Platelet Membrane Fluidity and Peroxynitrite Levels in Migraine Patients during Headache-Free Periods

Abstract

Previous investigations have suggested that changes in platelet activity may play a key role in the pathophysiology of migraine via mechanisms involving the nitric oxide (NO) pathway. Changes in platelet response and nitrite levels have recently been demonstrated during migraine attacks, while there is considerable uncertainty about NO activity in headache-free periods. A reactive oxidant produced from NO and superoxide anion at the site of inflammation, peroxynitrite (ONOO^-) has effects including changes in membrane activity and fluidity. The aim of the present study was to determine ONOO^- levels in the platelets of patients suffering from migraine during the headache-free period. Nitric oxide synthase (eNOS and iNOS) expression in platelets and the effects of ONOO^- on membrane Na⁺/K⁺-ATPase activity and membrane fluidity were also evaluated. Subjects were 57 patients suffering from migraine without aura and 35 controls. Blood samples were collected in the headache-free period. Platelet ONOO^- levels were determined using dichlorofluorescein acetate with steady-state fluorescence. Platelets were then probed for induction of eNOS and iNOS expression by western immunoblotting. Membrane Na⁺/K⁺-ATPase activity and fluidity were determined with the fluorescent probes TMA-DPH and DPH. In the presence of extracellular L-arginine(100 μmol/l), ONOO^- production was significantly greater in patients' platelets than in those of controls (P < 0.001). Western immunoblotting of platelet proteins evidenced higher iNOS expression in patients than in controls. In addition, platelet membrane Na⁺/K⁺-ATPase activity and membrane fluidity evaluated by TMA-DPH were significantly lower in patients (P < 0.001). In conclusion, migraine patients show intercritic changes in platelet membrane fluidity and activity that may be related to the oxidative stress caused by increased ONOO^- levels.

Introduction

The role of platelet dysfunction in the pathogenesis of migraine is a debated issue. Decreased platelet aggregability to collagen combined with increased platelet arginine levels (1) and increased concentrations of NO metabolites (nitrate and nitrite) in platelets during attacks have been reported in migraineurs (2). Other indirect measures of NO synthesis in platelets and serum have also provided evidence of NO pathway activation during attacks (3). Possible NO pathway alterations have been explored in different studies of migraine patients outside attacks. Although the results did not yield conclusive data, they did suggest that alterations involving NO pathway and platelet activity in the intercritical period might crucially affect periodic attack onset (3–5). NO synthesis from the terminal guanidino nitrogen of l-arginine is catalysed by nitric oxide synthases (NOSs) (6). Two isoforms are constitutive and calcium/calmodulin-dependent and release NO from endothelium (eNOS) and neurons (nNOS) (7); another NOS, inducible and calcium/calmodulin-independent (iNOS), is not normally found in large amounts but is expressed after stimulation by endotoxins and cytokines (8, 9). NO overproduction may stimulate generation of reactive free radicals, such as peroxynitrite (ONOO^–), in amounts sufficient to mediate processes involved in cellular dysfunctions. Investigation of aspects involved in NO activity, including expression of some enzyme activities and concentration and the effects of some NO products on platelets, can thus be an alternative method to the determination of NO levels with a view to evaluating the effect of NO pathway activation.

The present study was undertaken to investigate whether in migraine patients platelet oxidative status is involved in changes in their membrane activity during the headache-free period. To do this, we investigated platelet ONOO^– production and iNOS and eNOS expression in patients suffering from migraine without aura, and the effects of ONOO^– on platelet membrane Na⁺/K ± ATPase activity and membrane fluidity.

Subjects and methods

Patients

Fifty-seven migraine patients (12 men, 45 women, mean age: 39.23 ± 13.43 years (± SD)) with an attack frequency of less than 3 episodes per month (range 1–3) gave their informed consent to participate in the study. Diagnosis of migraine was based on International Headache Society criteria (10). None of the patients had been taking prophylactic medications, nor ergot alkaloids or sumatriptan for the treatment of acute attacks for at least a month. Exclusion criteria were therapy with nitrodilators or glucocorticoids in the last six months, a history of chronic liver or renal disease, haematological or autoimmune disorders, and systemic hypertension. Number of years from migraine onset ranged from 15 to 22. The control group consisted of 35 age- and sex-matched healthy volunteers (9 men, 26 women, mean age: 37.89 ± 6.33 years). Patients had not had migraine attacks for at least 48 h before and after blood collection.

