboxy (C)-termini of Fn were
detected by mAb-coupled probes using similar method to
the recognition of Fn adsorption by pAb probes. Results
show that Fn adsorbed from pure solutions showed more
C-terminus available compared to N-terminus in same
sample, and corresponded to higher bacterial adhesion,
while more N-terminus was measured in the presence of
HSA and with lower bacterial adhesion (Figure 3). Thus,
results suggest the orientation of protein Fn is important
in controlling S. epidermidis RP62A adhesion.
3.3. Binding Strengths of Bacterial Cell
Receptors and Protein Ligands
The interactions of plasma proteins (e.g., Fg or Fn) with
the bacterial cell surface are specific ligand/receptor type
interactions [43]. Characterization of the binding
strengths between cell and proteins as well as the distri-
bution of binding sites on cell surfaces can offer insight
into the mechanisms of bacterial adhesion. With a pro-
tein-modified probe, the interaction forces between pro-
teins and surfaces (cell or substrate) can be measured by
analysis of an array of force curves. Figure 4 illustrates a
representative low-resolution (32 × 32) height image of
bacterial clusters attached on polymer surface along with
the corresponding force map measured with an Fn-
modified probe. The warmer colors in the force map
represent a strong interaction force while the cooler col-
ors represent a weak force (Figures 4(c) and (d)). The
different colors in the force map of bacterial cell cluster
surfaces show the heterogeneous distribution of binding
sites on cell surfaces. Extracting the force data collected
from cell surfaces, the bindng strength distributions of i

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
492
(a) (b)
(c) (d)
Figure 2. Histogram of (a), (b) rupture forces and (c), (d) rupture lengths measured from PUU surfaces pre-adsorbed with
HSA and Fn.
Figure 3. Correlation between molecular Fn recognition, amino (N)-, carboxy (C)-termini of Fn, and bacterial adhesion on
PUU surfaces. Statistical analysis symbol # denotes the comparison of Fn recognition to surfaces adsorbed from pure Fn (0.01
mg/ml) solution, and * denotes comparing N- and C-termini on same sample.

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
493
Figure 4. (a) Height image of bacterial clusters on polymer surface, and (b) corresponding force map measured by Fn-probe
(32 × 32). Representative force curves measured on bacterial cell surface were shown as (c) weak interaction force corre-
sponding to cool color in (b) and (d) strong force corresponding to warm color in (b) (AFM image size: 5 × 5 µm2).
Fn or Fg were calculated and illustrated in Figure 5. To
recognize the protein receptors on bacterial cell surfaces,
HSA was used as a control and nonspecific interaction
forces between HSA and bacterial cell surfaces were
measured. The force distribution measured between
HSA-probe and bacterial cell surfaces produced a 95%
confidence limit at 0.36 nN. Binding strengths over this
limit were considered as protein ligand and cell receptor
interactions. There are approximately 48% of the meas-
urements showing bacteria-Fg interactions while around
69% of the measurements showing an interaction with
Fn-probes (Figures 5(a) and (b)). Results suggest more
Fn-receptor recognition events than Fg-receptor recogni-
tion events on the cell surface.
3.4. Interaction of Platelets and Bacteria in the
Presence of Plasma Proteins
Interaction of platelets and bacteria was illustrated from
the distribution of platelets and bacteria adhered on PUU
surfaces. Results show that bacteria adhered on surface
and aggregated to form clusters. Although fewer platelets
were observed compared to the number of bacteria, most
platelets were found to be either entrapped in bacterial
aggregates (green arrows in Figure 6) or adherent with
bacteria (red arrows in Figure 6), suggesting the forma-
tion of platelet-bacteria aggregates. The platelet-bacteria
aggregates on the PUU surface were analyzed and
counted when plasma proteins were added in bulk solu-
tion or proteins were just pre-adsorbed on PUU surfaces
(without proteins in bulk solution). Results show that Fn
leads to more aggregates than either Fg or albumin in
(a) (b)
Figure 5. Histogram of binding strengths between bacterial
cell surfaces and proteins (a) Fn, and (b) Fg.
Figure 6. Fluorescent images of S. epidermidis RP62A bac-
teria and platelets interactions on polyurethane surface, (a)
platelets and (b) bacteria. Red circles are drawn for com-
parison. (Image size: 226 μm × 169 μm).
both cases, and more aggregates formed when the protein
was present in bulk solution as opposed to just pre-ad-
sorbed on the polymer surface (Figure 7). It is interest-
ing to note that Fg showed a slight increase in aggrega-
tion, not significantly, compared to albumin or control.
The interactions of platelets and bacteria were further
imaged by AFM. Figure 8 shows the morphology of

