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Effects of Various Oxidants and Antioxidants on Fibrin Polymerisation

Margarita Lymbouris[1], School of Life Sciences, University of Westminster

 

Abstract

The role of oxidants in disease has become increasingly evident. Inflammatory responses bring about changes in circulation in disorders such as heart disease, asthma, Alzheimer's disease, Parkinson's disease and cancer. It is suggested that oxidants can target fibrin polymerisation, but the mechanisms by which oxidants affect fibrin polymerisation are still unclear. The aim of this study was to investigate the effects of oxidants on fibrin polymerisation and to determine whether plasma antioxidants have the ability to protect against it. The study also examined whether the observed effects were acting on fibrinogen or thrombin. Fibrin polymerisation was evaluated by changes of absorbance which enabled calculation of fibrin polymerisation rates. From the chosen oxidants, sodium hypochlorite, GSNO and SIN-1 affected fibrin polymerisation significantly; hydrogen peroxide did not have a significant effect on fibrin polymerisation. The strongest effect was observed for sodium hypochlorite and the strongest protection was observed with uric acid and glutathione. Further investigation confirmed that sodium hypochlorite was acting on fibrinogen. The findings of this study highlight the susceptibility of fibrinogen to oxidant attack and the ability of uric acid and glutathione to significantly protect against it as plasma antioxidants.

Keywords: Fibrin polymerisation, Oxidative stress, Plasma antioxidants, Sodium hypochlorite, Fibrinogen

 

Introduction

Haemostatic mechanisms are tightly regulated to maintain a balance in the initiation and termination of blood coagulation. This ensures adequate blood flow under normal physiological conditions and rapid coagulation when tissue injury occurs (Allford and Machin, 2004). Activation of blood coagulation leads to the formation of thrombin which converts fibrinogen into insoluble fibrin via the process of fibrin polymerisation. This conversion is crucial in haemostatic clot formation and is followed by fibrin polymer cross linking by factor XIIIα to stabilise the clot (Kamath and Lip, 2003).

Fibrinogen is a 340 kDa plasma protein made up of two subunits, each consisting of pairs of disulfide-linked polypeptide chains called Aα, Bβ and γ (Mosesson, 2005). The N-termini of these chains meet to form the central domain of the fibrinogen molecule called E domain, while the C-termini extend to form the D domains. The fibrin polymerisation process begins with the binding of thrombin to the E domain of fibrinogen and cleavage of fibrinopeptides A and B, leading to the exposure of the A-knob and the B-knob respectively (Wolberg, 2007). These knobs can interact with the a-holes and b-holes which are found within other fibrinogen molecules. When the A-knob of a fibrinogen molecule binds to the a-hole in the γC domain of an adjacent fibrinogen molecule, protofibrils form. Subsequent binding of the B-knob of a fibrinogen molecule to the b-hole of an adjacent fibrinogen molecule is suggested to have a role in strengthening lateral aggregation of the protofibrils (Okumura et al., 2007). Lateral aggregation of protofibrils leads to the formation of fibres (Mosesson, 2005).

Once fibrin polymerisation is complete, activated factor XIII leads to the stabilisation of the clot which in turn prevents blood loss (Kamath and Lip, 2003). It is therefore evident that fibrin polymerisation plays a central role in haemostatic mechanisms. Evidence suggests that the plasma proteins responsible for maintaining haemostatic mechanisms can be affected by oxidative and nitrosative stress. Studies demonstrate that alteration of these proteins leads to imbalances in the haemostatic process (Wolberg and Campbell, 2008), as they can alter the final structure of the fibrin network (Wolberg, 2010).

Oxidative and nitrosative stress causes damage in the form of structural changes to macromolecules, including proteins. These changes are increasingly being linked to many pathological conditions (Kedzierska et al., 2012). Reactive oxygen species and reactive nitrogen species are generated during inflammation and the inflammatory response, thus causing oxidative and nitrosative stress. Inflammation has been associated with diseases such as diabetes, cancer, asthma, Alzheimer's disease, Parkinson's disease and heart disease. These diseases exhibit functional and structural changes in circulation which are characteristic of inflammation (Kvietys and Granger, 2012). Fibrinogen has been shown to be a possible target for the nitrosative and oxidative attack explained above (Nowak et al., 2007).