The study was conducted in accordance with the principles of the Declaration of Helsinki as revised in 1996 and was approved by the local ethics commitee.

Methods

Platelet isolation

Platelets were isolated from peripheral venous blood mixed with anticoagulant citrate dextrose (36 ml citric acid, 5 mmol/l KCl, 90 mmol/l NaCl, 5 mmol/l glucose and 10 mmol/l ethylenediamine tetracetate, pH 6.8), according to Rao (11): briefly, after a preliminary centrifugation (200×g for 10 min) to obtain platelet-rich plasma, platelets were washed three times in antiaggregation buffer and again centrifuged as above to remove any residual erythrocytes. A final centrifugation at 2000×g for 20 min was performed to isolate platelets, which were used immediately.

Peroxynitrite production

Peroxynitrite production was determined by the 2,7-dichlorofluorescein (DCF) fluorimetric assay as described by Tannous et al. (12). Peroxynitrite oxidizes the nonfluorescent molecule 2,7-dichlorofluorescin diacetate (DCFDA) to the fluorescent DCF. A DCFDA-free base was prepared by mixing 0.05 ml of 10 mmol/l DCFDA with 2 ml of 0.01 N NaOH at room temperature for 30 min. The mixture was neutralized with 18.0 ml of 25 mmol/l phosphate-buffered saline (PBS), pH 7.4. This solution was maintained on ice in the dark until use. DCFDA-treated platelets were incubated in NO buffer with 100 µm l-arginine for 15 min at 37°C in a dark room. The mixture was washed with PBS pH 7.4 then centrifuged for 2 min at 214×g. Supernatant fluorescence was measured in a Perkin-Elmer MPF-66 spectrofluorometer at an excitation wavelength of 475 nm and emission wavelength of 520 nm.

Na⁺/K⁺-ATPase assay

Na⁺/K⁺-activated Mg²⁺-dependent ATPase activity was determined in platelet plasma membranes obtained using the method of Enouf et al. (13). ATPase activity was assayed by incubating membranes at 37°C in reaction buffer containing 5 mm MgCl₂, 140 mm NaCl, 14 mm KCl in 40 mm Tris-HCl, pH 7.7 as previously described (14). The ATPase reaction was started by adding 3 mmol/l Na₂ATP. The inorganic phosphate (Pi) hydrolysed from this reaction was measured according to Fiske and Subbarow (15). ATPase activity assayed in the presence of 10 mm ouabain was subtracted from the total Mg²⁺-dependent ATPase activity to calculate Na⁺/K⁺-ATPase activity (16). Protein concentration was determined with the Bradford Bio-Rad protein assay using serum albumin as a standard (17) and was expressed as µmol P_i/mg prot/h.

Fluorescence studies

Platelet plasma membrane fluidity was studied by determining the fluorescence anisotropy (reciprocal of fluidity) of probes TMA-DPH and DPH, which are incorporated at the lipid–water interface of the membrane bilayer. Cell incubation with TMA-DPH was performed as described by Sheridan and Block (18). Briefly, 3 µl of TMA-DPH and DPH (10⁻³ mol/l) were incubated for 5 min at room temperature (23°C) with 2 ml platelets in 50 mmol/l Tris-HCl buffer solution, pH 7.4. Fluorescence intensities (100 readings each) of the vertical and horizontal components of the emitted light were measured on a Perkin-Elmer MPF-66 spectrofluorometer equipped with two glass prism polarizers (excitation wavelength 365 nm, emission wavelength 430 nm). Steady-state fluorescence anisotropy (r) of TMA-DPH and DPH was calculated using the equation:

r = (I_VG − I_h)/(I_V + 2I_h)

where G is the instrumental factor that corrects the r-value for an unequal detection of vertically (I_V) and horizontally (I_h) polarized light. Sample temperature was maintained at 37°C using an external bath circulator (Haake F3).

