Plasma-deposited tetraglyme surfaces greatly reduce total
blood protein adsorption, contact activation, platelet
adhesion, platelet procoagulant activity, and in vitro
thrombus deposition
Lan Cao,1 Mark Chang,2 Chi-Ying Lee,1 David G. Castner,1,2 Sivaprasad Sukavaneshvar,3
Buddy D. Ratner,1,2 Thomas A. Horbett1,2
1
Department of Chemical Engineering, University of Washington, Seattle, Washington 98195
2
Department of Bioengineering, University of Washington, Seattle, Washington 98195
3
Utah Artificial Heart Institute, Salt Lake City, Utah 84103
Received 30 May 2006; revised 15 August 2006; accepted 29 August 2006
Published online 18 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31091
Abstract: The ability of tetraethylene glycol dimethyl ether
(tetraglyme) plasma deposited coatings exhibiting ultralow
fibrinogen adsorption to reduce blood activation was studied
with six in vitro methods, namely fibrinogen and von Willebrand’s factor adsorption, total protein adsorption, clotting
time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed
flow catheter model. Surface plasmon resonance results
showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in
the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the
intrinsic coagulation cascade. Platelet adhesion and thrombin
generation by adherent platelets were greatly reduced on tet-
raglyme-coated materials, compared with uncoated and Biospan1-coated glass slides. In the in vitro disturbed blood
flow model, tetraglyme plasma coated catheters had 50%
less thrombus than did the uncoated catheters. Tetraglymecoated materials thus had greatly reduced blood interactions
as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only
due to their reduced platelet adhesion and activation, but
also to a generalized reduction in blood interactions. Ó 2007
Wiley Periodicals, Inc. J Biomed Mater Res 81A: 827–837,
2007
INTRODUCTION
proteins are expected to have improved hemocompatibility.
Human blood contains a variety of protein types, and
the concentration of each type of protein varies greatly.
For example, albumin, IgG, and fibrinogen (Fg) are the
most abundant proteins in plasma, with a concentration
of about 40, 10, and 3 mg/mL, respectively, whereas the
concentration of von Willebrand’s factor (vWf) is only
about 0.01 mg/mL. Blood plasma proteins also have different adsorption affinities that vary with the surface
chemistry of materials. To distinguish which proteins
are the major players in mediating platelet-induced clotting events, our group has studied separately the role of
proteins in mediating platelet adhesion. We found that
Fg and vWf are the two major proteins mediating platelet attachment, depending on flow conditions.11,12 The
amount of adsorbed Fg needed to support full-scale platelet adhesion under both static and shear conditions11,12
was less than 10 ng/cm2, while even a very low adsorption of vWf (2 ng/cm2) was sufficient to greatly
increase platelet adhesion at higher shear rates.12
To reduce the reactivity of artificial materials with
human blood, many investigators have focused on preparing surfaces that would resist blood proteins adsorption.1–8 The rationale for this approach is that adsorbed
proteins can initiate the intrinsic coagulation cascade,
and more importantly, mediate platelet attachment and
activation, either of which will ultimately lead to thrombotic and thromboembolic complications.9,10 Therefore,
surfaces that can resist the adsorption of blood plasma
Correspondence to: T.A. Horbett; e-mail: horbett@cheme.
washington.edu
Contract grant sponsor: NIH; contract grant numbers:
HLR01, HL 67923
Contract grant sponsor: NIBIB; contract grant number: EB002027
' 2007 Wiley Periodicals, Inc.
Key words: tetraglyme; protein adsorption; platelet adhesion;
platelet activation; plasma deposition
828
Previous work by López et al.13,14 and the current
authors15,16 showed that a surface coating made by
radio frequency plasma deposition of tetraethylene glycol dimethyl ether (tetraglyme) was able to greatly reduce Fg adsorption, down to an ultralow level (<5 ng/cm2
from plasma). However, it was not clear whether the
high resistance to Fg adsorption was due to the preferential adsorption of other plasma adhesive proteins
such as fibronectin (Fn) and vitronectin (Vn), that might
outcompete Fg adsorption at the surface, or due to a
general reduction of adsorption of all proteins.17 Note
that at the levels Fn and Vn usually adsorb from plasma
they did not contribute to platelet adhesion.18 However,
it was still possible that the adsorption of these or
other proteins in plasma could be elevated on the tetraglyme plasma coatings due to the lower Fg adsorption,
and thus might play an increased role in platelet attachment. Bremmell et al.19 found Fg adsorption was substantially reduced on similar plasma-deposited poly
(ethylene oxide) (PEO)-like coatings from single component Fg solutions, which cannot be explained by increased adsorption of competing proteins, thus suggesting that a general reduction of adsorption might also be
responsible for lowered Fg adsorption to tetraglyme
coatings. Nonetheless, we wanted to confirm that the
adsorption of the other proteins in plasma was reduced
on tetraglyme coatings. Therefore, a study of total blood
protein adsorption on the tetraglyme plasma coating
was done with surface plasmon resonance (SPR).
