Published online before print August 7, 2008, doi: 10.1161/ATVBAHA.108.173815
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© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Apolipoprotein A-I Tryptophan Substitution Leads to Resistance to Myeloperoxidase-Mediated Loss of Function
From the Department of Cell Biology, Cleveland Clinic, Cleveland Ohio. Current address for D.-Q.P.: Department of Cardiology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 China.
Correspondence to Jonathan D. Smith, Department of Cell Biology, NC10, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail smithj4{at}ccf.org
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Abstract |
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Objective— Apolipoprotein A-I (apoAI) acts as an ABCA1-dependent acceptor of cellular phospholipids and cholesterol during the biogenesis of HDL, but this activity is susceptible to oxidative inactivation by myeloperoxidase. We tried to determine which residues mediated this inactivation and create an oxidant-resistant apoAI variant.
Methods and Results— Mass spectrometry detected the presence of tryptophan, methionine, tyrosine, and lysine oxidation in apoAI recovered from human atheroma. We investigated the role of these residues in the myeloperoxidase-mediated loss of apoAI activity. Site-directed mutagenesis and chemical modification were used to create variants of apoAI which were tested for ABCA1-dependent cholesterol acceptor activity and oxidative inactivation. We previously reported that tyrosine modification is not required for myeloperoxidase-induced loss of apoAI function. Lysine methylation did not alter the sensitivity of apoAI to myeloperoxidase, whereas site-specific substitution of apoAI methionine to valine increased the sensitivity of apoAI to myeloperoxidase. ApoAI tryptophan residues were identified as essential in apoAI function and oxidant sensitivity as substitution of all four apoAI tryptophan residues to leucine led to loss of function, but the conservative substitution to phenylalanine retained full function and was resistant to oxidative inactivation.
Conclusions— Tryptophan modification of apoAI is primarily responsible for the myeloperoxidase-mediated loss of the cholesterol acceptor activity of apoAI.
Key Words: dysfunctional HDL • oxidation • atherosclerosis
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Introduction |
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High levels of high density lipoprotein (HDL) and apolipoprotein A-I (apoAI), the major HDL protein, are associated with decreased risk for cardiovascular disease.1,2 The protection against cardiovascular disease afforded by apoAI and HDL may in part be attributed to their role in reverse cholesterol transport, although they have additional antiinflammatory, antioxidant, and endothelial cell regenerative properties that may also play protective roles. However, all HDL may not be functionally equivalent. Fogelman and colleagues have reported that HDL samples from patients with cardiovascular disease have a diminished capacity to inhibit LDL induced monocyte migration in a coculture system.3 Previously, work from our group and subsequently from the group led by Heinecke have found that apoAI serves as a preferred target for myeloperoxidase (MPO)-catalyzed oxidative modification, leading to chlorination and nitration of specific tyrosine residues and concomitant loss of ABCA1-mediated cholesterol acceptor activity.4,5,6,7 In addition, elevated content of chlorotyrosine and nitrotyrosine residues are observed in plasma apoAI4,8 and more highly in apoAI recovered from human atheroma.4,8
We also reported that the degree of tyrosine modification of plasma apoAI, isolated from cardiology patients, correlates with its cholesterol acceptor activity.4 Thus, tyrosine modification of apoAI by MPO is associated with loss of apoAI function as an ABCA1-dependent acceptor of cellular lipids and can serve as a fingerprint to monitor the extent of apoAI modification. However, our prior studies using a tyrosine-free apoAI derivative (7YF, all seven tyrosines substituted by phenylalanine) show that it is equally susceptible to MPO mediated loss of function compared to wild-type apoAI.9 We also demonstrated in vitro that MPO could modify apoAI lysine residues into lysine chloramines and aminoadipic acid, and apoAI tryptophan residues were converted into the mono- and dioxygenated derivatives.9 It has also been previously shown that MPO modification of HDL led to a time- and dose-dependent decrease in bulk tryptophan fluorescence.10 However, whether such modifications occurred in vivo and might be responsible for oxidative inactivation of apoAI cholesterol efflux activity remains unknown. In the current study we report the presence of multiple site-specific tryptophan, methionine, and lysine modifications in apoAI isolated from human atheroma. We thus sought to determine the effects of altering the MPO sensitive lysine, methionine, and tryptophan residues in apoAI and whether such modifications might account for the observed oxidative inactivation of apoAI in vivo. We report here that replacement of the four apoAI tryptophan residues with leucines led to loss of its cholesterol acceptor function, whereas the replacement of tryptophan with phenylalanines not only preserved apoAI function but rendered it resistant to MPO-mediated loss of cholesterol acceptor and lipid binding activities. The apoAI with tryptophan to phenylalanine substitutions, though it retained its activity, was still sensitive to MPO-mediated cross-linking and loss of 
-helical content. The present studies thus suggest that apoAI tryptophan residues are responsible for MPO-dependent oxidative loss of apoAI function in vivo.