Western blot analysis

Washed platelets were lysed in RIPA lysis buffer containing 1 × PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, aprotinin, 100 mm sodium orthovanadate and 4% protease inhibitor cocktails by microcentrifugation at 10,000×g for 10 min at 4°C. The supernatants were collected and treated with an equal volume of sample application buffer (125 mmol/l Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, 1%β-mercaptoethanol). The mixture was boiled for 5 min; 15 µl of each sample was applied to each well of an 8% SDS polycrylamide gel and electrophoresed for 1 h at 130 V along with a set of molecular weight markers (Broad Range, Sigma St. Louis, MO, USA). The resolved protein bands were then transferred onto PVDF membranes at 100 V for 60 min using a transfer buffer of 25 mmol/l Tris base, 192 mmol/l glycine, and 20% methanol. The blots were blocked overnight at 4°C with blocking buffer (5% nonfat milk in 10 mmol/l Tris pH 7.5, 100 mmol/l NaCl, 0.1% Tween 20). The blocking buffer was decanted and blots were incubated for 1 h at room temperature with eNOS and iNOS rabbit polyclonal antibody diluted 1 : 200 (both from Sigma). Positive controls were included in all experiments as recommended by the manufacturer to confirm antibody specificity. Blots were then washed using TTBS and incubated with goat antirabbit secondary antibodies conjugated to horseradish peroxidase (Sigma; 1 : 5000) for 1 h at room temperature following washes in TTBS. Peroxidase activity was revealed using 3,3′-diaminobenzidine (Sigma) as a substrate. Blots were then scanned on a Bio-Rad 620 densitometer.

Data analysis

Results are expressed as means ± standard deviation (SD). The unpaired t-test was used to compare patients (n = 57) and control subjects (n = 35). Differences were considered significant with P < 0.05.

Results

Production of l-arginine/NO-dependent ONOO^–(Table 1) was significantly higher in platelets from migraine patients than from controls (7.40 ± 0.06 vs.1.84 ± 0.01, respectively; P < 0.001).

Table 1

Na⁺/K ⁺ATPase activity (µmol P_i/mg prot/h), peroxynitrite production, anisotropy (r) of TMA-DPH and DPH in platelet membranes from migraneurs and control subjects

	Migraineurs(n = 57)	Controls(n = 35)
Na+/K+ATPase activity (µmol Pi/mg prot/h)	0.035 ± 0.001∗	0.224 ± 0.014
Peroxynitrite (Fluorescence numbers)	7.40 ± 0.06∗	1.84 ± 0.01
TMA-DPH (Anisotropy)	0.252 ± 0.004∗	0.228 ± 0.001
DPH (Anisotropy)	0.212 ± 0.029	0.215 ± 0.015

Values are expressed as means ± standard deviation.

∗

P < 0.001 migraine patients vs. control subjects.

Western blot analysis using anti-iNOS and eNOS monoclonal antibodies demostrated both isoforms in cell lysates. The eNOS antibody reacted with a 140 kD protein in the platelet lysate corresponding to eNOS. Band densitometric analysis (Fig. 1) evidenced slightly but not significantly higher eNOS protein levels in patients than in control subjects (0.077 ± 0.009 vs. 0.071 ± 0.005, respectively; P > 0.05). iNOS protein was detected at 135 kD in the platelet lysate. According to band densitometric analysis (Fig. 1), iNOS protein levels were significantly higher in the platelets of patients than of controls(0.145 ± 0.017 vs. 0.081 ± 0.009, respectively; P < 0.001).

Figure 1

Western immunoblots of eNOS and iNOS specific densities in platelets from patients and controls.

Membrane Na⁺/K⁺-ATPase activity (Table 1) was significantly lower in migraine patients than in controls (0.035 ± 0.001 µmol P_i/mg prot/h vs. 0.224 ± 0.014 µmol P_i/mg prot/h; P < 0.001).