The oxidants and antioxidants chosen for the study are present in human blood plasma at higher concentrations in the diseases mentioned above (Giustarini et al., 2008). It is therefore possible that the stress is acting on the step of blood coagulation investigated in this study, which is fibrin polymerisation. If this is the case, it is also essential to investigate whether antioxidants present in blood plasma are able to protect against the stress. Therefore the aim of the study was to investigate the effects of oxidative and nitrosative stress on the thrombin mediated fibrin polymerisation process, and establish the ability of plasma antioxidants to protect against this stress.

Materials

Bovine thrombin was purchased from Diagnostic Reagents (Oxfordshire). Hydrogen peroxide and sodium hypochlorite were purchased from Merck chemicals (UK). All other chemicals were purchased from Sigma-Aldrich (Poole, UK). Fibrinogen and thrombin solutions were prepared using Tris buffered saline (50 mM Tris and 150 mM NaCl, pH 7.3). Thrombin and GSNO were stored on ice during experiments to prevent thermal decomposition. All other solutions were prepared using MQ water and were kept at room temperature. Fresh solutions were prepared for each assay, and materials were stored as advised by the manufacturer.

 

Methods

Measurement of fibrin polymerisation

To measure fibrin polymerisation, thrombin (final concentration 1 U/ml) was added to bovine fibrinogen (final concentration 2 mg/ml) and absorbance values (350 nm) were measured. In each experiment the values were recorded every 15 seconds for 2 minutes. Once set up, the assays were carried out in triplicate. The values obtained were used to produce fibrin polymerisation curves from which fibrin polymerisation rates were calculated. A tangent was drawn at the steepest part of the curve to calculate the rate of polymerisation (Figure 1).

Figure 1: Fibrin polymerisation curve representing the calculation of the fibrin polymerisation rates from the assay values.

Figure 1: Fibrin polymerisation curve representing the calculation of the fibrin polymerisation rates from the assay values.


Optimisation of fibrin polymerisation assay

Thrombin was prepared according to the manufacturer's instructions (diagnostic reagents Oxfordshire) to produce final concentrations of 0.5 U/ml, 1 U/ml and 2 U/ml. Fibrinogen solution was prepared to final concentrations of 1 mg/ml, 2 mg/ml and 4 mg/ml. Measurement of fibrin polymerisation identified 1 U/ml thrombin and 2 mg/ml fibrinogen as the final concentrations to be used in the following assays. These concentrations produced curves with the most consistently measurable polymerisation rates (Figure 1).


Measurement of effects of oxidative and nitrosative stress on fibrin polymerisation

Fibrinogen samples (final concentration 2 mg/ml) were incubated with sodium hypochlorite (final concentration between 0.001-1 mM) or hydrogen peroxide (final concentration between 0.001-1 mM) for 5 minutes to allow possible oxidative stress effects to take place. Controls were not exposed to oxidative stress, therefore the oxidant was replaced with water to enable comparison with the samples exposed to stress. The same procedure was followed for nitrosative stress with SIN-1 or GSNO (final concentration 1 mM). After the incubation period, thrombin was added to measure fibrin polymerisation (as described) in triplicate. Sodium hypochlorite (final concentration 1 mM) was chosen for further investigation.


Measurement of effects of antioxidants against sodium hypochlorite effects

Antioxidants were prepared in deionised water and added to fibrinogen at 1 mM final concentration. The antioxidants used were cysteine, homocysteine, ascorbic acid, uric acid and glutathione. Fibrinogen was incubated for five minutes with the antioxidants only, before carrying out fibrin polymerisation, to determine whether they had any independent effect on fibrin polymerisation. No significant effects were observed, therefore all antioxidants were further investigated for their possible protection against oxidative stress. To investigate the potential ability of the antioxidants to protect against oxidative stress, three tests were set up. The first test was the control in which water was incubated with fibrinogen for five minutes. In the second test, sodium hypochlorite (final concentration 1 mM) was incubated with fibrinogen for five minutes (inhibiting control). In the third test, both the sodium hypochlorite (final concentration 1 mM) and the antioxidant (final concentration 1 mM) were incubated with fibrinogen for five minutes. After incubation periods at room temperature, thrombin was added to initiate fibrin polymerisation. The absorbance values were measured in triplicate for each test, as previously described, and fibrin polymerisation rates were calculated.


