Relationship between Vitamin D Binding Protein ... - ACS Publications

Relationship between Vitamin D Binding Protein and Aspirin Resistance in Coronary Ischemic Patients: A Proteomic Study Antonio J. Lo´ pez-Farre´ ,† Petra J. Mateos-Ca´ ceres,† Daniel Sacrista´ n,† Luis Azcona,† Esther Bernardo,‡ Teresa Perez de Prada,† Sergio Alonso-Orgaz,† Miguel Ferna´ ndez-Arquero,§ Antonio Ferna´ ndez-Ortiz,‡ and Carlos Macaya*,† Cardiovascular Research and Coronary Units, Cardiovascular Institute, and Immunology Department, Hospital Clıńico San Carlos, Madrid, Spain Received November 15, 2006

Our aim was to analyze the plasma proteome in aspirin (acetylsalicylic acid [ASA])-sensitive and ASAresistant coronary ischemic patients. Plasma from 19 ASA-sensitive and 19 ASA-resistant patients was analyzed. For the proteomic study, two-dimensional electrophoresis was performed. The expression of one isotype of the fibrinogen γ chain and three isotypes of haptoglobin was increased in ASAresistant patients. Three vitamin D binding protein isotypes were increased in ASA-resistant patients. In vitro incubation of vitamin D binding protein (DBP) with blood from healthy volunteers reduced the inhibitory effect of ASA on thromboxane A2 production. DBP may be a new regulator of the inhibitory effect of ASA on platelets. Keywords: Aspirin (acetylsalicylic acid [ASA]) ‚ plasma proteome ‚ platelets ‚ thromboxane A2

Introduction Aspirin (acetylsalicylic acid [ASA]) is the single most important drug in the secondary prevention of atherothrombotic disease. However, some patients experience recurrent vascular events despite treatment with aspirin, suggesting that these patients appear to be relatively resistant to the antiplatelet effect of aspirinsa phenomenon that has been called “aspirin resistance”.1,2 Aspirin exerts its main antithrombotic effect by inhibiting platelet cyclooxygenase-1 (COX-1) activity, thereby blocking thromboxane A2 (TxA2) synthesis in platelets.3 In this regard, laboratory aspirin resistance is defined as the failure of aspirin to inhibit platelet TxA2 production or TxA2-dependent platelet function (for a review, see ref 4). There are several different explanations for the limited efficacy of aspirin. Among others, it has been suggested that doses of aspirin higher than those currently used may be required in some patients to achieve the optimal antithrombotic effect of aspirin.5 However, low doses of aspirin block >95% of platelet COX-1 activity.6 There are other different explanations for the limited efficacy of aspirin in some patients, including genetic reasons and the expression of other cyclooxygenase isotypes.7,8 In addition, other factors such as the vascular functionality and the crosstalk between platelets and other cells, such as leukocytes, may also influence platelet reactivity.9,10 Therefore, it is quite possible that distinct molecular mechanisms that remain unex* To whom correspondence should be addressed. Dr. Carlos Macaya, Cardiovascular Research Unit, Cardiovascular Institute, Hospital Clıńico San Carlos s/n, Madrid 28040, Spain. E-mail: [email protected]. † Cardiovascular Research Unit, Cardiovascular Institute. ‡ Coronary Unit, Cardiovascular Institute. § Immunology Department. 10.1021/pr060600i CCC: $37.00

 2007 American Chemical Society

plored may contribute to the failure of aspirin to prevent the athrerothrombotic events. Two-dimensional electrophoresis (2DE) is a powerful technique to measure changes in expression of proteins and protein isotypes. We recently reported protein expression differences in plasma from patients during an acute coronary syndrome11 and from moderate hypercholesterolemic patients.12 This study is the first to use 2DE to compare expression changes in protein and protein isotypes in the plasma of aspirin-resistant to aspirin-sensitive coronary ischemic patients, and it identified several proteins whose expression was increased in aspirinresistant patients. These results may aid future development of more effective therapies for aspirin-resistant patients.