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
494
Figure 7. Bacteria-platelet aggregates on PUU surfaces ob-
served when proteins were present.
platelets and bacteria adhered on PUU surface. Both
non-activated (round) and activated (spread) platelets
were found on the surface when only platelets were pre-
sent in solution (Figure 8(a)), however, platelets were
found only activated and spread on the surface when
bacteria were present and interacted with platelets. Bac-
teria were seen to adhere with activated platelets and
form aggregates (Figure 8(b)), suggesting that bacteria
increase the platelets activation and aggregation of bacte-
ria-platelet. When plasma proteins were present in solu-
tion, HSA appeared to decrease the number of bacteria-
platelet aggregates on PUU surface (Figure 8(c)) while
Fg and Fn increased the formation of aggregates (Fig-
ures 8(d) and (e)). Furthermore, non-activated platelets
were observed on surface in the case of HSA while all
platelets were activated in the cases of Fg and Fn. The
images with large magnification show activated platelets
either adhered to bacteria or entrapped in bacterial clus-
ters (Figure 9).
4. Discussion
Bacterial adhesion is the first step in the development of
biofilm formation on implanted biomaterials. Factors that
influence bacterial adhesion on a polymeric surface in-
clude the nature of environment, type of microorganism,
and properties of material, and each one of these factors
is in turn affected by several other parameters [43].
When a material is implanted, plasma proteins rapidly
interact with the surface to form a layer of proteins. The
nature of adsorbed proteins is affected by the physico-
chemical properties of surface, and in turn moderates the
initial bacterial adhesion. S. epidermidis is a predominant
bacterial species contributing to cardiovascular implant
infection. In this study we measured the adhesion of S.
Figure 8. AFM images of platelet and bacteria interactions
on PUU surfaces, (a) platelet only, (b) platelet and bacteria
without plasma proteins, platelets and bacteria in the pres-
ence of (c) HSA, (d) Fg and (e) Fn. The bacteria-platelet
aggregates are indicated by circles. (Image size: 50 µm × 50
µm).
Figure 9. Aggregates of bacteria-platelets in the presence of
Fn showing platelet (a) adherent or (b) entrapped with bac-
teria. (Image size: 20 µm × 20 µm).
epidermidis and interaction with platelets on polyure-
thane biomaterial surfaces in the presence of plasma pro-
teins. The molecular scale measurement of protein ad-
sorption and interaction forces between protein and cells

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
495
were correlated to bacterial adhesion. Results provided
important information to understand the roles of plasma
proteins in bacterial adhesion and biological responses to
implanted biomaterials.
Albumin is the most abundant plasma protein. The ad-
dition of HSA to Fn solution decreased the adsorption of
Fn and subsequent bacterial adhesion to the PUU sur-
faces (Figures 1 and 3). No ligand/receptor binding
event was measured between albumin and the cell sur-
faces. Furthermore, the presence of albumin appeared to
decrease the activation of platelets and interactions with
bacteria.
Fibrinogen is the most third abundant plasma protein
in blood and plays a prominent role in development of
surface-induced thrombosis. It serves as a ligand, binding
to the platelet integrin receptor