Sephadex G25 separation to distinguish oxidant effects on thrombin or fibrinogen

Sephadex G25 separation was used to isolate the effects of sodium hypochlorite on fibrinogen from those on thrombin. Three assays were set up to distinguish these effects. In the first assay, fibrinogen was incubated with water for 5 minutes and was then separated. The eluate was then incubated with water for a further five minutes (control). In the second assay, fibrinogen was incubated with sodium hypochlorite (1 mM) for five minutes before separation to exert its effects on fibrinogen. The eluate was then incubated with water for five minutes. In the third assay, fibrinogen was incubated with water for five minutes and was then separated, after which the eluate was incubated with sodium hypochlorite (1 mM) for five minutes. After the incubation periods were complete, thrombin (final concentration 1 U/ml) was added to each test to measure fibrin polymerisation as described in triplicate.


Data analysis

Data were analysed using Microsoft Excel and SPSS. Values obtained from the experiments were expressed as mean±SD. Statistical analysis was carried out using one-way ANOVA and post hoc analysis (Bonferroni) when appropriate. P values less than 0.05 were accepted as statistically significant.

Results

Optimisation and reproducibility of fibrin polymerisation assay

To optimise the fibrin polymerisation assay, preliminary tests were carried out to choose the final concentration of thrombin and fibrinogen to be used in the study. This was achieved by using 0.5 U/ml, 1 U/ml and 2 U/ml of thrombin separately with 1 mg/ml fibrinogen. 1 U/ml thrombin was chosen and was then used to perform the fibrin polymerisation assay with 1 mg/ml, 2 mg/ml and 4 mg/ml of fibrinogen separately. It was decided that 2 mg/ml and 1 U/ml fibrinogen would be used in the study, since these produced the most reliable rate of polymerisation (Figure 1). Following this procedure, the fibrin polymerisation was measured as absorbance six times and was then expressed as polymerisation rates. The values obtained were used to calculate the coefficient of variation (CV) of the assay in terms of polymerisation rates. The CV was calculated to be 18%, representing the reproducibility of the fibrin polymerisation assay.


Effect of oxidative stress on fibrin polymerisation

Sodium hypochlorite and hydrogen peroxide were used separately to investigate the effects of oxidative stress on fibrin polymerisation as described. For both oxidants the final concentrations used were 0.001 mM, 0.01 mM, 0.1 mM and 1 mM. One-way ANOVA analysis was used to determine if there was a significant difference between the polymerisation rates of the oxidants compared to the control (Figure 2). No significant difference was found between the hydrogen peroxide concentrations and the control polymerisation rates (Figure 2). However, a significant difference between the sodium hypochlorite and control polymerisation rates was found by one-way ANOVA analysis and post hoc test. The final concentration of 1 mM of sodium hypochlorite resulted in the largest inhibition and the lowest standard deviation (Figure 3). As a result, 1 mM of sodium hypochlorite was chosen for subsequent experiments. These results demonstrate that exposure of fibrinogen to sodium hypochlorite reduces the rate of fibrin polymerisation.

Figure 2: Effects of hydrogen peroxide on the fibrin polymerisation rate expressed as mean polymerisation rate.

Figure 2: Effects of hydrogen peroxide on the fibrin polymerisation rate expressed as mean polymerisation rate.


Figure 3: Effects of sodium hypochlorite on fibrin polymerisation expressed as mean polymerisation rate.

Figure 3: Effects of sodium hypochlorite on fibrin polymerisation expressed as mean polymerisation rate.


Effect of nitrosative stress on fibrin polymerisation

GSNO and SIN-1 were both used separately at a final concentration of 1mM, in order to investigate the effects of nitrosative stress on the fibrin polymerisation process. Fibrin polymerisation was measured as described and the results were expressed as mean polymerisation rates (Figure 4). The effects of nitrosative stress compared to the control were statistically significant according to one-way ANOVA and post hoc test (p<0.0001). In contrast to the effects seen with sodium hypochlorite, GSNO and SIN-1 caused a slight increase in fibrin polymerisation rates.

Figure 4: Effects of nitrosative stress on the mean polymerisation rate. The values represent the mean±SD of three experiments in which a five minute incubation time was allowed before fibrin polymerisation was set up.

Figure 4: Effects of nitrosative stress on the mean polymerisation rate. The values represent the mean±SD of three experiments in which a five minute incubation time was allowed before fibrin polymerisation was set up.


Effect of plasma antioxidants against sodium hypochlorite effects

The antioxidants that were investigated were cysteine, homocysteine, glutathione, uric acid and ascorbic acid. In preliminary experiments none of these antioxidants were found to have an independent effect on fibrin polymerisation, asno significant difference was found between the antioxidant action and the control (p>0.05). Consequently, investigation of the potential protective effects of all antioxidants was carried out.