SPR has been extensively used to study the dynamics
and kinetics of protein adsorption.20,21 It offers the advantages of in situ, real time monitoring and avoids the
need for a large series of studies with numerous radiolabeled proteins. SPR detects adsorption through the
changes in refractive index at the surfaces, and since
most plasma proteins have similar refractive indices,
SPR measurements can be used to measure the total
protein adsorption.
The ability of ultralow Fg adsorption tetraglyme
plasma-deposited coatings to reduce interactions
with blood was also studied with five additional
in vitro methods, namely clotting time in recalcified
plasma, platelet adhesion and procoagulant activity,
and whole blood clotting in a disturbed flow catheter model. Although prior studies showed that platelet adhesion to tetraglyme-coated surfaces was lowered, it was possible that tetraglyme coatings would
activate the intrinsic coagulation cascade, and so a
clotting time study was used to test this possibility.
Similarly, if the low platelet adhesion to tetraglyme
was accompanied by a higher activation of adherent
platelets, tetraglyme coatings might be less bloodcompatible than expected; therefore, we assessed the
procoagulant activity of adherent platelets. Finally,
in vitro thrombus deposition was measured as an
end-point indicator of the hemocompatibility of tetraglyme coatings.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
CAO ET AL.
MATERIALS AND METHODS
Samples
Microscope cover glass slips (10-mm diameter, Fisherbrand1, Cat. no. 10 CIR-1, Fisher Scientific, Pittsburgh, PA)
were used as the substrate for Biospan1 samples. The glass
disks were cleaned by sonication for 15 min in Isopanasol (C. R.
Callen, Seattle, WA) solution with a ratio of 1:64 in DI water,
and then air-dried in a laminar flow hood and stored under
nitrogen until use. Biospan (Polymer Technology Group, Berkeley, CA) was dissolved in N,N-dimethylacetamide (Aldrich,
Milwaukee, WI) and made into 1% (wt) solutions. The solutions
were then filtered by syringe-driven filter units (Millex1 Millipore, Billerica, MA). The glass disks were coated using a commercial spin coating apparatus (Headway Research, Garland,
TX). About 20 mL Biospan solution was put onto one side of
glass disks, and they were then spun at 4000 rpm for 30 s. After
drying in the laminar flow hood for at least 45 min, the other
side of glass discs was coated by the same procedure.
Polyethylene (PE) samples are 0.5-mm-thick low-density
PE films (Laird plastics, Seattle, WA). IntramedicTM Clay
AdamsTM PE tubing (1.14 mm ID, 1.57 mm OD) was from Becton Dickinson (Sparks, MD) as catheter samples. SPR chips
used were SF14 glass slides (Schott Glass Technology, Durea,
PA) coated with 2-nm Cr and 50-nm Au (99.99%), and then
coated with tetraglyme plasma coating.
Plasma deposition
Tetraglyme was purchased from Sigma-Aldrich (Milwaukee,
WI). Plasma deposition was conducted using the system previously described.22 Flat-disk samples were placed onto a glass
substrate and coated on both sides in two deposition runs. For
the SPR substrates, a 100-nm-thick film was deposited onto the
gold-coated surface. Catheter samples were hung on a glass
rack, so that the outside surfaces of catheters were fully
exposed to the glow. Prior to deposition, argon etching at a
pressure of 350 mTorr, flow rate of 4.0 sccm (standard cubic
centimeters per minute), and power of 40 W was used to clean
the substrate and generate free radicals for subsequent plasmapolymerized grafting to the substrate. Tetraglyme vapor was
then introduced and maintained at an optimum flow rate (1.33
sccm) and pressure (350 mTorr). Power used during the tetraglyme deposition was 80 W for the first 1 min, 40 W for the
next 30 s, and 10 W for the final 10 min. Samples were rinsed
with DI water and blown dry with argon gas before and after
deposition. Rinsing was shown not to cause delamination of
the coating or to alter the chemical structure of coating. Tetraglyme plasma coated samples were analyzed by electron spectroscopy for chemical analysis (ESCA) as described previously,16 and all the test samples exhibited a C 1s ether (286.5
eV)-hydrocarbon (285.0 eV) ratio greater than 60%.
Buffers and biochemicals
Protein adsorption buffers included citrate phosphatebuffered saline (CPBS), which contained 10 mM Na2HPO4,
10 mM citric acid, and 120 mM NaCl. CPBS with azide
IMPROVED HEMOCOMPATIBILITY OF TETRAGLYME PLASMA COATINGS
(CPBSz) was CPBS with 0.02 % w/v added sodium azide.
CPBSzI was CPBSz with added 10 mM NaI. PBS contained
10 mM Na2HPO4 and 120 mM NaCl. Platelet suspension
buffer without metal ions (PSB-MIF) contained 5.5 mM dextrose, 4 mg/mL bovine serum albumin (BSA), 137 mM NaCl,
2.7 mM KCl, 0.4 mM sodium phosphate monobasic and
10 mM HEPES, and was sterile-filtered and supplemented
with 0.1 U/mL apyrase, grade V from potato (Sigma-Aldrich,
St. Louis, MO) prior to use. Platelet suspension buffer with
metal ions (PSB-MI) was PSB-MIF with added 2.5 mM CaCl2
and 1.0 mM MgCl2. All the buffers were at pH 7.4. The lactate
dehydrogenase (LDH) assay was done with a solution containing a proprietary mixture of lactate, NADH, iodotetrazolium
chloride, and catalyst from Boehringer Mannheim (Indianapolis, IN). Human thrombin, factor Xa, factor Va, and prothrombin were from Enzyme Research Labs (South Bend, IN). S-2238
was a chromogenic substrate specifically for thrombin from
Diapharma Group (Franklin, OH). The chemical structure of S2238 is H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline.
