I recently graduated from university with a BSc honours in biomedical science. I currently work voluntarily at a local youth centre, but for the future I am looking to work in the field of biosciences, preferably as a biomedical scientist or as a scientific researcher and next year I plan to do an MSc specialising in haematology. I specialise in all areas of biomedicine particularly haematology, clinical chemistry, medical microbiology and medical immunology

To determine the effects of the reactive nitrogen species, peroxynitrite, on the properties of fibrin polymerisation and lysis.

Introduction
The clotting system in human blood consists of many proteins and factors in a complicated system maintained in homeostasis in health. Blood is maintained in fluid state when circulating and in the event of a wound a clot is formed at the site of injury, preventing blood loss. Factors and proteins are also present which break down the clot and prevent spontaneous clotting. The numerous factors and proteins involved in this are all interlinked in a complex cascade, with loss of function in certain factors and proteins leading to irregularities in blood clotting, leading to either prothrombotic, or bleeding tendencies.

In health inactive forms of the clotting factors become activated during a bleed, leading to the cascade of reactions seen below in fig. 1. The clotting enzymes are proteases which carry out the cleavage of the clotting factors into active proteases. These activated factors then act to in turn cleave further clotting factors. This set of reactions is known as the coagulation cascade, with each reaction step amplified. Factor VII starts off the cascade, with a series of reactions leading to the formation of factor Xa, which cleaves the precursor prothrombin to the active thrombin (Berkner, 2001).

Thrombin then acts to convert fibrinogen to fibrin, an insoluble molecule, many fibrin molecules then crosslink and act to plug the wound. This cross linking is helped by the protein XIIIa which is cleaved from factor XIII by thrombin and help (Berkner, 2001). The clot however is not permanent and is lysed. Fibrin once formed activates a protein known as tissue plasminogen activator (tPA). tPA breaks down a precursor protein, plasminogen, into the active form plasmin, a serine protease that targets certain regions (mostly lysine residues) of the fibrin network so that the fibrin goes from being insoluble to soluble (Doolittle, 2001).

One group of molecules thought to have an affect on the clotting properties of blood and particular components of the system are reactive nitrogen species (RNS). These are formed when nitrogen oxide (NO) present in the body and produced by nitric oxide synthases (NOS). It is involved in many biological reactions and as a result RNS are formed as seen in fig 2. The particular RNS being investigated in the experiments is peroxynitrite (ONOO) which is produced by the reaction of NO with O2- (produced by xanthine oxidase and NADP(H)) (Patel et al, 2001).

The reactive nitrogen species, ONOO has been shown to be involved in many pathological conditions due to its high reactivity with certain proteins. Specifically it has been shown that ONOO causes oxidation of tyrosine residues of proteins, even though anti-oxidant agents are present in the body such as thiols and uric acid. The numbers of nitrated proteins in patients with health problems such as atherosclerosis and systemic sclerosis have been shown to be increased considerably compared to healthy individuals (Lupidi et al, 1999). Also in it has been shown that it depletes antioxidants and thus causes the oxidation of LDL (low density lipoproteins). This oxidation occurring in blood vessels may act to aggravate atherosclerosis (Halliwell, 1996). In this experiment therefore the effects of the reactive nitrogen species, ONOO, on the properties of blood coagulation will be tested.

The aim of this experiment was to study the affects of the reactive nitrogen species, ONOO, in vitro, on the properties of blood coagulation and lysis by measuring different aspects of the clotting system, namely fibrin polymerisation, fibrin lysis and fibrin compaction.

It was estimated that peroxynitrite would have an overall detrimental affect on all aspects of the clotting system. Specifically that ONOO would decrease the rate of polymerisation of fibrin from fibrinogen, decrease the zone of lysis of the fibrin clot and make the fibrin clot weaker and therefore increase fibrin % compaction.

METHOD:

Freeze dried fibrinogen and ß-Ala-Gly-Arg-p-nitroanilide (thrombin generating substrate) obtained from Sigma, Poole, UK. Bovine thrombin and freeze dried fibrinogen obtained from Diagen, Thame, UK.

Human citrated plasma obtained from national blood service which permission had been obtained for by Dr. Michael Gordge. Dr. Michael Gordge has also obtained ethical permission from the University of Westminster Ethics committee for permission to use human blood in the experiments (see appendix)

Preparation of peroxynitrite:

3 solutions were prepared as follows. Solution A: an equal mixture of 0.6M HCl and 0.7M of H2O2, solution B 0.6m NaNO2 and solution C 1.5M NaOH. A glass collection tube containing 500 mg of MnO2 was also prepared. All three solutions were put on ice to cool.