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Methods |
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Mass Spectrometry
Human atheroma derived apoAI was isolated by immunoaffinity chromatography as previously described.5 ApoAI was eluted in glycine buffer (pH 2.5) and subjected directly to trypsin digestion or first separated by SDS-PAGE and subjected to in gel trypsin digestion. Mass spectrometry was performed and collision-induced dissociation (CID) spectra were obtained, as previously described.5 Chlorotyrosine and 2-amino adipic acid analyses were performed after acid hydrolysis with heavy isotope internal standards as previously described4,9 using duplicate assays of apoAI from human atheroma or from plasma of healthy volunteers isolated by immunoaffinity chromatography.
Site-Directed Mutagenesis and Recombinant ApoAI Production
The pET-20b bacterial expression vector containing the cDNA of 6-His tagged recombinant human apoAI (rh-apoAI) was previously described.11 Point mutations to tryptophan (8,50,72,108) and methionine (86,112,148) residues were made using QuickChange Mutagenesis Kit from Stratagene and confirmed by DNA sequencing. Plasmids were transformed into Escherichia coli strain BL21 (DE-3) pLysS and apoAI expression and purification was performed as described previously.9 rh-ApoAI was extensively dialyzed against PBS or MPO reaction buffer (60 mmol/L sodium phosphate, 100 mmol/L sodium chloride, 100 µmol/L diethylenetriamine pentaacetic acid, pH 7.0) to remove any trace of imidazole, analyzed by SDS-PAGE, and found to be >95% pure. Because Trp and Met substitution alters the protein OD280 and reactivity to the BCA or Lowry protein assays, protein concentrations were determined based on free amines using the o-phthaldialdehyde (OPA) assay, with a human plasma-derived apoAI (Biodesign) standard, as previously described.12 Cleavage of the initial Met and His tag of rh-apoAI was performed by formic acid treatment,11 followed by fast protein liquid (FPLC) purification.
ApoAI Lysine Modifications
Human plasma-derived apoAI was dialyzed against PBS and diluted to 0.5 mg/mL. Lysine reductive methylation was performed as previously described.12 Extent of lysine modification was determined by the OPA assay. ApoAI was then dialyzed against MPO reaction buffer, and the protein concentration of lysine-modified apoAI was determined using the BCA reagent.
ApoAI MPO and Hypochlorous Acid Modifications
MPO at a final concentration of 57 nmol/L, prepared as previously described,4 was added to ApoAI at 100 µg/mL (3.5 µmol/L) that had been extensively dialyzed against MPO reaction buffer. The reaction was initiated by adding hydrogen peroxide at varying mole ratios to apoAI in 4 aliquots at 15-minute intervals at 37°C, and continuing the incubation for 90 minutes, at which time 2 mmol/L L-methionine was added to quench the reaction. For chemical modification of apoAI, sodium hypochlorite (NaOCl) was added to 100 µg/mL ApoAI in MPO buffer at varying concentrations in 4 aliquots at 15 minutes intervals at 37°C. After a total incubation time of 60 minutes, 2 mmol/L L-methionine was added to quench the reaction.