As shown in Figure 5, ascorbic acid did not protect against oxidative stress while the rest of the antioxidants did. Cysteine and homocysteine exhibited some protection but it was not statistically significant. The experiments demonstrated that uric acid and glutathione have the ability to protect significantly against oxidative stress according to one-way ANOVA and post hoc test (p<0.0001).

Figure 5: Effects of antioxidants against oxidative stress expressed as mean polymerisation rates as percentages of the control.

Figure 5: Effects of antioxidants against oxidative stress expressed as mean polymerisation rates as percentages of the control.


Sephadex G25 separation

Once the effects of oxidants on fibrin polymerisation were established, sephadex G25 separation was used to investigate whether the oxidants were acting on fibrinogen or thrombin. To achieve this, three tests where set up as described. Test one was the control in which fibrinogen was incubated with water before and after sephadex separation. In test two, sodium hypochlorite was incubated with fibrinogen for five minutes before separation; this test isolated the effects of the sodium hypochlorite on fibrinogen. In test three, fibrinogen was incubated with water for five minutes before separation and the eluate was incubated for five minutes with sodium hypochlorite before the addition of thrombin. This step allowed sodium hypochlorite to act on both fibrinogen and thrombin. The values obtained were expressed as mean polymerisation rates as a percentage of the control. As presented in Figure 6, test two and test three had equal mean rates of polymerisation. This observation leads to the conclusion that sodium hypochlorite (oxidant) exerts its effects on fibrinogen.

Figure 6: Fibrin polymerisation rates.

Figure 6: Fibrin polymerisation rates as a percentage of the control. The control represents zero oxidant stress before separation. Test 2 represents sodium hypochlorite action on fibrinogen before separation. Test 3 represents sodium hypochlorite action on both fibrinogen and thrombin after separation.

Discussion

There is increasing evidence that oxidative and nitrosative stress affects haemostatic mechanisms. The plasma proteins involved in the coagulation cascade can be altered by oxidants, leading to haemostatic imbalances (De et al., 2002). Oxidants are linked with vascular changes occurring during chronic inflammation (Wolin, 2009). Studies have also identified fibrinogen as being highly susceptible to oxidant attack (Nowak et al., 2007) and have highlighted the importance of antioxidants in reducing the effects of oxidants (Giustarini et al., 2008). Therefore, the study aimed to investigate the effects of oxidants on fibrin polymerisation induced by thrombin, a key step in the coagulation cascade, and the potential protective effects of antioxidants. As a final aim it investigated where the oxidants exerted their effects.

The fibrin polymerisation process was measured in terms of polymerisation rate as described above. Before beginning the experiments, the final concentrations of fibrinogen and thrombin needed to be selected in order to ensure polymerisation rates were measurable. In addition to this, the concentrations were important because fibrin clot structure can be affected by a number of variables including the concentration of fibrinogen and thrombin (Wolberg and Campbell, 2008). After a series of experiments 2 mg/ml fibrinogen and 1 U/ml thrombin were chosen, as they produced the most measurable fibrin polymerisation rate (Figure 1). The values were also relevant because human plasma levels of fibrinogen are 2-4 mg/ml (Wolberg and Campbell, 2008). This suggested that the results of the study are relevant to human plasma.

Sodium hypochlorite (0.001-1 mM) was shown to have a significant effect on fibrin polymerisation. This is consistent with a previous study showing that sodium hypochlorite decreases clotting ability (Stief et al., 2000). Fibrinogen has also been identified as an antioxidant itself, with the capacity to inhibit myeloperoxidase activity which is linked to sodium hypochlorite levels (Olinescu and Kummerow, 2001). In contrast, this study found that hydrogen peroxide did not affect fibrin polymerisation. However, it has been suggested that hydrogen peroxide could have a possible role in the overall thrombus formation and data regarding this role are controversial. While some support its role in enhancing platelet aggregation (Praticò et al., 1991), others do not (Ohyashiki et al., 1991). Both sodium hypochlorite and hydrogen peroxide are generated by phagocytes, specifically neutrophills (Belisario et al., 2000).