The ice cold solutions were loaded onto the three 10 ml syringes and placed into a three-way tap above the pre-cooled glass tube containing MnO2. Solutions A and B were injected (at the same time) at a slow rate of approximately 10 seconds for 5 ml. Once the solutions reached the third syringe, solution C was injected at the same rate.

The mixture was vortexed to remove any oxygen generated and then freeze fractionated by incubating the tube at -20°C for 5 minutes. The product was allowed to just thaw before the top layer (yellow) was removed, which contained a relatively higher concentration of ONOO.

Concentration of peroxynitrite was estimated from its absorbance using a co-efficient of 1670 M-1 cm-1 at 302nm:
ONOO diluted in NaOH (10 mM) 100 fold gave an absorbance reading of 0.658
S302= 1670 M-1 cm-1
0.658/1670 x 1000 = 0.394 mM
Concentration in the original solution was therefore 0.394 x 100=39.4 mM
This concentration was stored at -20°C in 0.5 mL aliquots.

Preparation of fibrinogen:

50 mg of fibrinogen (Sigma, Poole, UK) was weighed out and diluted in 5 ml of TBS saline to make a concentration of 10 mg/ml. 1 ml aliquots were stored at -20°C prior to use.

For experiments using fibrinogen supplied by Daigen (Thame, UK) fibrinogen was prepared according to manufacturers instructions.

Preparation of TBS:

To make 20 mM of Tris (final volume 250 ml):
605 mg of Tris (to make 20 mM) and 2175 mg of NaCl (to make 150mM)
pH was adjusted 7.4 with HCl and NaOH using a pH meter.

Preparation of thrombin:

Thrombin (Diagen, Thame, UK) was diluted in 1mL of water according to manufacturers’ instructions to give a final concentration of 200 U/ ml. This was further diluted in TBS to give 50 U/ml. When used in experiments thrombin was added at 0.5 U/ml final concentration.

Preparation of decomposed ONOO:

1 ml aliquots of ONOO were left at room temperature for 3 days to allow for decomposition of the ONOO. When used the decomposed ONOO was diluted in 10 mM of NaOH, in a similar way to the active ONOO.

Dilution of ONOO for use:

Peroxynitrite was diluted in 10 mM NaOH to give stock solutions containing 4mM. 2 mM, 1 mM, 0.5 mM and 0.25 mM were added into experiment samples at 1/20 dilutions to give final concentration of 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM respectively.

Fibrin polymerisation (Lupidi et al, 1999):

ONOO (0-200µM) was incubated with fibrinogen for 5 minutes at room temperature. 980 µL from each tube was then transferred to a cuvette and placed into the spectrophotometer. 20 µL of thrombin (final concentration 0.5 U/mL) was added to each cuvette and the absorbance at 350 nm was measured every 15 seconds. Experiments were performed in quadruplicate.

Thrombin generation substrate:

A further experiment was carried out to determine if changes observed in the polymerisation experiment were due to the effects of ONOO on either fibrin or thrombin:

The thrombin substrate, ß-Ala-Gly-Arg-p-nitroanilide (Sigma, Poole, UK) was dissolved at a concentration of 5 mm in 10:1 water/methanol. 90 µL of this substrate was then added to 860 µL of TBS in 6 different LP4 tubes. ONOO (0-200 µM) was then added and the absorbance measured at 405 nm over a period 150 minutes. Graphs of absorbance against time were plotted to investigate the effect of ONOO on the action of thrombin.

Fibrin plate lysis (Lewis, 2001):

ONOO (0-200 µM) was added to 1 mg/ml fibrinogen in 13.5 mL TBS. 75 µL of 200 mM CaCl2 (25 mM) and 50 µL of thrombin (final concentration 0.5 U/mL) were then added to each dish and the contents mixed. The plates were left on a level workbench for 30 minutes to allow the clot to form.

A euglobulin fraction was prepared from human plasma by dispensing 3.6 mL of 2.7 mM acetic acid into a centrifuge on ice. 400 µL of plasma was added to the tube. The tube was then left for 15 minutes to allow the euglobulin fraction to precipitate after which the tube was centrifuged at 2500 rpm for 10 minutes. The supernatant was poured off and drained onto tissue and the white precipitate was redissolved in 400 µL of ice-cold TBS.

30 µL of this precipitate was added to the clotted plates, 5 times, and left for 30 minutes before transferring to a 37ºC incubator:

The plates were left in the incubator for 24 hours before the zones of lysis for each spot were measured. The entire procedure was repeated and a graph plotted of the mean zones of lysis (mm) against concentration of experimental substrate (ONOO).