Surface plasmon resonance
The SPR liquid sensing system used in this study has been
described and characterized in detail elsewhere.23 Briefly, the
SPR system was set up with a planar prism (Kretschmann) configuration. A 50-nm gold layer on top of a 2-nm chromium adhesion layer on the glass slide was electron-beam evaporated at
a pressure below 1 10–6 Torr. The glass side of the gold-coated
substrate was refractive index-matched to the prism, while the
tetraglyme-coated gold surface was mechanically pressed
against a milled Teflon flow cell. A polychromatic light beam
was passed through the prism and the backside of the goldcoated substrate, to excite surface plasmon waves at the
metal-dielectric interface. The wavelength at which resonant
excitation occurred depended on the refractive index of the
analyte in the proximity of the SPR surface. Thus, the amount
of adsorbed analyte at the surface can be quantified by measuring the shift in the resonant wavelength induced by the refractive index change. The reflected light was analyzed with a
spectrograph, which determined the intensity versus wavelength at a fixed angle. During SPR measurements, the gold
substrates were first equilibrated with degassed water followed by PBS buffer solution. After establishing a stable baseline, blood plasma was delivered to the flow cell at a flow rate
of 50 mL/min at room temperature. Protein adsorption at the
surface was observed by monitoring the wavelength shift of
the SPR reflected minimum. The index of refraction of the various bulk solutions was measured with a spectronic Abbe-3L
refractometer (Thermo Electron Corporation, Waltham, MA).
Protein radioiodination and adsorption
Purified human fibrinogen (Fg) and human von Willebrand’s factor (vWf) were purchased from Enzyme Research
Laboratories. Human plasma was obtained from George King
Bio-Medical (Overland Park, KS) and kept at –808C until use.
BSA fraction V (Sigma-Aldrich, St. Louis, MO) was used to
block nonspecific platelet adhesion.
Radiolabeling of Fg with 125I was done with a modified ICl
method using a 2:1 molar ratio of ICl to Fg, as previously
829
described.11 Radiolabeling of vWf with 125I was performed
using iodo-beads (Pierce Biotechnology, Rockford, IL) as
described.24 D-Salt desalting columns (Pierce Biotechnology)
were used to remove excess unincorporated 125I from the iodinated proteins. Radiolabeled Fg and vWf were stored at –
808C and used within 2 weeks. The effect of radioactive decay
was taken into consideration for all calculations.
Protein solutions with specific activity of at least 100 cpm/
ng were prepared by adding 125I protein to 1% human plasma.
The amount of added 125I protein was enough to provide the
desired specific activity. The added 125I protein did not significantly change the concentration of the protein in the plasma,
because of the high specific activity of the radiolabeled protein
stock, that is, the mass of added 125I protein was less than 1%
of the amount of the protein in the plasma. Before adsorption,
samples were incubated with CPBSzI buffer for 45 min. Then
protein solution containing 125I protein was added to the
buffer solution in which the samples were still submerged,
thus avoiding exposure of the samples to the air–protein solution interface. To adsorb proteins to tubular samples prepared
for the catheter studies, the ends of the samples were sealed
during adsorption, and protein was not allowed to adsorb to
the inside surface of the tubes. Protein adsorption lasted for
1.5 h at 378C. Unbound protein was rinsed away by repetitively dipping sample disks or catheters into a beaker containing fresh CPBSzI buffer. The rinsing buffer was changed several times, until no radioactivity was detected in the rinse
buffer. The radioactivity of surface-adsorbed proteins was
then measured using a Cobra1 II Series Auto-Gamma1
Counting system (Packard Instrument, Meriden, CT). The
amount of protein adsorbed onto the surface was calculated
from the retained radioactivity, corrected for background, divided by the specific activity of the protein solution and the
planar surface area of the sample.
Platelet adhesion
Platelets were collected by sequential centrifugation (180g for
20 min to collect platelet rich plasma (PRP), followed by 1500g
for 15 min) of acid citrate dextrose (ACD) anticoagulated (1:9
v/v) human whole blood. The platelet pellets from the second
centrifugation were resuspended in PSB-MIF. Platelet concentration was determined with a Cell-Dyn1 3700 cell counter
(Abbott Laboratories, Abbott Park, IL) at the University of
Washington Medical Center. Platelet concentration for adhesion studies was adjusted to be 1 108/mL in PSB. Metal ions
(Ca2þ and Mg2þ) were added to the platelet suspension immediately prior to platelet adhesion experiments, to achieve a final
concentration of 2.5 mM CaCl2 and 1.0 mM MgCl2. Flat samples were put into 24-well plates and preadsorbed with 1%
human plasma for 2 h at 378C and blocked with BSA. The use
of BSA for ‘‘blocking’’ on tetraglyme coating samples was to
make the platelet adhesion condition comparable for the noncoated and tetraglyme-coated samples. After preadsorption
with 1% human plasma, there should be negligible amount of
BSA adsorbed to tetraglyme coatings (as suggested by SPR
study), and so the BSA blocking will not affect the platelet adhesion results. After rinsing to remove unbound proteins, a platelet suspension was introduced and incubated at 378C for 1.5
h. After the incubation, unbound platelets were rinsed away
with PBS buffer. The adherent platelets were lysed by adding
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
830
CAO ET AL.