Materials and Methods Patients. The study was performed in 38 clinically stable coronary ischemic patients taking acetylsalicylic acid (ASA) (100 mg/day). Patients were divided into aspirin-resistant (n ) 19) and aspirin-sensitive (n ) 19) patients according to the PFA100 assay (see below). The patients had been taking aspirin for at least the last 9 months, and the coronary acute event occurred at least 9 months before the inclusion. Patients were excluded if they were on other antithrombotic drugs or nonsteroid anti-inflammatory drugs within 30 days before inclusion. Blood samples were obtained in the morning 2-4 h after aspirin by antecubital venipuncture, and the initial 3-4 mL of blood was discarded to avoid spontaneous platelet activation. Blood samples were collected in citrate tubes for both the PFA100 assay and platelet-rich plasma isolation and in EDTA for plasma isolation. Moreover, all patients received another dose Journal of Proteome Research 2007, 6, 2481-2487

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research articles of ASA (100 mg), and 1 h afterward an additional blood sample was obtained to perform again the PFA-100 assay. The study was approved by the Ethical Committee of the San Carlos University Hospital, and all patients gave informed consent. Classification of Aspirin-Resistant and Aspirin-Sensitive Patients. ASA-resistant patients were classified by using the PFA-100 assay (Dade Behring, West Sacramento, CA) as reported.13 The PFA-100 assay uses disposable test cartridges that have a collagen-coated membrane infused with epinephrine (10 µg). A syringe aspirates citrate whole blood under high shear flow conditions (5000-6000 s-1) through a small aperture (150 µg) cut into a membrane placed in the test cartridge. The time necessary for the occlusion of the aperture, defined as the closure time (CT), is indicative of platelet function for the whole blood sample. According to the manufacturer, CT ranges from 94 to 193 s with epinephrine cartridges in aspirin-resistant patients.14 In aspirin-sensitive patients, epinephrine CT is prolonged, and after 300 s, the process automatically terminates. Two-Dimensional Electrophoresis. For 2-DE, 500 µg of protein contained in the plasma was diluted in 350 µL of 8 mol/L urea, 2% CHAPS w/v, 40 mmol/L dithiothreitol, 0.2% Bio-Lyte ampholyte (Bio-Rad), and 0.01% w/v bromophenol blue. The samples were loaded on immobilized gradient IPG strips (pH 4-7), and isoelectric focusing was performed using a Protean IEF cell system (Bio-Rad), as reported.11,12 The gels were actively rehydrated at 50 V for 60 h, followed by rapid and linear voltage ramping steps. In the second dimension, the proteins from the strips were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels using a Protean II XL system (BioRad Labs). The gels were fixed in a solution containing a Fixative Enhance Concentrate solution (Bio-Rad Labs), methanol, and acetic acid in distilled water for 20 min. After two washings in distilled water, gels were silver stained (Silver Stain Plus Kit, Biorad), according to the manufacturer’s instructions. Staining was stopped with 5% acetic acid, and the gels were finally washed in distilled water. At least two different gels were run per patient. Image Acquisition and Analysis. The stained gels were scanned in a UMAX POWERLOOK III scanner operated by the software Magic Scan V 4.5. Intensity calibration was carried out using an intensity stepwedge prior to gel image capture. Image analysis was carried out using PD Quest 6.2.1 and Quantity One 4.2.3 (Bio-Rad). Image spots were initially detected, matched, and then manually edited. Each spot intensity volume was processed by background subtraction, and total spot volume was normalized by the corresponding spot volume of albumin for each patient. Mass Spectrometry. As reported,11 the spots of interest were manually excised from the gels using biopsy punches. To identify the spots of interest by mass spectrometry, spots from two different gels were obtained. The spots were washed twice with water, shrunk with 100% acetonitrile, and dried in a Savant SpeedVac. The samples were then reduced with 10 mmol/L dithiothreitol in 25 mmol/L ammonium bicarbonate and subsequently alkylated with 55 mmol/L iodoacetamide in 25 mmol/L ammonium bicarbonate. They were digested with 12.5 ng/µL sequencing grade trypsin (Roche Molecular Biochemicals) in 25 mmol/L ammonium bicarbonate (pH 8.5) overnight at 37 °C. After digestion, the supernatant was collected, and 1 µL was spotted onto a MALDI target plate and allowed to airdry at room temperature. Then, 0.4 µL of a 3 mg/mL R-cyano4-hydroxy-transcinnamic acid matrix (Sigma) in 50% acetoni2482