Experimental treatment of plasma:

To determine whether the results seen in the lysis experiment was due to the action of ONOO on fibrinogen or the plasminogen activator this second experiment was carried out.

3 plates (triplicate) were made up with 10.5 mL of TBS, fibrinogen (final concentration 1mg/ mL), (Diagen, Thame, UK), CaCl2 (final 25 mM) and thrombin (Diagen, Thame, UK) (final concentration 0.5U/mL)). The plates were allowed to clot for 30 minutes on a level workbench.

The euglobulin fraction was prepared as follows: 570 µL of human plasma and 30 µL of ONOO (0-200 µM) were incubated together for 1 hour, before 400 mL of this was added to 3.6 mL of acetic acid (2.7 mM) and chilled on ice for 15 minutes to allow for protein precipitation after which the tube was centrifuged at 2500 rpm for 10 minutes. The supernatant was poured off and drained onto tissue and the white precipitate was redissolved in 400 µL of ice-cold TBS.

The plates were left for 24 hours in a 37°C incubator and the zones of lysis measured. A graph of zone of lysis against concentration of ONOO was plotted to investigate the effect of ONOO on plasminogen activator.

855 µL of human plasma and 45 µL of ONOO (0-200 µM) were placed into appropriately labelled eppendorf tubes. 50 µL of CaCl2 (final concentration 10 mM) and 50 µL of thrombin (Diagen, Thame, UK) (final concentration 1 U/mL) were added to each eppendorf tube and left for 4 hours to allow formation of the clot.

The clotted samples were centrifuged at 8000g for 45 seconds. The volume of liquid expelled from the fibrin network was removed and the volume calculated as a percentage.

The compaction % was then plotted against the concentration of ONOO to determine the effect of ONOO on the fibrin network.

Factor XIII:

This experiment was carried out to determine whether the results from the compaction assay were due to the effect of ONOO on fibrinogen or an effect on factor XIII.

885 µL of human plasma were added to 14 tubes, along with 45 µl of ONOO (0-200µM), 50 µL of CaCl2 (final concentrations 25 mM) to the first set of 7 tubes, with 50 µL of EDTA (final concentration 10 mM) to the second set of 7 tubes. 50 µL of thrombin (Diagen, Thame, UK) were added to all tubes and left to clot over 4 hours before being centrifuged for 45 seconds at 8000g.

The volume of liquid expelled from the fibrin network was removed and the volume calculated as a percentage. The compaction % was then plotted against the concentration of ONOO to determine the effect of ONOO on factor XIII.

All results are expressed as the mean ± SD. Analyses of the effects of peroxynitrite on blood coagulation proteins/factors on all variables were conducted with one-way analysis of variance (ANOVA) with a P value of <0.05 considered significant.

RESULTS

Preliminary tests were done for optimisation of the assay and estimation of reproducibility to determine what concentration of thrombin to use. By using 5 different concentrations of thrombin the varying rates of polymerisation (i.e. rates of reaction) were determined. Using the graph as shown in figure 4 it was then decided to use 0.5 U/ml of the thrombin which produced the best rate of reaction. All concentrations below 0.5 U/ml had rates of reaction that were too slow, and the 1 U/ml thrombin concentration produced a rate of reaction that was too fast which would make it difficult to measure accurately, especially at the steepest point (fastest rate of reaction).

The coefficient of variation, which measures the reproducibility of the results, was calculated. The mean rate of polymerisation and standard deviations were calculated from the rates of polymerisation. The mean rate of polymerisation was 0.00396 /s and the standard deviation was 1.873 x 10-4. From these the coefficient of variation was calculated as being 4.72%. The rate of polymerisation was calculated each time by drawing a tangent at the steepest part of the graph. An example of one of the graphs used is shown below in fig. 5, with results drawn from column 1 in table 1.

	Absorbance (350 nm)
Time	1	2	3	4	5	6
0	0.001	0.002	0.001	0.001	0.001	0.003
15	0.014	0.015	0.020	0.018	0.016	0.020
30	0.074	0.073	0.072	0.069	0.072	0.071
45	0.112	0.110	0.109	0.111	0.108	0.110
60	0.141	0.140	0.138	0.139	0.138	0.138
75	0.162	0.160	0.163	0.162	0.160	0.161
90	0.178	0.179	0.177	0.179	0.180	0.181
105	0.193	0.192	0.189	0.195	0.193	0.195
120	0.203	0.202	0.199	0.206	0.205	0.207
Table 1: Replicate measurements of change in A350 during fibrin polymerisation. This was performed to determine reproducibly. Results from highlighted column are shown fig. 5.