50 mL of 1% Triton in PSB to each well. The number of adherent
platelets was determined by LDH assay of the Triton lysate,
converting the observed LDH activity to the number of platelets
with a calibration curve. Scanning electron microscopy (SEM)
was also used to observe the morphology of adherent platelets.
Thrombin generation
Thrombin generation was measured using the previously
published prothrombinase assay.25,26 Samples preadsorbed
with 1% plasma and blocked with BSA were incubated with a
platelet suspension for 1.5 h to allow platelets to adhere, using
the same protocol described earlier in the adhesion experiments. After rinsing away the unbound platelets, the sample
disks with adherent platelets were incubated with a mixture
of human factor Va (6 nM) and Xa (150 pM) in PSB for 15 min,
to allow prothrombinase to form on the platelet surface. Prothrombin (600 nM) was then added, and the solution was
mixed well. After 0, 3, and 6 min, an aliquot (25 mL) was collected and put into separate wells of a 24-well plate containing
EDTA (50 mL, 80 mM). The EDTA chelates Ca2þ and, therefore, stopped the conversion of prothrombin to thrombin. To
assay the amount of thrombin formed, triplicate portions
of the solution was transferred from each well of the 24-well
plates to separate wells of a 96-well plate and then s-2238
(420 mM) was added to each well. The absorbance at 405 nm
was recorded over a 5-min period, and mOD405 nm/min was
calculated by the microtiter plate reader. Thrombin concentration was determined by dividing the mOD405 nm/min by the
slope of a calibration curve made by plotting mOD405 nm/
min versus thrombin for a series of known thrombin solutions.
Finally, the thrombin generation rate by the samples was calculated from the thrombin concentrations measured after 0, 3,
or 6 min of incubation with the prothrombin solution.
Recalcified plasma clotting time
The clotting time protocol was similar to that reported by
Grunkemeier et al.27 Human platelet poor plasma (citrate-anticoagulated) was brought to 20 mM CaCl2 by addition of calcium from a 1M stock solution. The plasma was then quickly
mixed well by vortex, and 0.25 mL plasma was immediately
added to the wells of a 24-well plate. Falcon 24-well nontissue
culture treated polystyrene (PS) plates were used. Prior to
adding plasma, the different surfaces to be tested had been
placed into the wells. The surfaces used were 10-mm-diameter
circular glass coverslips and fluorinated ethylene propylene
(FEP) disks, either unmodified or tetraglyme-treated. Twentyfour-well plates were placed in a 378C shaking water bath,
and the clotting time of the plasma was determined visually.
The plasma clotting time was measured as the time it took for
the plasma to undergo gelation, detected by loss of movement
of the plasma in response to the rotation and shaking.
Blood interaction studies in an in vitro
catheterization model
An in vitro model that incorporated disturbed blood flow
was utilized to assess the efficacy of tetraglyme-coated catheJournal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
Figure 1. Schematic illustration of the in vitro model to measure thrombus deposition on catheters. Catheters were either
untreated or tetraglyme plasma treated 1-mm internal diameter PE tubes, sealed at the ends. The tetraglyme coating was
on the outside surface of the PE tubes.
ters in reducing catheter thrombosis.28 The samples evaluated
were plain 1.57 mm OD PE tubes, or PE tubes coated on the
outside surface with tetraglyme.
Fresh bovine blood was obtained by inserting a cannula
directly into the heart of stunned cows from a local abattoir.
Approximately 4–10 L of blood was collected into a collapsible
reservoir and anticoagulated with heparin (1.5 U/mL final
concentration). Autologous 111indium-labeled platelets were
added to the blood, and the blood from a single animal was
divided into two separate 1-L blood reservoirs. To make the
radiolabeled platelets, 200 mL of autologous blood was collected prior to the commencement of experiment in ACD and
centrifuged at 350g for 15 min to sediment the red cells. The
supernatant platelet-rich plasma was separated and centrifuged at 1000g for 15 min to sediment platelets. After decanting the plasma, the sedimented platelets were resuspended in
5 mL ACD-saline (0.25 mL ACD þ 4.75 mL of 0.9% NaCl) to
which 50–200 mCi 111indium oxine (Intermountain Radiopharmacy, Salt Lake City, UT) was added and incubated at 378C
for 20 min.