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Mateos-Ca´ ceres et al.

trile was added to the dried peptide digest spots, which were again allowed to air-dry at room temperature. MALDI-TOF MS analyses were performed in a Voyager-DE STR instrument (PerSeptives Biosystems), a model fitted with a 337 nm nitrogen laser and operated in reflector mode, with an accelerating voltage of 20000 V. All mass spectra were calibrated externally using a standard peptide mixture (Sigma). Peptides from the autodigestion of trypsin were used for the internal calibration. The analysis by MALDI-TOF mass spectrometry produced peptide mass fingerprints, and the peptides observed can be collated and represented as a list of monoisotopic molecular weights. MS/MS sequencing analyses were carried out using the MALDI-tandem time-of-flight mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Peptides with a signal-to-noise greater than 20 were considered. For protein identification, Mascot database 1.9 (http://www.matrixscience.com) was used as an algorithm to match the peptides obtained by mass spectrometry. In all protein identification, the probability scores were greater than the score fixed by Mascot as significant with a p-value less than 0.05. The peptide matched and the sequence peptides matched for each of the plasma protein and plasma protein isotypes analyzed using mass spectrometry as previously described.12 TxB2 Determination and Platelet-Rich Plasma Isolation. Whole blood was obtained from seven male healthy volunteers (38 ( 4 years) who, at least for 15 days, had not taken aspirin nor any other antiplatelet or antiinflamatory drugs. Whole blood was divided in aliquots of 1 mL/tube, and then the aliquots were incubated in the presence and in the absence of 0.33 mmol/L aspirin (acetylsalicylic acid, SIGMA-Aldrich) and 15 µmol/L vitamin D binding protein (DBP) (Calbiochem, Merck Biosciences). The aspirin concentration used was similar to that detected in the serum of patients taking low-doses of aspirin.10 The DBP concentration used was similar to that reported to affect actin clearance.15 DBP was added to whole blood 5 min prior to aspirin. Similar experiments were performed in platelet-rich plasma (PRP). PRP was obtained from whole blood obtained from the same healthy volunteers and collected in citrate tubes, which were centrifuged at 800g for 15 min as reported.16 The samples (1.25 × 108 platelets/tube) were incubated at 37 °C with continuous stirring (800g) for 10 min. At the end of the incubation period, the samples were centrifuged and the supernatant was recovered for TxB2 determination. TxB2 is the major metabolite of TxA2 and was determined by a commercial enzyme immunoassay kit (Amersham Biosciences, Uppsala, Sweden). The inter-assay and intra-assay variabilities were 11.9% and 5.6%, respectively. Statistical Analysis. Results are expressed as means ( SD. To determine statistical significance, we used the MannWhitney test for the proteomic study and Wilcoxon’s test for the in vitro experiments. A p-value of 300 >300

100 33.3 59.0 16.7 57.9 61.5 111.4 ( 21.6 125.8 ( 27.9

a ACE: angiotensin-converting enzyme. CT: closure time. CT is the time necessary for the occlusion of the aperture in the PFA-100 cartridge and is indicative of platelet function. The upper limit of the test is 300 s. Results are represented as mean ( SD.

