The effects of increased nitric oxide levels on platelet ...



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

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 fibrinogen-coated 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 μL.

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, 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 μm plastic sheets separated by a 200 μm 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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(B)[pic]

(C)[pic]

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.

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 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 16-gauge 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 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 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.

[pic]Figure 3. QCM results for collagen assembly

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Figure 4. QCM results for fibrinogen assembly

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.

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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.

Tests were performed using L-arginine concentrations varying from 1-25 μL. Pearson’s correlation test (p ................
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