As shown in Figure 1, uncoated and tetraglyme plasma
coated 15 cm 1-mm ID PE tubing was deployed one at a time
inside 3.2 mm ID PVC Tygon1 tubing (Saint-Gobain Performance Plastics, Akron, OH) by inserting them through the wall
of the PVC tubing, to simulate catheter deployment in a blood
vessel. The tubing segments with the devices were connected
IMPROVED HEMOCOMPATIBILITY OF TETRAGLYME PLASMA COATINGS
Figure 2. SPR measurements of total protein adsorption
from various concentrations of human blood plasma to
uncoated and tetraglyme plasma coated SPR gold chips. For
all curves, the solution was changed from buffer to plasma
and then to buffer again. (a) 1% plasma on bare gold; (b) 1%
plasma on tetraglyme; (c) 10% plasma on tetraglyme; (d) 50%
plasma on tetraglyme; (e) 100% plasma on tetraglyme. Curves
for tetraglyme have been corrected for the effect of the refractive index change caused by the overlayer of tetraglyme film
(see text).
to the blood reservoirs (maintained at 378C). A roller pump
was used to induce blood flow at 75 mL/min for 1 h. At the
end of the experiment, the test devices were retrieved, and
radioactivity associated with the thrombi on the device was
measured with a gamma counter (Minaxi 5000, Packard).
Statistical analysis
831
that the proteins adsorbed on bare gold was strongly
bound.
When the tetraglyme sample was exposed to higher
plasma concentrations, there was an increased wavelength shift (18 nm for 10% plasma, 22 nm for 50%
plasma, and 71 nm for 100% plasma) in the presence of
plasma. After the buffer rinse, the wavelength shift was
greatly reduced. Even after exposure to 100% plasma,
the wavelength shift after the rinse step was <1 nm
(equivalent to a total mass of adsorbed proteins of
24.1 ng/cm2). This indicated that even at a high protein concentration, the tetraglyme coatings were still
able to keep most proteins from adsorbing. The greater
wavelength shift in the presence of plasma was probably due to the coupling of the evanescent wave with
the high concentration of bulk phase protein.
It should be noted that the layer of tetraglyme plasma
coatings changed the sensitivity of measurements of
wavelength shifts, because the attenuation of the evanescent wave by protein adsorption to tetraglyme is less
than for protein adsorbed to gold. Thus, the refractive
index change caused by protein adsorption on the tetraglyme-coated gold surface will be less than on a bare
gold surface. Using the equation by Jung et al.,23 we
estimated that, on a typical 100-nm-thick tetraglyme
film (measured by AFM, images not shown here), the
shift caused by an adsorbed protein film would be 35%
less than that by the same protein on gold. All wavelength shifts on the tetraglyme plasma coated sample
presented here were therefore corrected to account for
this attenuation.
The total mass of adsorbed proteins can be approximately estimated using the following two equations
developed by Jung et al.:23
Coated gold: R ¼ mðZa Zs Þ½1 expð2da =ld Þ
Comparison between two groups were made by the Student’s t test. Values of p < 0.05 were considered significant.
RESULTS
Surface plasma resonance
The SPR studies showed that tetraglyme plasma coating greatly reduced total protein adsorption from human
blood plasma. As shown in Figure 2, incubation of the
tetraglyme-coated sample with 1% plasma resulted in a
negligible SPR wavelength shift (0.2 nm, equivalent to
1.3 ng/cm2; see later). After buffer rinsing to remove
the loosely bound proteins, the wavelength shift
became even smaller and returned to the baseline level.
In contrast, incubation of 1% plasma with bare gold
resulted in a SPR wavelength shift of 14 nm. After
buffer rinsing of this sample, the wavelength shift
was still 14 nm (equivalent to 244 ng/cm2), suggesting
expð2db =ld Þ
Bare gold: R ¼ mðZa Zs Þ½1 expð2da =ld Þ
where DR was the SPR wavelength shift; m was the sensitivity factor expressed as the slope of the linear function of wavelength shift versus refractive index change;23
ld was the characteristic decay length calculated from
the Maxwell’s equation23 ld ¼ (l/2p)/Re{Z4eff/(Z2eff þ
emetal)}1/2 (where l was the light wavelength at the SPR
minimum, Zeff was the effective index of refraction, and
emetal was the complex dielectric constant of the metal
at that wavelength deduced from the paper of Innes
et al.29); Za was the average refraction index of plasma
protein: 1.6; Zs was the refraction index of the plasma
solution; db was the average thickness of deposited
tetraglyme films, as measured by AFM. Adsorbed protein thickness da was calculated by substituting values
for all other parameters and then da was converted to
mass by using the specific volume 0.77cm3/g or density
1.3 g/cm3 of serum albumin.30 As it can be seen from
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
832
Figure 3. Fg adsorption from 1% human plasma to untreated
and tetraglyme plasma treated Biospan, glass, and PE disks.
Open bars, untreated samples; solid bars, tetraglyme plasma
treated samples. Error bar represents SD. (N ¼ 5, *p < 0.01,
Student’s t test).
the equation, the wavelength shift will decrease exponentially with the increase of deposited film thickness.
If the thickness of deposited polymer film equals the
characteristic decay length, the sensitivity would be
dropped by a factor of 7. In our case, the thickness of
tetraglyme coating (100 nm) was less than 25% of the
characteristic decay lengths for all situations; therefore,
it did not significantly affect the sensitivity. The detection limit for tetraglyme-coated SPR system was 0.0056
nm in thickness or 0.7 ng/cm2 in mass based on the
equations mentioned earlier.