1). This work is included within a greater study to analyze genetic causes associated with aspirin-resistance, and the 38 patients included were chosen based on the fact that CT ranges were extreme for each of the two aspirin responsiveness conditions (see Table 1). In the plasma proteomic map different spots were densitometrically analyzed, and some of them were identified by comparison with those found in the SWISS-2D database of the plasma map (http://www.expasy.ch/ch2d). Densitometric analysis of each spot was calculated using albumin as a reference value, as reported.17 In previous works, we and others have identified by mass spectrometry some of the here-reported proteins.12,18,19 However, in order to confirm the identity of some of the here-measured plasma proteins, we have identified again some of these proteins by mass spectrometry. Therefore, in the present work we have identified by mass spectrometry the following proteins: three isotypes of the fibrinogen γ chain, three isotypes of vitamin D binding protein, five isotypes of haptoglobin, and one albumin precursor isotype (Figure 1A,B). We detected three isotypes of the fibrinogen γ chain (Figure 2). The fibrinogen γ chain isotype 1 was enhanced in plasma from aspirin-resistant patients compared with that from aspirinsensitive patients (Table 2). Moreover, five isotypes of haptoglobin were also identified in the plasma (Figure 2). Haptoglobin isotypes 1, 2, and 3 reached a statistically significant increase in the plasma from aspirin-resistant patients compared with that from aspirin-sensitive patients (Table 2). Apolipoprotein A-IV (Apo-AIV) was also identified by comparison with the SWISS-PROT 2D. There was no difference in the Apo-AIV plasma level between the two groups of patients (Table 3). Moreover, a spot identified by mass spectrometry as albumin precursor showed no differences between the two groups of patients (Table 3). We have also identified three spots that by mass spectrometry analysis demonstrated to be vitamin D-binding protein. The expression of the three DBP isotypes was significantly increased in the plasma from aspirin-resistant patients compared with that from aspirin-sensitive patients (Table 3). A number of spots that have not yet been identified in the SWISS-PROT 2D plasma database were also measured [(1) kDa/ pI: 38.8/5.37. (2) kDa/pI: 38.5/ 5.56. (3) kDa/pI: 38.6/5.57. (4)

kDa/pI: 38.6/5.59. (5) kDa/pI: 36.9/5.40. (6) kDa/pI: 36.9/5.58. (7) kDa/pI: 36.6/5.9. (8) kDa/pI: 36.7/6.12. (9) kDa/pI: 34.0/ 6.05. (10) kDa/pI: 34.0/6.14. (11) kDa/pI: 32.0/6.06]. However, the expression of any of these proteins was different between aspirin-sensitive and aspirin-resistant patients. Effect of DBP on TxA2 Production. We analyzed if DBP could modify the ability of aspirin to inhibit COX-1 activity. As shown in Table 4, aspirin reduced TxB2 levels in whole blood from healthy donors. DBP by itself failed to modify TxB2 levels in whole blood but significantly reduced the ability of aspirin to prevent TxB2 production (Table 4). We then analyzed if the effect of DBP on aspirin-inhibitable TxA2 production could be related to a direct effect of DBP on platelets. Then, experiments were performed in PRP obtained from the same healthy donors from whom the whole blood was obtained. Aspirin reduced TxB2 levels in PRP although with lesser ability than in whole blood (Table 4). DBP by itself did not modify TxB2 levels in PRP but significatively reduced the inhibitory effect of aspirin on TxB2 levels (Table 4). Moreover, when aspirin was added 5 min before DBP to PRP, the protective effect of DBP on platelet COX-1 activity was not observed (TxB2 in pg: 27.1 ( 14 pg, p > 0.05 with respect to aspirin alone). It was remarkable that the protective effect of DBP on TxB2 production in ASA-incubated PRP was of lesser magnitude than that observed in whole blood (p ) 0.03). In this regard, the protective effect of DBP in whole blood, in terms of difference in TxB2 production between the presence of ASA and ASA + DBP, was 18.2 ( 26.4 pg while in PRP this difference was 8.3 ( 11.1 pg (p < 0.05 between them).

