The Effects of Increased L-Arginine Concentrations on Platelet Thrombus Formation
over Fibrinogen and Collagen
Kimberly Schipke, Biomedical Engineering
Mississippi State University
Faculty Advisor: Steven A. Jones, Associate Professor, Louisiana Tech University
Randa Eshaq, Biomedical Engineering, Louisiana Tech University
August 2005
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I. Abstract
Myocardial infarctions result from thrombus formation within the coronary artery.
Platelets are responsible for arterial thrombosis, and they produce and secrete inhibitors
that prevent their function in a control mechanism. L-arginine converts to nitric oxide and
L-citruline within the platelets when catalyzed by nitric oxide synthase. The role of
platelet-synthesized nitric oxide remains unclear; therefore, a need exists for a clinical
device for that can assess the amount of platelet activation, aggregation, and adhesion in
an individual’s blood with increasing amounts of L-arginine.
Experiments were performed to determine how the variation of L-arginine
concentrations affects platelet adhesion when exposed to collagen and fibrinogen. A
previously designed platelet analyzer was used to test adhesion patterns. Platelet-rich
plasma with differing L-arginine levels was pumped across fibrinogen-coated and
collagen-coated channels at a shear rate of 1500/s. The channels were stained with
acridine orange and observed under fluorescent microscopy. Images were processed
through a MATLAB program to determine the percentage of platelet adhesion.
The results from the experiment show that an increase in L-arginine production of
nitric oxide is directly proportional to the decrease in platelet adhesion on fibrinogencoated channels. The percent adhesion initially decreased on the collagen-coated
channels but the increase of L-arginine had little effect in concentrations higher than
1 L.
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II. Introduction
Almost all myocardial infarctions are associated with the thrombotic occlusion of
the coronary artery. (Prentice) Platelets’ main function is hemostasis, and they are major
contributors of arterial thrombosis. They produce and secrete their own inhibitors, the
most effective being nitric oxide. Nitric oxide (NO) is produced within the platelets when
NO synthase catalyzes the conversion of L-arginine into L-citruline. Due to its small size,
NO has a high diffusivity constant; therefore, it can perfuse through the thrombus in a
short period of time, making it an effective inhibitor. (Freedman et al.)
Initiation of thrombosis occurs when circulating platelets are activated by exposed
collagen and fibrinogen, allowing the accumulation of a single layer of platelets that
supports thrombin generation and the formation of platelets into aggregates. Under static
conditions, collagen is able to activate platelets and cause shape change, aggregation, and
secretion without the assistance of cofactors. The p65 platelet receptor for type I collagen
appears to be linked to the generation of nitric oxide. (Michelson, p197-198) As for
fibrinogen, its interaction of flowing platelets is predominantly instantaneous and
irreversible. Usually less than 10% of platelets that become attached to fibrinogen move
from the initial contact site by a distance greater than their own diameter. (Michelson,
p216)
For these reasons, the adhesion patterns of platelets over fibrinogen and collagen
with increasing amounts of L-arginine were investigated. The percent adhesion was
expected to decrease with increasing amounts of L-arginine due to an increase of nitric
oxide production. The role of platelet-synthesized nitric oxide remains unclear; therefore,
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experiments were performed to examine its function and compare the amount of adhesion
between two different substrates.
III. Materials and Methods
A. Experimental Design
A perfusion system was developed to monitor thrombus formation in a simple,
well-controlled manner. The model replicated a blood vessel and allowed the analysis of
the effects of specific proteins on platelet thrombus formation. The model was composed
of double sealed Plexiglas® plates that bound microchannels in place to prevent leakage.
Teflon nuts of outer diameter 1/16" were inserted into threaded holes of diameter 1/4.28"
in the upper plate. Ferrules with an outer diameter of 1/16" were fixed at the bottom of
the nuts, and FEP Teflon® tubes with the same diameter were inserted into ferrules to
provide inlets and outlets for the injection of blood, as shown in Figure 1. A UV-curable
sealant was used to hold the tubing, ferrules, and nuts together. To secure the two plates
together, screws were inserted surrounding the microchannels to further prevent leaks.
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Figure 1.(A) Side view of micro-fluidic system, (B) top view of micro-fluidic system
The microchannels were cut out of plastic silicone elastomer sheets (McMaster)
and coated with specific proteins that served as a replica of a blood vessel. Each channel
had a width of 2mm, a length of 5cm, and a height of 180µm. The silicone elastomer
sheets were composed of two 110 m plastic sheets separated by a 200 m silicone sheet.
In order to construct the channels, the top two layers were cut out and removed, leaving a
layer of plastic to deposit collagen and fibrinogen. The top sheet of plastic was then
removed to expose silicone so that it could act as a sealant between the channels and
Plexiglas plates to prevent leakage shown in Figure 2.