All the rates of polymerisation for the replicates were calculated as shown in table 2 with the standard deviation and overall mean rate of polymerisation shown below:

Rate of polymerisation (abs/s)
1	2	3	4	5	6
0.00404	0.00394	0.00363	0.00419	0.00404	0.00394
Mean rates of polymerisation calculated from gradient of graphs as shown in fig. 5

Mean rate of polymerisation: 0.00396 abs/s
Standard deviation: 1.873 x10-4
CV: 4.72%

Mean rate of polymerisation: 0.00396 abs/s
Standard deviation: 1.873 x10-4
CV: 4.72%

Polymerisation results:

The polymerisation experiment was repeated four times to allow for maximum accuracy and the graph below, fig 6, was plotted on the mean results of all four experiments. It can be clearly seen from this graph that with increasing concentration of the experimental substrate, peroxynitrite. The standard deviation was taken for the mean results and the results plotted as y-error bars on the graph, with the 100 µM concentration results showing the greatest deviations.

Figure 7 shows a graph showing the mean rates of polymerisation of fibrin in peroxynitrite (0-200µM). One way analysis of variance was carried out to determine whether or not there was a significant difference between the calculated mean rates of polymerisation for the increasing concentrations of peroxynitrite.

An F value of 3.807 was calculated (p=0.005) and the critical F value determined as 2.409 (df1=5, df2=48). Because the calculated F value is greater than the critical F value, it was concluded that there was a significant difference between the different concentrations and there effects on the relative rates of polymerisation of fibrin, measured as absorbance over time.

Further fibrin polymerisation tests:

A further experiment was carried out to determine whether this difference in rate of polymerisation was due to the experimental compound peroxynitrite was due to its effect on fibrinogen or thrombin. An effect on thrombin by the peroxynitrite would yield similar results as an affect on fibrin so in the next experiment, fibrin was excluded and the effect of peroxynitrite on thrombin was determined. This was done by using ß-Ala-Gly-Arg-p-nitroanilide diacetate, which when cleaved by thrombin produces a yellow pigment. The rate of production of this pigment was used to determine the rate of action of thrombin in the presence of peroxynitrite. 0µM (using TBS), 0µM (using decomposed ONOO) to determine if the chemicals present in ONOO may be having an affect rather than the active ONOO, and 200µM of peroxynitrite. The results of the rate of action of thrombin are plotted below in fig. 8.

The graph shows no clear distinction between the rates of action of thrombin in the presence of any of the three substrate concentrations. To confirm this one way ANOVA was carried out. The F factor was calculated as 0.0019 (between the concentrations) and the critical F value was calculated as 3.68 (df1=2, df2=15), which shows no significant difference between the concentrations and there affect on the action of thrombin.

Fibrin plate lysis:

Preliminary experiment carried out for this part of the experiment was a measure of reproducibility, i.e. calculating coefficient of variation. By using TBS saline as the control a clot of fibrin was formed on a plate using thrombin and fibrinogen. Once the clot formed, the euglobulin fraction of plasma (containing plasminogen and plasminogen activator) was placed onto the plates (5 spots) and left to lyse over 24 hours. The zone of lysis was measured in mm of the 5 different spots on the plate. The measurements were made bidirectionally, horizontally and vertically, to gain a more accurate zone of lysis. The results are shown in table 3, with the SD, mean and CV below:

Table 3: Zones of lysis measured twice for each spot to get an average size. Repeated 5 times to obtain a coefficient of variation (CV) n=5.
	Size of zone of lysis (mm) after 24 hours		Mean (mm)
1	7	7	7
2	6	6	6
3	6	6	6
4	7	6	6.5
5	7	6	6.5
	Mean	6.4 mm
	Standard Deviation	0.418
	CV	6.54%

Another preliminary test done for the lysis experiment was the determination of whether to continue the use of Sigma fibrinogen, which had been shown to produce poor results in previous failed lysis experiments. To solve this issue of poor results another brand of fibrinogen, Diagen, was used. The experiment was set up to directly compare the two types, Sigma and Diagen, and their varying zones of lysis with the euglobulin fractions.

	Size of zone of lysis (mm) after 24 hours
Fibrin Type	1		2		3		4		5		Average
Sigma	7	7	6	7	7	6	14	8	6	6	7.4
Diagen	5	5	5	6	5	4	5	4	6	5	5.0
Table 4: Results of the two types of fibrinogen with the euglobulin fractions and there resultant zones of lyses n=2.

The mean zones of lysis for both types of fibrinogen were quite similar so an unpaired t-test was carried out. The observed t value (3.04) was greater than the critical t value of 2.23 so the means were significantly different (p=0.005), and that the Sigma fibrinogen gave better results. It was concluded that Sigma fibrinogen was to be used for all further experiments.