Discussion In the present study we analyzed, for the first time using proteomics, changes in different proteins expressed in plasma from aspirin-sensitive and aspirin-resistant coronary ischemic patients, defined by their platelet functionality. We found an increased plasma expression in three DBP isotypes in aspirinresistant patients. Moreover, in vitro experiments demonstrated that DBP reduced the inhibition of aspirin on TxA2 production. Despite the demonstrated clinical benefit of aspirin, the absolute risk of recurrent vascular events among patients taking aspirin remains relatively high. Therapeutics resistance to aspirin might explain a portion of this risk. Since there are many unanswered questions regarding the biological mechanisms associated with aspirin resistance, we here used proteomics to detect proteins and protein isotypes whose expression may be changed in aspirin-resistant compared with aspirin-sensitive patients. In the plasma proteome three isotypes of fibrinogen γ chain were first analyzed. The plasma level of the fibrinogen γ chain isotype 1 was found increased in aspirin-resistant patients. This fibrinogen γ chain isotype 1 has also been found increased in patients during an acute coronary syndrome.11 In this regard, fibrinogen participates in platelet aggregation and inflammation. A different alternative splicing of fibrinogen γ chain messenger ribonucleic acid has recently been demonstrated that results in fibrinogen γ chain variants that alter fibrin formation and structure and could be associated with an increase of thrombotic risk.20 Accordingly, a greater incidence of cardiovascular and cerebrovascular events is associated with aspirin resistance.21,22 Another protein whose expression was different in plasma from aspirin-resistant patients compared with that from aspirinsensitive patients was haptoglobin. Haptoglobin is an acute Journal of Proteome Research • Vol. 6, No. 7, 2007 2483

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Figure 1. (A) Representative MS/MS spectrum of fibrinogen γ chain isotype 1 (1), haptoglobin isotype 3 (2), and MS of albumin precursor (3). Panel B shows the MS and MS/MS spectrum of the vitamin D binding protein isotype 3. In the spots submitted to MS/MS, multiple charged peptides observed in the MS mode were selected for fragmentation (MS/MS). 2484


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Figure 2. (Left) Representative two-dimensional gel electrophoresis of a plasma proteome from a resistant patient using a pH range between 4 and 7. Five areas were analyzed (A-E) in each group of patients. In areas A-C there were statistical differences. No statistical differences were observed in areas D and E, corresponding to apolipoprotein A-IV and albumin precursor, respectively. (Right) Representative patterns of the different proteins identified in the plasma proteome (areas A-C) of aspirin-resistant and aspirin-sensitive patients. Albumin was identified and used to normalize total spot volume in each of the other areas. Table 2. Identification and Expression of the Fibrinogen γ Chain and Haptoglobin Isotypesa protein

fibrinogen γ chain fibrinogen isotype 1 fibrinogen isotype 2 fibrinogen isotype 3 haptoglobin haptoglobin isotype 1 haptoglobin isotype 2 haptoglobin isotype 3 haptoglobin isotype 4 haptoglobin isotype 5

experimental mass (kDa/pI)

identification method

ASA-sensitive group (A.U.)

ASA-resistant group (A.U.)

49.4/5.6 49.4/5.4 49.5/5.3

plasma map, MS plasma map, MS/MS plasma map, MS/MS

1.4 ( 0.7 1.4 ( 0.6 0.9 ( 0.7

2.3 ( 1.3* 2.01 ( 1.2 1.1 ( 0.5

41.41/5.21 41.49/5.08 42.22/4.97 43.24/4.88 44.52/4.81

plasma map plasma map plasma map plasma map, MS/MS plasma map

5.0 ( 1.2 4.4 ( 1.2 2.8 ( 1.0 1.0 ( 0.6 0.3 ( 0.3

7.0 ( 1.4* 6.3 ( 2.0* 4.0 ( 1.6* 1.6 ( 0.9 0.5 ( 0.4

a pI: isoelectric point. A.U.: arbitrary units. MS: mass spectrometry. MS/MS: tandem mass spectrometry. Database for plasma map: SWISS-PROT 2D database. Results are represented as mean ( SD. An asterisk (*) indicates p < 0.05 with respect to aspirin-sensitive patients.