Based on the aforementioned equations, the calculated mass of total adsorbed proteins from 1% human
plasma on bare gold was 244 ng/cm2, whereas it was
1.3, 4.8, 17.3, and 24.1 ng/cm2 total adsorbed proteins
from 1, 10, 50, and 100% plasma, respectively, on tetraglyme-coated gold surfaces.
CAO ET AL.
Figure 4. vWf adsorption from 1% human plasma to
untreated and tetraglyme plasma treated Biospan, glass, and
PE disks. Open bars, untreated samples; solid bars, tetraglyme
plasma treated samples. Error bar represents SD. (N ¼ 5. *p <
0.01, Student’s t test).
Platelet adhesion and procoagulant activity
Figure 5 shows that tetraglyme coating of Biospan,
glass, or PE greatly decreased platelet adhesion when
compared with the uncoated materials (p < 0.01). For
tetraglyme-coated samples, the lowest platelet adhesion
was on coated PE, while the highest was on coated Biospan. For the uncoated materials, glass had the most
adherent platelets, and Biospan had the lowest platelet
adhesion.
Activation of surface-adherent platelets was assessed
by measuring the rate of formation of thrombin in prothrombin solutions incubated with the adherent platelets.
Thrombin generation is affected by both the number of
surface-adherent platelets as well as the degree of activation of adherent platelets. As shown in Figure 6, all the
Protein adsorption from plasma
As shown in Figures 3 and 4, Fg and vWf adsorption
from 1% plasma were both decreased on tetraglyme
plasma coated Biospan, glass, and PE when compared
with uncoated controls (p < 0.01). Uncoated PE had the
most Fg adsorption (75 ng/cm2), while uncoated glass
had the most vWf adsorption (1 ng/cm2). Uncoated
Biospan had the least Fg and vWf adsorption, showing
that Biospan was more protein-resistant than hydrophobic PE and glass. On the other hand, the chemical
nature of the substrates seemed to have little effect on
the protein resistance once they were coated with tetraglyme. For example, Fg adsorption was similar on all
tetraglyme-coated materials (3 ng/cm2), and vWf
adsorption was around 0.2 ng/cm2 for all tetraglyme
coatings. These data are consistent with our previous
results that showed the high resistance of the plasmadeposited tetraglyme coating to Fg adsorption.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
Figure 5. Platelet adhesion from washed platelets suspensions to untreated and tetraglyme plasma treated Biospan,
glass, and PE disks. Open bars, untreated samples; solid bars,
tetraglyme plasma treated samples. Error bar represents SD.
(N ¼ 5. *p < 0.01, Student’s t test).
IMPROVED HEMOCOMPATIBILITY OF TETRAGLYME PLASMA COATINGS
833
when compared with uncoated glass or FEP (p < 0.01).
This showed that tetraglyme coatings activated the
intrinsic coagulation cascade less than uncoated controls. Glass had the shortest clotting time, suggesting
that it was potent in activating the intrinsic coagulation
cascade.
Thrombus deposition in an in vitro model
of catheterization
Figure 6. Thrombin generation by surface adherent platelets
to untreated and tetraglyme plasma treated Biospan, glass,
and PE disks. Open bars, untreated samples; solid bars, tetraglyme plasma treated samples. Error bar represents SD. (N ¼
5, *p < 0.01, Student’s t test).
tetraglyme-coated samples showed decreased thrombin
generation when compared with uncoated samples. PE
had the most thrombin generation while Biospan had
the least thrombin generation. It is notable that although
Biospan had much lower platelet adhesion than glass
and PE (Fig. 5), there was not much difference in thrombin generation for Biospan, glass, and PE (Fig. 6).
Recalcified plasma clotting times
As shown in Figure 7, tetraglyme coating of glass or
FEP extended the clotting time of recalcified plasma
The importance of fluid dynamics in the thrombotic
process, especially disturbed flow that enhances interplatelet collisions and transport of thrombotic components to the biomaterial surface, makes models that
incorporate blood flow particularly relevant to the
assessment of device thrombosis. Thus, an in vitro
model that incorporated disturbed blood flow was utilized to assess the efficacy of tetraglyme-coated catheters in reducing catheter thrombosis.28
As seen in Figure 8, the amount of end-point thrombus (measured as adherent 111In platelets) on the tetraglyme-coated devices was significantly less than the
uncoated control (p < 0.05, paired t-test, n ¼ 5), as measured in this bovine in vitro flow model (Fig. 1). SEM examination of the sample corroborated the quantitative
data, and showed that the tetraglyme-coated devices
had substantially less thrombus on the surface compared with the uncoated control (Fig. 9).
DISCUSSION
Thrombosis and embolism remain major challenges
for medical devices that are used in contact with blood.
Therefore, anticoagulant therapy is usually given to
patients with cardiovascular implants. However, difficulties occur with the use of anticoagulants (e.g. bleeding episodes). Thus there is still a great need for materials that have reduced reactivity with blood. Since pro-
Figure 7. Clotting time of recalcified platelet poor plasma in the presence of different materials. All samples were put in the 24well nontissue culture treated PS plate. ‘‘Polystyrene’’ sample means the well plate only. (N ¼ 6, *p < 0.01, Student’s t test).