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(A)
(B)
(C)
Figure 2. Side view of microchannels (A) Uncut elastomer sheet (B) Channels
cut and removed (C) Layered with substrate and silicone exposed.
To ensure that layers were assembled, the fibrinogen and collagen were tested
using a quartz crystal microbalance (QCM). The QCM has a piezoelectric quartz crystal
in between a pair of electrodes. The frequency shift of the quartz crystal resonator is
directly proportional to the added mass on the electrode (Jonnalagadda et al.). Thus, the
change in the frequency was used to assess the change in the thickness of each added
layer of mass.
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20mM Tris Buffer was made by dissolving 1.21g of TRIS buffer (20% by weight)
in 500mL of DI water. TRIS buffer is stable at pH 7.5; therefore, the following polyion
solutions and proteins were altered to pH 7.5 as well. To make 3mg/mL of Poly-Sodium
4-Styrene-Sulfonate) (PSS), 45mg of PSS were dissolved in 15mL of TRIS buffer.
1.473mL of 100% Poly Ethylene Imine (PEI) were added to 150mL of TRIS buffer to
yield a 1mg/mL concentration. In order to obtain a 3mg/mL concentration of Poly
Dimethyl-Diallyl-Ammonium chloride (PDDA), 10.5mL of PDDA (20% by weight)
were added to 100 mL of DI water. 1.42g of sodium phosphate were dissolved in 100mL
of DI water to make 0.1M Phosphate Buffered Saline (PBS). To make 1mg/mL
concentration of fibrinogen, 100mg of fibrinogen were dissolved in 100mL of TRIS
buffer. 50mL of collagen (100% by weight) were added to an acetic acid solution
comprised of 1.1mL of glacial acetic acid (99.5% by weight) added to 98.9 mL of DI
water. Acridine Orange (AO), the fluorescent dye, was prepared by dissolving 100mg of
AO in 100mL of DI water to attain a concentration of 1mg/mL. 1mM L-Arginine
solution was prepared by dissolving 1.742mg of L-Arginine in 10mL of DI water.
Layer-by-Layer (LbL) Self-assembly was the technique used to immobilize
fibrinogen and collagen onto the microchannels. LbL is a process of layering by
alternating oppositely charged polyion solutions onto a substrate. PDDA, a strong
polycation, and PSS, a weak polyanion, were used to form a negatively charged layer, in
which the positively charged collagen adsorption could occur. The polycation, PEI, and
PSS were used to form a positively charged layer to adsorb the negatively charged
fibrinogen. In order to coat the channels with fibrinogen, the channels were immersed for
10 minutes, a time optimized for the adsorption of a single layer (1nm in thickness), in
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PEI and PSS solutions, then rinsed in TRIS buffer and dried with nitrogen gas, alternating
six times to allow adsorption of six foundation layers. The positively charged channels
were then immersed in fibrinogen for 20 minutes, alternating four times with PEI.
Channels were coated with collagen in the same manner by alternating PDDA and PSS to
form six bilayers with four terminal bilayers composed of PSS and collagen.
The resonator was cleaned in a solution comprised of 50% DI water, 49% alcohol,
and 1% potassium hydroxide for two minutes. It was then dried with a steady stream of
nitrogen and placed in the QCM to measure the initial frequency. The resonator was
immersed in PDDA for 10 minutes, washed in TRIS buffer for 30 seconds, and dried
with nitrogen. The resonator was placed on the QCM to measure frequency to ensure the
deposit of layers. It was then immersed in PSS for 10 minutes, washed, dried, and
measured. The previous steps were repeated to deposit six bilayers of PDDA and PSS.
PDDA was replaced with collagen and submerged for 20 minutes, repeating steps to form
four bilayers ending in collagen. Using the previous method of layering collagen, six
primary bilayers of PEI and PSS were deposited. PSS was replaced with fibrinogen and
immersed for 20 minutes, resulting in four bilayers of PEI ending with fibrinogen.
Blood was collected from cow #818 at the Louisiana Tech Dairy Farm. Blood was
obtained from the same cow to ensure consistent results. The cow was stuck with a 16gauge needle in the milk vein. Blood was allowed to flow freely for three seconds before
being collected in conical tubes. Sodium citrate was added to blood in a 1:9 ratio. The
blood and sodium citrate mixture was immediately turned over four times to a
homogeneous mixture of the anticoagulant. Blood was centrifuged at 1500 rcf for 15
minutes at 25 ˚C using a Hermle Labnet Z323K. The blood separated and the top layer of
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platelet-rich plasma (PRP) was extracted. PBS was added to PRP to obtain the original
concentration. L-arginine was added to 1mL of PRP in portions of 1 μL, 5 μL, 10 μL, 15
μL, and 25 μL, leaving a sample of PRP unaltered to be used as a control.