phase reactant protein that binds free hemoglobin and removes it from the circulating blood. By binding free hemoglobin, haptoglobin exerts antioxidant properties.23 Haptoglobin consists of two different polypeptide chains: the R and β chains. The β chain (40 kDa) is heavier than the R chain.24 In our study we detected five haptoglobin isotypes with an apparent experimental mass of 40 kDa, which suggests the presence of the β chain haptoglobin isotype. Plasma expression of three haptoglobin isotypes was significatively increased in ASAresistant patients compared with that from aspirin-sensitive patients. In this regard, different works have suggested that low doses of aspirin reduce oxygen free radical damage and even reduce the production of free radicals by platelets.25,26 There-

fore, in aspirin-sensitive patients, aspirin may be more efficient to prevent oxidative stress than in aspirin-resistant patients who may require greater activation of the endogenous antioxidant machinery to protect them against oxidative damage. We also failed to detect differences in the plasma levels of Apo-AIV between aspirin-sensitive and aspirin-resistant patients. Apo-AIV is a protective vascular protein since its overexpression reduces aortic atherosclerotic lesions.27,28 Taken together, all these results may suggest that neither the vascular damage nor the inflammatory status seem to be different by the different platelet response to aspirin. However, further specific experiments are needed to analyze this hypothesis. Journal of Proteome Research • Vol. 6, No. 7, 2007 2485

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Table 3. Identification and Expression of Apolipoprotein-AIV (Apo AIV), Vitamin D Binding-protein (DBP) Isotypes, and Albumin Precursora exptl mass (kDa/pI)

protein

Apo-AIV DBP DBP isotype 1 DBP isotype 2 DBP isotype 3 albumin precursor

ASAsensitive group (A.U.)

identification method

ASAresistant group (A.U.)

43.7/5.2

plasma map, MS 0.5 ( 0.4 0.6 ( 0.5

54.52/5.4 54.5/5.3 54.4/5.1 36.9/5.55

MS MS, MS/MS MS, MS/MS MS, MS/MS

0.9 ( 0.3 0.7 ( 0.3 0.3 ( 0.1 2.3 ( 1.1

2.1 ( 0.8* 1.5 ( 0.6* 0.8 ( 0.5* 2.8 ( 1.7

a pI: isoelectric point. A.U.: arbitrary units. MS: mass spectrometry. MS/ MS: tandem mass spectrometry. Database for plasma map: SWISS-PROT 2D database. Results are represented as mean ( SD. An asterisk (*) indicates p < 0.05 with respect to aspirin-sensitive patients.

Table 4. Effect of DBP on TxB2 Productiona basal

ASA

DBP

DBP + ASA

whole blood 29.3 ( 12.7 3.30 ( 0.6* 32.0 ( 21.6 ( 9.9† PRP 41.8 ( 7.7 28.0 ( 14.2* 42.4 ( 2.0† 6.1 ( 11.1*, † 6.8†

a TxB2 production (pg) was studied in whole blood and platelet-rich plasma (PRP) from healthy volunteers. The whole blood and PRP were incubated in the presence and in the absence of ASA (0.33 mmol/L) and DBP (15 µmol/L). Results are mean ( SD. An asterisk (*) indicates p < 0.05 with respect to basal. A dagger (†) indicates p < 0.05 with respect to ASA alone.

The plasma expression of albumin precursor, a nutritional marker,29 was not different between the two groups of patients, which suggests that the nutritional status was similar between the two groups. The plasma expression of a number of not-yet-identified proteins was not changed in aspirin-sensitive patients compared with aspirin-resistant patients. However, a remarkable finding was that the expression in plasma of three DBP isotypes was significatively increased in aspirin-resistant patients compared with aspirin-sensitive patients. In this regard, the existence of three DBP isotypes has been demonstrated, and they are distinguished by different combinations of amino acid substitution in the protein.30 The main function of DBP is to bind and transport vitamin D analogous (for a review, see ref 31). However, more recently DBP has been described as a leukocyte activator.32 Moreover, a previous study from Vasconcellos and Lind has postulated that depletion of DBP may allow actin released from injured tissues to stimulate purinergic receptors on platelets.15 On the contrary, we have observed that plasma from aspirin-resistant patients contained greater amounts of the three DBP isotypes than that from aspirin-sensitive patients. Then we analyzed if DBP may modify the ability of aspirin to inhibit platelet COX-1 activity by determining TxB2 levels in ex vivo incubated blood. In this regard, Zimmerman et al. have shown that, in coronary artery bypass graft patients who did not respond to aspirin, the in vitro addition of thromboxane synthase and TxA2 receptor inhibitors reduced platelet aggregation, which suggested that persistent thromboxane production may be involved in aspirin resistance.33 In our study, in vitro addition of DBP to whole blood significantly reduced the inhibitory effect of aspirin on TxA2 production. Previous works have demonstrated that leukocytes may be involved in the antiplatelet effect of aspirin.10 Moreover, different studies have demonstrated that DBP can enhance neutrophil chemotactic activity.34,35 We further analyzed if the 2486