Fibrin lysis results

The results for the lysis experiment were averaged from the 5 spots on the plate to give a mean zone of lysis over 24 hours, twice, to give two sets of results. These were plotted on a graph, as shown in fig 9 with the standard deviations plotted as y-error bars.

There was no significant change seen in the zone of lysis between the varying concentrations of ONOO exposed to fibrinogen. To confirm this, a one way ANOVA was carried out to determine if there was any significant difference between the results for each concentration of ONOO and the zones of lyses. The calculated F value between the different concentrations was 0.342857 (p=0.89) with the critical F value being 3.866 (df1=6, df2=7), indicating that because the calculated F value is smaller than the critical F value, there is no significant difference between the zones of lyses seen for each concentration of ONOO. This suggests that peroxynitrite has no significant effect on the lysis of fibrin.

fig9

Further tests:

To determine if any possible effect seen in the lysis experiment will be due to effect of peroxynitrite on fibrinogen or plasminogen activator. To do this the plasminogen was treated with the experimental substrate (as opposed to the fibrinogen). The results of the treated plasminogen are shown in fig. 10.

To determine if there was any significance between the results, one way ANOVA was carried out, comparing the variation between the different concentrations. The F value was calculated as 2.472 and the critical value was greater at 2.758(df1=4, df2=25), indicating no clear difference between the different concentrations of the experimental substrate treated plasminogen activator and its effect on the zone of lysis. This in turn means that the substrate, ONOO has no clear affect on the properties of plasminogen activator.

fig10

Fibrin compaction:

A preliminary test was carried out to determine whether to use blood plasma or pure fibrin as the basis of the compaction experiment. Experiments were carried out simultaneously with both plasma and fibrin being exposed to the varying concentrations of the experimental compound, peroxynitrite (ONOO). The percentage compaction was then calculated for each by measuring the volume of liquid plasma/fibrin not clotted, and this was expressed as the percentage of the original volume to calculate the percentage compaction. The two sets of results were plotted on one graph, fig 11 shown below.

The results showed a clear difference in compaction between the two, with the plasma showing higher compaction results, and therefore easier to measure test results when compared to the fibrin, which was found to have very little volume of liquid left after clotting, making its measurement harder. It was concluded therefore to use plasma for the remainder of the compaction experiment.

fig11

Another preliminary test done for the compaction experiment was the calculation of the coefficient of variation, to determine reproducibility. This was done by clotting the plasma with the control TBS and thrombin, ten times and the subsequent percentage compactions were calculated as in table 4.

	Weight (g)		Volume (µl)
	100 µl	Total	Total	%Compaction
1	0.102	0.829	812.7	81.30
2	0.106	0.816	769.8	77.00
3	0.103	0.762	739.8	74.00
4	0.102	0.727	712.7	71.00
5	0.099	0.839	847.5	84.80
6	0.099	0.819	827.3	82.70
7	0.101	0.752	744.6	74.50
8	0.104	0.821	789.4	78.90
9	0.099	0.825	833.3	83.30
10	0.103	8.829	804.9	80.50
Mean compaction = 78.8%
Standard deviation =4.49
CV = 5.69%
Table 4: results from the compaction assay controls n=10

Coefficient of variation

The CV was calculated for each experiment, and an ideal coefficient of variation of 5% was aimed for. The first experiment, measuring polymerisation, had a CV of 4.72%, indicating that the dispersion is low between replicate results. The CV for the second experiment, lysis, and third experiment, compaction, were 6.54% and 5.69% respectively. These results indicate that the dispersion is a little higher in these two experiments, but only slightly. With these values it can be said with some certainty that any dispersion seen will be due to the experimental compound, peroxynitrite, rather than a general dispersion of the assays themselves.

Fibrin compaction results:

The results of the compaction assay were three times and a mean of all the results were plotted in a graph as seen in fig. 12. A clear trend can be seen, with increasing ONOO concentration there is a decrease in the percentage compaction of the fibrin.

fig12

To statistically validate this trend, a one way ANOVA was carried out. The F value was calculated as 70.17639 with the critical F value much lower at 2.85 (df1=6, df2=14), indicating that the differences seen in compaction between the different concentrations of ONOO are significantly different. This shows that the substrate actually strengthens the fibrin network and prevents it from compacting

Further Tests:

To determine whether this effect of decreasing compaction with increasing concentration of ONOO is due to the substrates affect on the fibrin or an affect on factor XIII, another experiment was carried out which involved using EDTA, instead of Ca2+. Calcium ions are required by factor XIII to function. The graph below in fig. 9 shows the two curves of the experiment carried out, one in the presence of CaCl2, and one in the presence of EDTA and the results plotted in figure 13.

fig13

To determine if there was a significant difference in results between the EDTA and CaCl2 assays a one way ANOVA was carried out. An F value of 0.444 was calculated with a higher F critical value of 3.866 (df1=6, df2=7), showing no significant difference between the two sets of results and each concentration (i.e. no significant difference between compaction % for CaCl2 and EDTA for ONOO concentration of 25 µM).This would lead to the conclusion that the negative correlation seen in compaction assay, between concentration and compaction % is not due to the effect of ONOO on factor XIII.