ABCA1-Dependent Cholesterol Efflux Assay
RAW 264.7 murine macrophage cells were labeled with [3H]cholesterol and treated with 0.3 mmol/L 8Br-cAMP to induce ABCA1 activity, as previously described.13,14 The cells were washed and chased for 4 hours in serum-free medium in the presence of 0.3 mmol/L 8Br-cAMP and the presence or absence of various apoAI preparations. The radioactivity in the chase media was determined after brief centrifugation to pellet debris. Radioactivity in the cells was determined by extraction in hexane:isopropanol (3:2) with the solvent evaporated in a scintillation vial prior to counting. The percent cholesterol efflux was calculated as 100x(medium dpm) / (medium dpm+cell dpm).
Lipid Binding Activity Assay
Lipid binding activity of apoAI was assessed via the inhibition of phospholipase C (PLC)-mediated aggregation of human low density lipoprotein, performed as previously described.12 We have previously shown that this assay give results similar to those observed by the DMPC dispersion clearance assay, but it is more sensitive and requires less apoAI.12 The final concentration of apoAI used in this assay was 12.5 µg/mL, which was sufficient to decrease the initial rate of LDL aggregation by 
75%.
Detection of ApoAI Cross Links
250 ng of apoAI per lane was denatured in an SDS sample buffer, run on a 10% Tris glycine gel in the presence of SDS, and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed sequentially with goat anti-human apoAI primary antibody (1:1,000 dilution, DiaSorin) and rabbit antigoat-HRP conjugated antibody (1:10,000 dilution), and apoAI was visualized with an enhanced chemiluminescent substrate.
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Results |
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ApoAI Modifications in Human Atheroma
We previously demonstrated the presence of nitro- and chlorotyrosine in apoAI isolated from human atheroma tissue.4 We extended these studies to determine whether we could also identify modified tryptophan, methionine, and lysine residues in apoAI isolated from human atheroma, all modifications which we or others have previously identified after in vitro MPO treatment of apoAI.9,15 Using tandem mass spectrometry, we were able to detect monohydroxytryptophan residues at all four tryptophan positions, 8, 50, 72, and 108, and dihydroxytryptophan at position 108 within apoAI (Figure 1A through 1E). We previously identified mono- and dihydroxytrytophan at residue W72 in in vitro MPO-modified apoAI.9 In addition we detected methionine sulfoxide at residues 48 and 112 (Figure 1D and 1E). To look for lysine modification by MPO-generated HOCl we used stable isotope dilution high-performance liquid chromatography (HPLC) with online tandem mass spectrometry to quantify 2-aminoadipic acid, an end product of lysine oxidation by the MPO generated oxidant. 2-Aminoadipic acid levels were low but detectable in apoAI isolated from the plasma of 6 healthy volunteers, whereas the mean levels were strikingly elevated
16-fold in apoAI isolated from 6 human atheroma samples (Figure 2, P=0.005 by a 2-tailed t test). Thus, tryptophan, methionine, and lysine oxidation of apoAI occur physiologically within human atheroma. We therefore sought to determine which of these modifications was responsible for yielding dysfunctional apoAI with a diminished capacity to accept cellular cholesterol.