IIb

3, leading to platelet
immobilization, activation, and aggregation [44,45]. It is
also found to promote S. aureus adhesion to material
surfaces [17]. The increase in adhesion of S. aureus with
Fg was identified to be due to the Fg-binding MSCRA-
MM clumping factor on cell surface [27]. However, the
presence of Fg shows no significant increase in S. epi-
dermidis RP62A adhesion compared to HSA in this
study. Lower bacterial adhesion was measured on poly-
mer surfaces in the presence of Fg compared to Fn, al-
though the solution concentration of Fg (0.3 mg/ml) is
10-times higher than that of Fn (0.03 mg/ml) (Figure 1).
Results suggest that S. epidermidis RP62A cell surface
has fewer binding sites to Fg than Fn, as evidenced by
the molecular scale measurement of protein binding sites
on cell surfaces, where approximately 48% of the meas-
urements showed bacteria-Fg interactions while ~69% of
the measurements showed an interaction with Fn-probes
(Figure 5).
Fibronectin is one of the main plasma proteins respon-
sible for forming a conditioning film on implanted bio-
materials. It can bind a variety of extracellular molecules
including fibrin, heparin, and collagen, and plays a key
role in cell adhesion and proliferation [46,47]. Numerous
studies have found that Fn facilitates bacterial adhesion
to biomaterials including S. epidermidis [48-50]. In this
study, the macroscale measurements of bacterial adhe-
sion show higher adhesion of bacteria on surfaces pre-
adsorbed with Fn compared to Fg or HSA, and consistent
with the amounts of molecular Fn detected by anti-Fn pAb
probe on polymer surfaces (Figure 3). Results strongly
suggest that Fn plays an important role in adhesion of S.
epidermidis to polymer surfaces. Higher adhesion of
bacteria on PUU surfaces bearing Fn is believed to be
due to the interaction of Fn with MSCRAMM on S.
epidermidis RP62A cell surfaces. William et al. identi-
fied a giant Fn-binding protein, extracellular matrixbind-
ing protein (embp), from S. epidermidis cell surface [28].
Christner et al. further demonstrated that embp mediates
binding of S. epidermidis to solid phase attached Fn,
constituting the first step of biofilm formation on condi-
tioned surfaces. Embp is also a multifunctional cell sur-
face protein that mediates attachment to host extracellu-
lar matrix, biofilm accumulation and escape from pha-
gocytosis, promoting biomaterial-associated infections
[32]. Although the Fn-binding proteins on S. epidermidis
RP62A cell surface were not identified in this study, such
proteins are expected to be present on cell surface, as
evidenced by the larger binding strengths and increased
binding events measured on cell surfaces by Fn-probe
(Figure 5(a)). The binding between Fn and cell surface
can be considered as a ligand/receptor interaction. Bus-
tanji et al. measured the energy landscape of this bind-
ing/unbinding process through dynamic force spectros-
copy under different loading rates, and revealed the mo-
lecular mechanism of Fn in bacterial adhesion [51].
The orientation of Fn influences bacterial adhesion. Fn
is a dimer of two similar polypeptides linked by disulfide
bonds at the carboxyl terminus, possessing several func-
tional domains that bind to a variety of extracellular
molecules such as heparin and collagen [52]. There are
two particularly important relevant binding sites for S.
epidermidis, which are located at the N-terminus and the
C-terminus of Fn [39]. The immuno-AFM measurements
show that C-terminus is much more available than
N-terminus of Fn molecules when adsorbed from pure Fn
solution. The presence of albumin appears to influence
the orientation of Fn, with more N-terminus available
although the total amount of Fn adsorbed appears low.
Bacteria in blood can interact with platelets. This in-
teraction appears mediated by plasma proteins. Albumin
appears to inhibit the bacteria-platelet interaction and
activation/aggregation of platelets, while Fg and Fn
promote the interactions of bacteria and platelets along
with platelet activation, leading to bacteria/platelet ag-
gregation (Figure 8). Interactions between bacteria and
platelets are characterized as the binding of bacteria to
platelets either directly through a bacterial surface pro-
tein or indirectly by a plasma bridging molecule that
links bacteria and platelet receptors [53]. S. epidermidis
induced platelet activation and aggregation of bacteria
and platelets in the absence of plasma proteins, showing
the direct mechanism mostly involved (Figure 8(b)),
while more bacteria-platelet aggregates were observed in
the presence of Fg and Fn, suggesting that both mecha-
nisms may be involved in interaction of bacteria and
platelets (Figures 8(d) and (e)). More aggregates of bac-
teria-platelet were measured on PUU surface when Fn or
Fg were added in bulk solution compared to the case of
proteins only pre-adsorbed on surface (Figure 7). This
suggests that Fg or Fn may serve as linker in interaction

Effects of Plasma Proteins on Staphylococcus epidermidis RP62A Adhesion and
Interaction with Platelets on Polyurethane Biomaterial Surfaces
496
of bacteria and platelets.
Fn leads to more bacteria adhering to platelets and
forming more aggregates than Fg does. This may be due
to the different functions of MSCRAMM involved in
bacteria-platelet interactions. Different MSCRAMM on S.
aureus cell surface have been identified including clump-
ing factors (Clf) and Fn binding proteins (FnBP), and
they were shown to promote bacterial adhesion to and
activation of platelets [11,43]. Both Clf and FnBP bind
Fg, allowing an interaction with platelet GPIIb/IIIa,
leading to platelet adhesion. However, less information
on the interactions of platelets and S. epidermidis is
available. Recently Brennan et al. reported that Fg-bind-
ing serine-aspartate repeat protein G (SdrG) from S. epi-
dermidis supports adhesion to platelets and aggregation
through both a direct interaction with platelet integrin
receptor IIb3, and an indirect mechanism by a bridge of
Fg [33], however, the roles of other MSCRAMM, e.g.,
the giant Fn-binding protein (embp) from S. epidermidis
cell surfaces, have been identified in bacterial adhesion
and biofilm formation, but without report of interactions
of bacteria-platelet.
5. Conclusion
Bacterial adhesion to polyurethane biomaterial surfaces
as well as interactions with platelets is complex and can
be mediated by plasma proteins. This study demonstrated
the roles of plasma proteins (albumin, fibrinogen, and
fibronectin) in the adhesion of bacteria, S. epidermidis
RP62A, on polyurethane biomaterial surfaces. Results
show that Fn leads to increased bacterial adhesion, with
the order of effectiveness being Fn Fg > albumin. A
correlation between molecular scale Fn adsorption and
macroscale bacterial adhesion was observed, with in-
creased numbers of Fn-receptor recognition events meas-
ured on cell surfaces as compared to Fg-receptor recog-
nition events, suggesting Fn is an important protein in
bacterial adhesion. Interactions between bacteria and
platelets induced platelet activation and bacteria-platelet
aggregation. Albumin inhibited bacteria-platelet interact-
tions and platelet activation, while both Fg and Fn appear
to serve as a linker, promote the adhesion of bacteria to
platelets and platelet activation, resulting in bacteria-
platelet aggregation.

6. Acknowledgements
Financial support was from a Department of Surgery
Feasibility Grant at the Penn State College of Medicine.
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