Platelets from patients showed significantly increased TMA-DPH fluorescence anisotropy (r) compared with controls (0.252 ± 0.040 vs. 0.228 ± 0.013; P < 0.001) (Table 1). TMA-DPH provides information on the more superficial region of the plasma membrane, having a polar part anchored at the lipid–water interface and the hydrocarbon tail in the lipid part of the membrane. Because anisotropy is inversely related to the fluidity of the probe's microenvironment, the present results indicate a lower fluidity of the external surface of patients’ platelet plasma membrane; when fluidity was probed with DPH (Table 1), which provides data on the deeper, hydrophobic part of the membrane, differences were not significant (0.212 ± 0.029 vs. 0.215 ± 0.015, respectively).

Discussion

Changes in platelet functionality in patients suffering from migraine have been described many years ago. In particular, spontaneous platelet aggregation was observed during and between attacks (19) and changes in platelet membrane viscosity due to differences in lipid composition have been reported (20). Mounting evidence suggests that NO release is an important molecular trigger mechanism in primary headache, and that platelets may have a physiopathological role in migraine via NO pathway activation (3, 21, 22). However, studies exploring NO levels and platelet activity in migraine patients during headache free-periods have yielded conflicting results (3–5).

Peroxynitrite is a reactive oxidant produced from NO and superoxide. It may be considered a marker of oxidative stress and its action involves a variety of biomolecules including proteins, lipids, and DNA. Further activities are initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, and inactivation of glyceraldehyde-3-phosphate dehydrogenase. Thus, ONOO^– induces inhibition of membrane Na⁺/K⁺-ATPase activity and calcium-activated potassium channels (23–26). The present study showed that increased platelet ONOO^– levels are not exclusively related to migraine attacks, but are also detected during headache free-periods. In fact, increased ONOO^– production was observed after addition of l-arginine at concentrations up to 100 µmol/l. Since, of the three NOSs, iNOS is the one producing the most NO and O^- ₂, it has been implicated in ONOO^– production. It is thus conceivable that the significantly greater amount of ONOO^– produced in the platelets of our patients than in those of controls may result from induction of iNOS by migraine-related factors. Indeed, in patients iNOS expression was also significantly greater than eNOS expression.

The lower fluidity of the external platelet membrane surface in migraine patients has potential physiopathological relevance because the membrane surface is responsible for the interaction between platelets and circulating molecules, and for cell adhesion (27). The low membrane Na⁺/K⁺-ATPase activity and decreased membrane fluidity detected in migraineurs can be regarded as secondary effects of increased ONOO^– levels. For this reason, ONOO^– is likely to have a central role in the overall ion imbalance and consequent membrane instability. In fact, the Na⁺/K⁺-ATPase enzyme system is an important contributor to cellular ionic homeostasis, and because it is an integral membrane protein with enzyme activity, it requires the presence of phospholipids, pointing to a close relationship with membrane's physicochemical structure (28). A large body of evidence indicates that increased membrane fluidity is associated with increased ATPase activity, and vice versa (24). On the other hand, peroxynitrite anion reduces both membrane fluidity and ATPase activity, as demonstrated in our patients. It is well known that ATPases are sensitive to changes in the physical parameters relating to membrane fluidity, and that Na⁺/K⁺-ATPase activity is affected by small modifications in the motional properties of surrounding lipids (29). The change in Na⁺/K⁺-ATPase activity observed in our patients might stem from subconformational changes in the protein depending on its microenvironment, indirectly reflecting changes in surrounding lipids and a different interaction between protein and lipids and/or within the protein subunit.

The results of the present study do not provide clear indications on the role of peroxynitrite in the physiopathology of migraine. In particular, they do not allow to draw conclusions on whether the changes described in migraineurs are to be interpreted as a cause of attacks or as a consequence of their onset. On the other hand, the present findings may contribute to a better understanding of the molecular mechanisms generating platelet changes in migraine. Increased ONOO^– production appears to be a significant cause of platelet damage and may have therapeutic implications. Indeed, the results of pharmacological modulation of the effects of peroxynitrite have been encouraging. In an in vivo model of myocardial ischemia, coadministration of ascorbic acid and glutathione methyl ester (GSHme) markedly enhanced the protective effects of GSHme (30). Furthemore, administration of melatonin and phytopharmacons (plant-derived antioxidant agents) was protective against the action of ONOO^– (31).

Further research is needed to explore the potential of antioxidant agents to modulate ONOO^– production in migraine and to control its effects.