prevention exerted by DBP on the inhibitable effect of aspirin on platelet TxA2 production was dependent on the presence of blood cells other than platelets. In PRP, the presence of DBP also reduced the effect of aspirin on TxA2. The protective effect of DBP on TxA2 production in aspirin-incubated PRP was lower than that observed in the experiments performed in whole blood. These results suggest that DBP may protect COX-1 activity from aspirin by directly acting on platelets, although an effect mediated by the other blood cells could be also involved. In this regard, in addition to inhibiting TxA2 synthesis by platelets, aspirin reduced platelet activation throughout, stimulating nitric oxide release from leukocytes.10 Accordingly, in the present study it was observed that the basal release of TxB2 was higher in whole blood than in PRP, and the inhibitory effect of aspirin on TxB2 was also greater in the experiments performed in whole blood than those in PRP. It has been previously demonstrated that nonsteroidal antiinflammatory drugs may competitively reduce the capacity of low doses of aspirin to cause inhibition of COX-1 in platelets.36 While addition of DBP prior to aspirin reduced the ability of aspirin to inhibit COX-1 in platelets, the addition of aspirin 5 min before DBP failed to modify the inhibitory ability of aspirin on COX-1 in platelets. There are different possibilities to explain this finding. As occurs with nonsteroidal drugs, DBP may reduce the accessibility of aspirin to the acetylation site in COX1.36 An alternative hypothesis is that DBP, through its ability to bind cytoskeleton proteins such as actin and profilin, may modify the structural conformation of COX-1.37 A structural conformational change in COX-1 may also reduce the accessibility of aspirin to its acetylation site in COX-1. Accordingly, microinjection of plasma DBP to nonmuscle cells has been shown to cause disruption of microfilament organization.38 Moreover, Esteban et al. have demonstrated that human lymphocytes bound and internalized DBP, localizing it in the cytoplasm.39 However, with the present experimental design we cannot dilucidate the exact molecular mechanism by which DBP protected COX-1 activity from aspirin. Therefore, further studies are now needed and warranted to know how DBP affects the inhibitory action of ASA on COX-1 activity. In addition, further studies are warranted to assess whether the determination of the three identified plasma DBP isotypes in patients taking aspirin might be used as markers to gain insights in the efficacy of aspirin. Several considerations about the present study should be made. First, we have performed the experiments in patients with a previous history of coronary ischemia. In this regard, it has been reported that patients with a history of coronary artery disease had nearly twice the odds of being aspirin resistant.40 Second, a patient could be showing aspirin resistance due to reduced patient compliance with aspirin therapy. Indeed, up to 40% of patients with cardiovascular disease do not comply with aspirin therapy.41 In this regard, we administered an additional aspirin dose to confirm the result of the PFA-100 assay, and we only included in the study patients that demonstrated a similar CT range in response to epinephrine at inclusion and 1 h after the additional 100 mg aspirin administration. It is also important to remark that several techniques are available to determine aspirin responsiveness. In the present study, aspirin-resistant and aspirin-sensitive patients were classified by their platelet functionality determined by the PFA100 assay. Whether the here-reported proteomic plasma differences between aspirin-sensitive and aspirin-resistant pa-

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tients would be comparable using methods other than PFA100 is unknown, and it is out of the scope of the present study. In conclusion, the here-reported results provide strong evidence that aspirin-resistant patients have higher plasma levels of three DBP isotypes than aspirin-sensitive patients, which was associated with the failure of aspirin to prevent both TxA2 production and platelet activation. DBP prevents the inhibitory effects of aspirin on COX-1 activity, opening a new undescribed mechanism that could be involved in the aspirinresistant phenomenon.

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Journal of Proteome Research • Vol. 6, No. 7, 2007 2487

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