B. Testing
PRP was pumped via a syringe pump at a shear rate of 1500 s‾ ¹, corresponding to
the shear rate found in arterioles as well as the optimal shear rate of platelet adhesion.
The altered PRP was purged through 1/16" FEP Teflon® tubing into six parallel
microchannels, each of which contained a small area that served as the model blood
vessel. This area consisted of a silicone elastomer sheet coated with the structural
proteins, fibrinogen and collagen. After 30 seconds of blood flow through each
microchannel, the channels were allowed to incubate for 10 minutes, the substrates were
washed in PBS for 30 seconds to remove excess platelets and allowed to air dry. The
microchannels were then dyed with fluorescent acridine orange (AO) for 20 minutes.
Excess acridine orange was then washed off in PBS for 15 seconds and allowed to air
dry.
The microchannels were then analyzed with a Nikon TS100 Eclipse Epi
fluorescence microscope with a FITC B-2A filter. Digital images were taken of platelet
adhesion along the middle of the microchannel. An image processing MATLAB program
was used to determine the percent adhesion of platelets on each microchannel. The image
was sent through a two-dimensional median filter that removed noise present in the
images. The program then performed background subtraction and thresholding to
eliminate any non-uniform illuminated objects. The RGB image was then converted to
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grayscale and then to a binary image, where the platelet coverage was determined by the
number of “on” pixels found.
C. Statistical Analysis
Numerical data were expressed as the mean ± standard deviation. The statistical
analysis was performed using linear regression analysis correlating L-arginine levels with
platelet adhesion. Probability values less than 5% were considered to indicate
significance (p< 0.05).
IV. Results
QCM
As previously stated, the LbL assembly of collagen and fibrinogen were tested
using the QCM to ensure deposition of layers. The initial layers of PDDA and PSS of
collagen had a steady decrease in frequency which proved the addition of layers because
the change in frequency and the change in thickness are directly proportional. After
collagen was added, the frequency increased which suggests that several layers were
stripped off. The frequencies from collagen to collagen layer continued to decrease which
proved that collagen was successfully assembled as shown in Figure 3.
QCM analysis for fibrinogen was also performed to guarantee the deposition of
layers. As with collagen, the frequency continued to decrease on the foundation layers of
PEI and PSS. Layers were also stripped away once fibrinogen was added, but the
frequencies decreased on the fibrinogen to fibrinogen layers. Therefore, the adsorption of
layers was successful. The QCM results for fibrinogen are shown in Figure 4.
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Layer-by-Layer Self Assembly of Collagen on QCM
180
10,000
160
8,000
120
7,000
6,000
100
5,000
80
4,000
60
3,000
40
2,000
20
1,000
la
ge
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C
ol
P
SS
la
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ol
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SS
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D
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A
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SS
P
D
D
A
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SS
P
D
D
A
P
al
-
-
In
iti
Thickness (nm)
140
D Frequency (Hz)
9,000
Layers Deposited on Substrate
Figure 3. QCM results for collagen assembly
Layer-by-Layer Self Assembly of Fibrinogen on QCM
70
4,500
3,000
40
2,500
30
2,000
1,500
20
1,000
10
500
Figure 4. QCM results for fibrinogen assembly
og
en
EI
br
in
Fi
br
in
P
og
en
EI
Fi
br
in
Layers Deposited on Substrate
P
og
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In
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Thickness (nm)
3,500
50
D Frequency (Hz)
60
4,000
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L-arginine Tests
Varying concentrations of L-arginine were added to bovine PRP, and platelet
adhesion over fibrinogen and collagen were compared at a shear rate of 1500/s. Results
of these tests are shown in Figure 5.
Percent Adhesion on Fibrinogen and Collagen
6
Percent Adhesion (%)
5
4
Fibrinogen
3
Collagen
2
1
0
Control
1 uL
5 uL
10 uL
15 uL
25 uL
Concentration of L-Arginine (L)
Figure 5. Average percent adhesion of fibrinogen and collagen
The L-arginine standard was a 1 M solution (1.742 mg L-arginine in 10 mL H2O).
V. DISCUSSION
Myocardial infarctions are associated with high platelet reactivity and
coagulation within blood vessels. NO production reduces the amount of platelet adhesion,
and is produced within the platelets by L-arginine. (Freedman et al.) Therefore, the study
of the effects of increased L-arginine levels on platelet adhesion to fibrinogen and
collagen was performed to test whether the platelet analyzer was able to differentiate
between the two substrates.
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Tests were performed using L-arginine concentrations varying from 1-25 L.