DISCUSSION

The experiment compromised three parts, the first experiment tested the polymerisation of fibrin from fibrinogen under the effects of increasing concentrations of ONOO and the rate of polymerisation calculated as the absorbance change over time. This allowed the measurement of the rate of polymerisation indirectly, via the cloudy formation of fibrin and its change in the absorbance. A decrease in the rate of fibrin polymerisation in the ONOO treated fibrinogen compared to the control plasma would support the hypothesis. Any affect on thrombin was measured by using the substance ß-Ala-Gly-Arg-p-nitroanilide, which when cleaved by thrombin produces a yellow pigment. The rate of formation of this pigment allowed the rate of thrombin action to be measured by the rate of change of absorbance over time.

The second experiment was carried out to determine the affect of peroxynitrite on the properties of fibrin lysis in ONOO. This was done by measuring the zones of lyses of fibrin using plasminogen and tPA obtained from human plasma. A decreased zone of lysis in the presence of ONOO compared to the control would suggest that ONOO has a damaging affect on the coagulation system and would support the hypothesis. To determine if any affect seen in the lysis of fibrin in the presence of ONOO, was due to fibrinogen or tPA, a further experiment was carried out. This experiment involved treating the euglobulin fraction containing the tPA and plasminogen with ONOO as opposed to the fibrinogen. This treated euglobulin fraction was then used to lyse the clotted plates. A decrease in the zone of lysis in the presence of ONOO as opposed to the control would support the hypothesis.

The final part of the experiment involved measuring the affect of ONOO on the stability of the fibrin network once a clot had formed. This was measured by treating plasma with varying concentrations of ONOO and clotting then clotting the plasma. The clot was centrifuged and any liquid expelled from the network was measured. A strong network of fibrin would expel less liquid than a weakly formed fibrin clot, therefore an increase in the volume of liquid expelled from the ONOO treated plasma compared to the control would support the hypothesis.

The results obtained from the fibrin polymerisation experiment show a distinct difference in the rate of polymerisation with varying concentrations of ONOO. This suggests then that the rate of polymerisation of fibrin in blood may be affected by the by product of NO reactions in vivo, producing ONOO. To confirm the results a one way ANOVA was carried out which showed there was a significant difference between the different concentrations of ONOO and there effects on the relative rates of polymerisation of fibrin. From this it can be concluded that increasing concentrations of ONOO has a negative effect (slows down rate) on the formation of fibrin from fibrinogen.

This affect of ONOO on the polymerisation of fibrin was in fact its affect on the fibrin precursor fibrinogen. This was determined by the next experiment which tested the affect of ONOO on thrombin which if affected by the ONOO would also slow down the rate of polymerisation. This experiment however showed no apparent affect of varying concentrations of ONOO on thrombin, with the highest concentration of ONOO (200 µM) and no ONOO (TBS) showing very similar rates of polymerisation of fibrin. The same is also shown in the presence of decomposed ONOO, with the rate of polymerisation not affected. This shows that the individual components of ONOO do not affect the thrombin and therefore the rate of fibrin polymerisation. A one way ANOVA test was carried out which determined that there was no significant difference in the rate of action of thrombin and the concentration of ONOO. From this it can be concluded that ONOO has no affect on the rate of polymerisation through the action of thrombin, i.e. ONOO has no affect on thrombin.

The results obtained form the fibrin lysis experiment showed no distinct difference in the zones of lysis in the presence of varying concentrations of ONOO. This can be seen in figure 6, where the zones of lysis are very much the same for all the concentrations of ONOO tested and were confirmed using one way ANOVA, which showed no significant difference in the zones of lysis of fibrin in the presence of varying ONOO concentrations. This showed that ONOO had no significant affect on fibrinogen and fibrin.