The investigation of nitrosative stress identified that fibrin polymerisation is also susceptible to GSNO and SIN-1 as fibrin polymerisation rates increased (Figure 4). However previous studies have shown that GSNO inhibits fibrin polymerisation when fibrinogen is exposed to it (Geer et al., 2008). Relative plasma levels of GSNO are 100-300 nM (Bateman et al., 2012) whereas the concentrations used in this study were 1 mM. A recent study supported that GSNO alters the fibrin clot structure in a dose dependent manner. It was found that the fibrin fibre thickness gradually increased from 0.01 to 1 mM (GSNO) and then became thinner as the concentration of GSNO increased to 2.5 mM. This may suggest that the in vivo values of GSNO concentration affect its function (Bateman et al., 2012). GSNO is a carrier of nitric oxide and is being investigated to understand its cellular function because of its increased levels in diseases such as asthma (Staab et al., 2008). Lung cancer patients and smokers have also been shown to present high levels of oxidised fibrinogen in their lungs (Nowak et al., 2007), thus identifying the need for further investigation and potential prevention of these effects. Studies investigating SIN-1 identified its roles as a peroxynitrite donor that has been reported to oxidise proteins (Tackey et al., 2001). Further investigation identified that exposure of fibrinogen to peroxynitrite alters its structure and functional properties. This results in alteration of the fibrin structure and thus inhibition of thrombus formation (Nowak et al., 2007).

This study also investigated whether fibrin polymerisation could be protected by antioxidants. The antioxidants chosen for the study are found in blood plasma and are commonly linked to diseased states. This study showed that cysteine and homocysteine did not significantly protect against oxidative stress; these findings contradict reports that red blood cells synthesise cysteine and homocysteine in order to protect against oxidative and nitrosative stress (Giustarini et al., 2008). Ever since its discovery, ascorbic acid has been identified as an important antioxidant amongst other functions (Arrigoni and De Tullio, 2002); however, this study did not show significant protection against oxidative stress by ascorbic acid. Conversely, uric acid and glutathione protected against the inhibition of fibrin polymerisation, consistent with studies showing the protective capacity of uric acid and glutathione against oxidative stress. Uric acid has been suggested to have protective effects against thrombosis and atherosclerosis (Nieto et al., 2000). Glutathione, in contrast with the findings of this study, has been shown to interfere with fibrin formation as it reduced the fibrin polymerisation rate (Geer et al., 2008).

To provide a greater understanding of the study findings, G25 sephadex separation was carried out to determine the site of action of sodium hypochlorite. The findings indicate that sodium hypochlorite acts principally on fibrinogen. Consistent with this indication, studies have identified fibrinogen as highly susceptible to modification. It has been identified that interactions within fibrinogen molecules are essential for thrombin induced fibrin polymerisation (Geer et al., 2008).

In summary this study shows that sodium hypochlorite inhibits fibrin polymerisation significantly. It identifies fibrinogen as the target of the oxidative stress. In addition, uric acid and glutathione can protect significantly against the action of sodium hypochlorite, thus highlighting the importance of these antioxidants in protecting the haemostatic mechanisms. The results provide a starting point in understanding disease in terms of oxidative stress. Future work could enhance the understanding of the effects of oxidants and antioxidants on fibrinogen and their role in pathological conditions. Further work could include repeating the study under flow conditions, in order to gather information which is closer to the human model. Studies to find the exact location of the oxidant action on fibrinogen also represent an important aspect of future work.


List of figures

Figure 1: Fibrin polymerisation curve representing the calculation of the fibrin polymerisation rates from the assay values.

Figure 2: Effects of hydrogen peroxide on the fibrin polymerisation rate expressed as mean polymerisation rate.

Figure 3: Effects of sodium hypochlorite on fibrin polymerisation expressed as mean polymerisation rate.

Figure 4: Effects of nitrosative stress on the mean polymerisation rate.

Figure 5: Effects of antioxidants against oxidative stress expressed as mean polymerisation rates as percentages of the control.

Figure 6: Fibrin polymerisation rates as a percentage of the control.

 

Notes

[1] Margarita Lymbouris has recently completed a first class honours degree in Human and Medical Science at the University of Westminster. She is currently studying Medicine at Norwich Medical School at the University of East Anglia, and hopes to continue research once she has completed training in her desired specialisation field.

 

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To cite this paper please use the following details: Lymbouris, M. (2013), 'Effects of various oxidants and antioxidants on fibrin polymerisation', Reinvention: an International Journal of Undergraduate Research, Volume 6, Issue 1, www.warwick.ac.uk/reinventionjournal/archive/volume6issue1/lymbouris Date accessed [insert date]. If you cite this article or use it in any teaching or other related activities please let us know by e-mailing us at Reinventionjournal at warwick dot ac dot uk.