Pearson’s correlation test (p<0.05) was used to determine the statistical significance
between L-arginine levels and platelet coverage. The p-value shows a significant
decrease in platelet coverage on fibrinogen with an increase in L-arginine concentration.
The L-arginine concentrations had an initial effect on platelet coverage over collagen, but
subsequent increases in L-arginine concentrations had little to no effect. The results of
these experiments show that the percentage of platelet coverage to fibrinogen-coated and
collagen-coated surfaces at a shear rate of 1500/s decreases with an increase in L-arginine
concentration.
VI. CONCLUSION
The results show that platelets adhere to fibrinogen at a higher percentage than
collagen. The adhesion pattern for fibrinogen shows that with increasing concentrations
of L-arginine, less adhesion is seen. The increase of adherence at 1 L and 25 L are
believed to be caused from the incubation of platelets within the channel, along with the
staining of AO to fibrinogen. Adhesion of platelets to collagen initially decreased and
leveled off with increasing amounts of L-arginine. These results show that platelet
adhesion to collagen reaches a steady state with concentrations of L-arginine higher than
1 L.
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VII. FUTURE WORK
The main objective of these experiments is to devise an instrument that can
measure the critical parameters of platelet function including platelet activation,
aggregation, and adhesion. To continue this endeavor, further testing of platelet function
over different substrates seems to be the most logical next phase of experimentation. The
experiments will be repeated using the previously designed platelet analyzer over
fibronectin and von Willebrand factor. Fibronectin and von Willebrand factor are other
proteins found within the blood vessel wall that promote platelet activation and adhesion.
Once these experiments are performed, the percent adhesion with increasing
concentrations of L-arginine can be directly compared between all four substrates.
VIII. ACKNOWLEDGEMENTS
First and foremost, I would like to thank my parents for being supportive in every
aspect of my life. I would also like to thank my siblings for being good role models and
motivating me to succeed. I thank the NSF for realizing the funding for research is crucial
to our future. I owe many thanks to Dr. Steven A. Jones for allowing me to participate in
this prestigious program. This experience has allowed me to apply the knowledge that I
have accumulated over the past three years and motivates me to learn more. I would like
to thank Erich Stein for helping me use the MATLAB program and Javeed Shaikh
Mohammed for showing me how to use all the different machines required to perform my
experiments. I would also like to thank the IfM staff for tending to my every need.
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IX. REFERENCES
Freedman J, Loscalzo J, Barnard M, Alpert, Keaney J, Michelson A. Nitric Oxide
Released from Activated Platelets Inhibits Platelet Recruitment. J. Clin. Invest.
1997; 100:350-356.
Jonnalagadda, K. Platelet Adhesion in Microchannels: Effects of Protein Coatings and
Glucose Concentrations, 2004. Thesis, Department of Biomedical Engineering,
Louisiana Tech University.
Michelson, Alan D. Signal Transduction During the Initiation, Extenstion, and
Perpetuation of Platelet Plug Formation. Platelets. 2002. p197-198.
Michelson, Alan D. Platelet Thrombus Formation in Flowing Blood. Platelets. 2002.
p216.
Prentice, Colin R.M. Pathogenesis of Thrombosis. Haemostasis. 1990. 20:50-59.
X. APPENDICES
The following is the MATLAB program that was used to calculate the percent
adhesion of platelets within the microchannels. The program was derived by Kanvasri
Jonnalagadda.
for x=02:1:99
X=num2str(x); //Converts a number to a string
fileName=strcat(strcat('DSCN00',X),'.jpg'); //Concatenate strings
BW1=imread(fileName); //Read image from graphics file
background= imopen(BW1,strel('disk',30)); //Performs morphological
operation
S=imsubtract(BW1, background); //Subtracts the background of the
image
[l,m,n]=size(S) //Determines the size of the image
W=zeros(l,m,n);
r=S(1:l,1:m,1);
g=S(1:l,1:m,2);
b=S(1:l,1:m,3);
//Performs thresholding and segmentation
for i=1:l
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for j=1:m
if r(i,j)<55
W(i,j,1)=0;
else
W(i,j,1)=256;
end
if
g(i,j) < 80
W(i,j,2)=0;
else
W(i,j,2)=256;
end
if(b(i,j) < 72 )
W(i,j,3)=0;
else
W(i,j,3)=256;
end
end
end
%figure,imshow(uint8(W))
g=rgb2gray(uint8(W)); //Converts an RGB image to a grayscale image
MF=medfilt2(g); //Performs 2-D median filtering by using 3x3
neighborhood
%figure,imshow(MF) //Shows the image
%pixval
T = roicolor(g,76,255); //Select region of interest based on color
and converts to a binary image
%figure,imshow(T)
u=bwarea(T); //Computes the area of objects in binary image
z=l*m
p=(u/z)*100
PercentArea(x-01)=p
end