However to determine if ONOO has an affect on plasminogen activator, tPA, the second experiment involved treating the euglobulin fraction of the plasma, containing tPA, with varying concentrations of ONOO. The results show a slight difference in the zones of lysis between the different concentrations of ONOO, with an overall increase in the zone of lysis fro increasing ONOO concentrations. However although the zone of lysis increases with increasing ONOO concentration, the highest concentration of ONOO, 200 µM, shows a slight decrease in zone of lysis compared to 100 µM. So although the results show a trend of increasing ONOO with increasing zone of lysis, suggesting that ONOO in fact acts to help plasminogen activator, tPA, as opposed to hindering it, the highest concentration of ONOO does not follow this trend. This may be because only very low concentrations of ONOO help tPA, therefore increasing the zone of lysis. One way ANOVA was carried out on the results to and it was determined that there was no significant difference in the increasing concentration of ONOO and the resulting zone of lysis. This means that ONOO has no effect on tPA and its subsequent cleaving of plasminogen to plasmin.

For this experiment a clear set of results were obtained showing a negative trend between % compaction of the fibrin clot and ONOO concentration (fig. 9). With increasing ONOO concentration, % compaction decreases. One way ANOVA was carried out on the results and showed that there was a significant difference between the concentrations of ONOO and there effect on % compaction. Therefore it can be concluded that in the presence of ONOO the strength of the fibrin network is increased. This is in direct opposition to the hypothesis that ONOO acts to increase the compaction % i.e. decrease the strength of the fibrin clot.

To determine if this affect of the ONOO was on the fibrinogen or factor XIII, which is involved in the linking of fibrin monomers a further test was carried out involving CaCl2 and EDTA. The results as seen in figure 10, show the experiment carried out in the presence of EDTA (no factor XIII action) and in the presence of CaCl2 (factor XIII in action). The results however show no distinct difference between the % compaction in the presence of CaCl2 and in its absence, therefore ONOO has no direct affect on factor XIII, and the effects seen in the previous experiment is due to the affect of ONOO on fibrinogen.

Peroxynitrite showed considerable inhibition of the polymerisation of fibrin from fibrinogen and this has been seen in other experiments such as one carried out by Nielsen et al. they showed that peroxynitrite decreases the function of fibrinogen in vitro, and they concluded that ONOO decreases the function of fibrinogen via its nitration by the reactive nitrogen species (Nielsen et al, 2004). The same was seen in the experiment carried out by Lupidi et al, where it was discovered that the clotting activity of fibrinogen was fully inhibited in vitro by peroxynitrite even at very low concentrations, much the same as this experiment where a concentration of 25-50 µM of ONOO showed considerable decrease in the rate of polymerisation of fibrin (Lupidi et al, 1999). The decrease in polymerisation seen in the above experiments and the experiment carried out it is clear that ONOO has some clear affect on fibrinogen and this is most likely due to the reactive nature of ONOO as a free radical reacting with and causing structural changes in fibrinogen. In one experiment, fibrinogen, along with other blood proteins, was treated with peroxynitrite and using western blotting it was discovered that in the presence of ONOO, fibrinogen showed nitrated tyrosine residues (Minetti et al, 1998). This nitration of the fibrinogen would mean that the cleaving by thrombin would be inhibited and hence the decrease in the rate of polymerisation of fibrin.

Peroxynitrite showed considerable inhibition of the polymerisation of fibrin from fibrinogen and this has been seen in other experiments such as one carried out by Nielsen et al. they showed that peroxynitrite decreases the function of fibrinogen in vitro, and they concluded that ONOO decreases the function of fibrinogen via its nitration by the reactive nitrogen species (Nielsen et al, 2004).

The same was seen in the experiment carried out by Lupidi et al, where it was discovered that the clotting activity of fibrinogen was fully inhibited in vitro by peroxynitrite even at very low concentrations, much the same as this experiment where a concentration of 25-50 µM of ONOO showed considerable decrease in the rate of polymerisation of fibrin (Lupidi et al, 1999).

The decrease in polymerisation seen in the above experiments and the experiment carried out it is clear that ONOO has some clear affect on fibrinogen and this is most likely due to the reactive nature of ONOO as a free radical reacting with and causing structural changes in fibrinogen. In one experiment, fibrinogen, along with other blood proteins, was treated with peroxynitrite and using western blotting it was discovered that in the presence of ONOO, fibrinogen showed nitrated tyrosine residues (Minetti et al, 1998). This nitration of the fibrinogen would mean that the cleaving by thrombin would be inhibited and hence the decrease in the rate of polymerisation of fibrin.

Peroxynitrite had no significant effect on the lysis of fibrin at any concentration measured. This was both in the treatment of fibrinogen and tPA, with ONOO. However other experiments carried out on ONOO and its affect on tPA do show affects. In one experiment it was shown that tPA activity was reduced considerably in the presence of ONOO in vitro (Nielsen et al, 2004). Another experiment however showed that nitration by reactive nitrogen species on fibrinogen, causing it to become modified, demonstrated no difference in the rate of plasmin-induced clot lysis. So although in both the firbin lysis and polymerisation experiments the concentrations of ONOO used were the same, and therefore the level of nitration, there was no significant affect on the lysis of fibrin. This may due to the decreased affinity of thrombin to fibrinogen due to its nitration, decreasing the rate of polymerisation.