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
834
Figure 8. Thrombus accumulation on surfaces studied in the
catheter model. The results are expressed as a percent of the
uncoated control. The tetraglyme-coated sample showed a
statistically significant reduction in thrombus accumulation
(N ¼ 5, *p < 0.05, Student’s t test).
tein adsorption onto biomaterials takes place prior to
cellular interaction with the surfaces, and is known to
promote platelet adhesion and activation, control of the
CAO ET AL.
amount and composition of the adsorbed protein is a
good strategy to improve blood compatibility.
Our previous studies have indicated that Fg and vWf
are the most important plasma proteins in mediating
platelet adhesion.12,18 Thus a surface that could resist
the adsorption of Fg and vWf would be expected to
have reduced platelet adhesion. On the other hand,
reduction of Fg and vWf adsorption should not come at
the price of allowing increased adsorption of other platelet adhesive plasma proteins. Adhesive proteins such
as Vn and Fn, when present on surfaces in high
amounts (e.g., by preadsorption with pure solutions of
these proteins), were able to mediate platelet adhesions
as well.18,26
Driven by these considerations, we tested whether
plasma-deposited tetraglyme coatings previously
shown to be highly Fg and vWf resistant would also
resist the adsorption of other blood proteins. Results
from SPR suggested that plasma-deposited tetraglyme
coating greatly reduced total protein adsorption, even
when exposed to 100% human blood plasma.
Figure 9. SEM images of surfaces in the catheter model. (a,b): Magnification 27; (c,d): Magnification 200. (a,c): Tetraglyme
plasma coated PE catheter; (b,d): Uncoated PE catheter.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
IMPROVED HEMOCOMPATIBILITY OF TETRAGLYME PLASMA COATINGS
The reason why plasma-deposited tetraglyme coatings
are highly protein-resistant is not fully understood. We
believe the ability of plasma-deposited tetraglyme coatings to tightly structure water is the reason that they exhibit strong inhibition of protein adsorption. This ability
is thought to be associated with two factors. First, the
ether linkages in the tetraglyme coatings bind and tightly
structure the water into ‘‘ice-like’’ forms. Second, the
plasma deposition process creates relatively short ethylene oxide (EO) chains that are able to fill in and cover the
surface and achieve a high surface density of EO units. In
addition, the conformation of the EO chains in the tetraglyme coating may also play a role in inhibiting protein
adsorption. Li et al.31 found that there was an optimal
packing density of oligo ethylene glycol self-assembled
monolayers (OEG-SAMs) to resist protein adsorption. For
the most tightly packed OEG-SAMs, protein adsorption
was higher than on less densely packed OEG surfaces,
apparently because the high-packing made it more difficult for the access of water molecules to the OEG. Since
the tetraglyme chains produced from the deposition process do not self assemble, but are more likely arranged in
a random orientation to the surface with many chains
parallel to the surface plane, the high surface density of
EO chains is unlikely to limit the accessibility to water
molecules. AFM force measurements by Bremmell et al.19
showed a steric repulsive nature of similar PEO-like coatings. Thus it is possible that tetraglyme plasma coatings
exhibited similar steric–entropic barrier nature.
Antonsen and Hoffman32 and Tanaka et al.33–35
showed that ‘‘freezing bound water’’ appeared to be
critical in making polyethylene glycol and poly(2methoxyethyl acrylate) (PMEA) repel protein. The EO
functional group in the tetraglyme glow discharge-deposited coating may cause a similar freezing bound
water effect and make it highly protein resistant. If protein adsorption is driven by the entropic increase associated with ‘‘melting’’ by displacement of surface-bound
water, and if EO units create tightly bound ‘‘frozen’’
water, then the EG units may prevent melting, thus
eliminating the driving force for protein adsorption.
Tanaka et al.33–35 also suggested that adsorbed proteins on PMEA retained a more native conformation
than when they were adsorbed on poly 2-hydroxyethylmethacrylate (PHEMA). Even though the hydrophobicity of PMEA and PHEMA are similar, the Fg bound to
PMEA was not only lower in amount, but was less
active in binding platelets, and retained more of its
native conformation. On the surface of tetraglyme coatings exhibiting very low Fg adsorption, the tight binding of water may allow adsorbed Fg to be in a more
native conformation with consequent lower platelet
reactivity. However, investigation of the conformation
and orientation of adsorbed proteins on tetraglyme
coatings, for example, by probing the availability of platelet binding epitopes36 is difficult, because the total
amount of adsorbed protein was very low.
835
Although the tetraglyme plasma coating greatly inhibited total protein adsorption, there were still some
platelets adherent to the surface. Whether these sparsely
adherent platelets are bound to the residual adsorbed
Fg or vWf, or possibly by other platelet adhesive proteins such as Vn and Fn, is still not clear. However, it
seems mostly likely that the residual platelet adhesion
is due to the small residual Fg adsorption, given the extreme sensitivity of platelets to adsorbed Fg. Further
studies to identify all the proteins adsorbed from plasma
onto tetraglyme glow discharge deposited surfaces may
provide more information and insights about the relationship between protein adsorption and blood cells
attachment and activation.37 For example, in a recent
report by Elbert and coworkers,38 it has been shown that
a plasma protein named ‘‘serum amyloid P’’ was found
to be critical in mediating monocyte adhesion, which
was not detected before by other techniques.
On the other hand, assuming that all the platelet adhesion related proteins have been identified, it is still a
challenge to make a material that resists the adsorption
of all the adhesive proteins. This requires a further
understanding of the mechanism of protein–material
interactions. We believe that the residual adsorption to
our ultralow protein adsorption tetraglyme coatings is
due to two factors. First, there are probably small areas
of the substrate that are not well covered by the plasma
coating. Second, the small content of hydrophobic fragments on the tetraglyme coating that are inevitably generated during the plasma deposition process may also
adsorb proteins.
Although it is known that platelet adhesion is mainly
mediated by adsorbed Fg under static situations, a comparison of Figures 3 and 5 showed that the highest platelet adhesion did not occur on the surface that had the
highest Fg adsorption (uncoated PE), but on uncoated
glass. This was probably due to the fact that the potency
of adsorbed Fg in supporting platelet adhesion depends
on both the surface it is adsorbed to and the adsorbed
amount, which is associated with variable exposure of
the platelet binding epitope that can be measured with
monoclonal antibodies.36
Platelets adherent to Biospan were in a more procoagulant state than platelets on PE and glass. The rates of
thrombin formation on tetraglyme-coated Biospan,
glass, or PE in the presence of adhered platelets were
much lower than on the uncoated surfaces (Fig. 6), but
the reduction in thrombin formation by the tetraglyme
coating was not as large as the reduction in platelet adhesion. These results differed from that from previous
thrombin formation studies we did with tetraglymecoated tubes,16 in which the thrombin formation was
much lower than reported here. We believe that the difference may be due to the use of a PS well to contain the
flat surfaces in the current studies, whereas in the tube
studies the only surface present was the inside wall of
the tetraglyme-coated tube.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
836
In the clotting time study, glass had the shortest clotting time when compared with other surfaces. This was
expected, as it is well known that surfaces such as glass
initiate contact activation by adsorbing prekallikrein
(PK), high-molecular-weight kininogen (HMWK), factor XI, and factor XII. In contrast, the tetraglyme coatings, comprised mostly of short ethylene glycol chains,
are highly protein-resistant and they probably inhibit
the adsorption of PK, HMWK, factor XI, and factor XII,
thus decreasing the activation of the intrinsic coagulation cascade. PS had a longer clotting time than FEP.
This is probably due to the differences in their adsorption of clotting factors.
In vitro models that incorporate disturbed blood flow,
such as the one used in the current study, provide a
more realistic assessment of device-induced thrombosis.
The conditions (e.g., blood flow, hemostasis) in in vitro
models are relatively more controlled than in vivo models. This allows the experiment to be focused on a specific variable (e.g. surface chemistry), with other relevant parameters (e.g. disturbed flow) being present but
remaining relatively constant. However, it is important
to note that there are differences between in vitro and in
vivo models that make it difficult to directly extrapolate
in vitro results to in vivo situations. Items that are present in vivo but absent in vitro include (i) a more dynamic
and comprehensive hemostatic system (e.g. thrombolytic pathway) and (ii) less activated blood components
(e.g. platelets and clotting factors). Further, in vitro models require at least some anticoagulation of blood to prevent clotting of the entire pool of blood during the
experiment, while this is not necessarily in the case of in
vivo situations. Also, the in vitro system allows for the
assessment of acute-phase thrombosis (*hours), but
does not provide any information on chronic thrombogenicity (*days or *months). Thus, while the thromboresistance exhibited by the tetraglyme-coated catheters under disturbed flow conditions in vitro is encouraging, evaluation in long-term implants in vivo is
required to reveal the ability of tetraglyme coatings to
resist chronic thrombus formation.
In studies published using a long-term ex vivo shunt
model of platelet interaction, platelet consumption was
noted to increase with increasing equilibrium water
content of radiation-grafted hydrogels.39,40 PEO-like
polymers are associated with high water contents.
However, we believe our present results are not contradictory to this previously published water content
trend. First, our tetraglyme coating is so tightly crosslinked that it exhibits little swelling and, in fact, appears
neither highly hydrophobic nor hydrophilic by contact
angle measurement.41,42 A study by Bremmell et al.19 by
AFM force measurements suggested that their similar
PEO-like plasma coatings possessed a steric repulsion
force indicative of hydrogel compression. Thus tetraglyme coatings may exhibit the steric repulsive nature
to some extent. Second, PEO has no hydrogen bond doJournal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
CAO ET AL.
nor groups, whereas all the polymers assessed in the
report by Hanson et al.39,40 did contain hydrogen bond
donor groups. Hydrogen bond donor groups were
postulated to be responsible for blood interactions.43,44
We thank Winston Ciridon for assistance with plasma deposition, Jim Hull for the AFM study, and Dr. Steve Golledge,
Dr. Lara Gamble, and Dr. Dan Graham for ESCA operation
and analysis. The SPR, ESCA, and AFM experiments were
performed at the National ESCA and Surface Analysis Center
for Biomedical Problems. We thank the many individuals
who donated blood for our studies.
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