The polymerisation results showed a slight increase in the strength of the fibrin clot network in the presence of increasing ONOO concentrations excluding the highest concentration of ONOO. This may be because the nitration of the fibrinogen and its eventual cleavage to fibrin, acts to strengthen the cross linking. One experiment found that fibrinogen exposed to nitrating oxidants significantly increased factor XIII cross-linking (Vadseth et al, 2004) and although the compound they used to nitrate the fibrinogen was not ONOO, nitration did occur in the fibrinogen. This could explain why with increasing ONOO concentrations there was an increase in the strength of the fibrin network. This experiment by Vadseth et al also used electron microscopy and found that the fibrin fibres formed from fibrinogen exposed to nitrating compounds, were thinner than fibrin fibres formed from untreated fibrinogen. Under electron microscopy the fibrin network (from the nitrated fibrinogen) also showed more twisted fibres than the normal fibrin. This may explain why the compaction of the fibrin exposed to ONOO in this experiment was higher than the control, with the twisted, nitrated fibrin fibres forming a more solid clot (Vasdeth et al, 2004)

Although the results obtained were done so with the accuracy and precision in mind, there are still sources of error that may have occurred including human error in the cases of measuring the volumes of solutions and substances used.

One aspect of the experiment that may have caused shortcomings in the results is the peroxynitrite in use, although it was stored in a freezer and only removed when in use; the peroxynitrite was left to incubate with the fibrinogen for a period of time in some stages of the experiment. This included the fibrin lysis experiment, where the fibrinogen was allowed to incubate with the ONOO at room temperature for 30 minutes or more. This introduces the possibility that the peroxynitrite decomposed before any lysing was done with the euglobulin fraction. This may be the reason for the poor results seen in the lysis results, which showed no distinction between the zones of lysis for each ONOO concentration.

Measurement of lysis: Another shortcoming that could be improved upon in future experiments is the method of measurement of the zones of lysis. This was done using a simple millimetre measuring ruler, in the future this could be improved upon by using a

The one area where the results were poor was in the lysis experiment. The procedure, yielded zones of lyses that were overall quite small, with the main problem encountered being the initial clotting of the fibrinogen in the plate. With thrombin added the plate seemed not to clot completely in the 30 minutes it was left for, and this may be why the zones of lyses were so small. This could be improved upon in the future by increasing the concentration of thrombin used, although this may improve the clot of the fibrin and thus the zones of lysis.

With these shortfalls in mind the final conclusion may be held in some doubt especially concerning the lysis of fibrin.All the experiments carried out were done so in vitro, and although ONOO is produced in vivo, it is not certain whether the effects seen in vitro will be the same as any affects seen in vivo. Further experiments could involve conditions very similar to what goes on in vivo, such as carrying out the experiment at 37°C, or in animal models.Further experiments that could be done would be to test more aspects of the clotting system, including ONOO affect on other factors, enzymes and co-factors.Another area that could be included in the experiment for the future is investigating molecules and substances that reduce the nitrating affects of ONOO such as bilirubin and CO2. One such as experiment carried out by Minetti at al discovered that bilirubin was an effective antioxidant against ONOO and its affects on blood components (Minetti et al, 1998). This could be expanded to include many other substances with antioxidant properties and used in the presence of ONOO and fibrinogen to see if the effects are still apparent in the presence of the antioxidant.

In previous experiments and literature it has been demonstrated that reactive nitrogen species are involved in many pathological diseases and in this experiment the conclusion drawn on the full affects of the particular reactive nitrogen species, ONOO, on blood coagulation as a whole are inconclusive. In one particular aspect of blood coagulation, specifically fibrin polymerisation, ONOO had a definite detrimental affect on fibrinogen and slowed down the rate of polymerisation; however in the lysis experiment there was no conclusive evidence that ONOO has any affect on the lysis of fibrin. In the compaction assay the results pointed at ONOO having a positive effect on the fibrin network, making it more stable. So in two of the three aspects of blood coagulation there was an affect, one negative and one positive. This leads to the conclusion that ONOO although having a pathological role in blood clotting; the same cannot be said of fibrin lysis, or fibrin compaction.

References

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Nair C H, Shats E. A. 1997. Compaction as a method to characterise fibrin network structure: Kinetics studies and relationship to cross linking. Thrombosis research; Volume 88: pp 381-387.