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ApoAI Lysine Modification
In the amphipathic structure of apoAI, the 21 lysine residues overwhelming reside on both sides of and adjacent to the hydrophobic face.16,17 Lysine modification by MPO is an attractive candidate to be responsible for MPO-induced loss of apoAI function as we previously demonstrated that apoAI lysine residues can undergo modification by MPO,9 and that extensive chemical modification of apoAI lysine residues that alter its positive charge led to loss of apoAI cholesterol acceptor activity.12 However, we also found that lysine modification by reductive methylation, which retains the lysine positive charge, led to only modest reductions of apoAI function.12 ApoAI was subjected to reductive methylation, leading to 92% lysine modification, or control incubation and dialyzed extensively against MPO reaction buffer. Modification reactions were performed using catalytic amounts of MPO and increasing molar ratios of H2O2:apoAI. The reaction products were assayed for cholesterol acceptor activity using cholesterol-labeled RAW264 macrophages that had been pretreated with a cAMP analogue to induce ABCA1. In the absence of H2O2 in the modification reaction, the methylated and nonmethylated control apoAI had robust and equivalent ABCA1-dependent cholesterol acceptor activity. With increasing doses of H2O2, the cholesterol acceptor activity of both the methylated and control apoAI samples declined in a similar fashion (supplemental Figure I, available online at http://atvb.ahajournals.org). In addition, the alpha helix content of these preparations was estimated by CD, and both methylated and control apoAI preparations were similarly susceptible to the MPO/H2O2 dose dependent reduction in alpha helix content (supplemental Table I). Because reductive methylation of primary amine of lysine into a tertiary amine decreases its chemical reactivity but did not lead to protection of the function of apoAI, apoAI lysine modification by MPO is unlikely to be responsible for apoAI’s loss of function.
ApoAI Methionine Substitution
We then turned our attention to the 3 apoAI methionine residues, which were previously implicated by Heinecke and colleagues in the MPO induced loss of apoAI function.15 To substitute valine for all 3 methionines, we used recombinant human apoAI (rh-apoAI), which adds an additional methionine initiation codon and a 6-His tag to the N terminus. We and others have previously demonstrated that rh-apoAI behaves similarly to plasma-derived apoAI in its cholesterol acceptor activity, lipid binding activity, and its susceptibility to MPO-mediated loss of function.11,15,18,9 Using site-directed mutagenesis, we created an apoAI expression construct encoding a protein with the 3 internal methionines converted to valine (rh-apoAI 3MV). One cannot substitute for the initiating methionine; however, this methionine and the His tag can be chemically cleaved by formic acid.11 We determined that the rh-apoAI 3MV, regardless of whether the initiating methionine and His tag were intact or removed, had similar ABCA1-dependent cholesterol acceptor activity compared to wild-type rh-apoAI. In addition, the rh-apoAI 3MV, with or without the N-terminal methionine, and wild-type rh-apoAI were equally susceptible to a high dose (H2O2: apoAI=15:1) MPO-mediated loss of cholesterol acceptor activity (Figure 3A). MPO modifications of rh-apoAI and the 3MV variant were performed at varying and modest molar ratios of H2O2:apoAI, and the 3MV variant was paradoxically more sensitive to loss of cholesterol acceptor activity at low molar ratios (Figure 3B). For example, at an H2O2:apoAI ratio of 1.4, wild-type apoAI had a negligible loss of cholesterol acceptor activity, whereas the 3MV variant lost approximately half of its cholesterol acceptor activity. Thus, we conclude that the methionine residues in apoAI, instead of playing a role in oxidative impairment of apoAI function, actually play a protective role by absorbing oxidants, a function previously suggested by Stocker and colleagues.19
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ApoAI Tryptophan Substitutions
We altered all 4 tryptophans to either leucine (rh-apoAI 4WL) or phenylalanine (rh-apoAI 4WF) for functional characterization and oxidant sensitivity testing. Our data revealed that the aromatic or bulky nature of the tryptophan residues seemed to be crucial for the cholesterol acceptor activity of apoAI, as the 4WL variant lost the majority of this activity over a wide range of apoAI doses, whereas the 4WF variant retained this activity (Figure 4A). Thus, the 4WF variant was competent for physiological lipidation by cellular ABCA1. In addition, the 4WF variant was equally competent compared to wild-type rh-apoAI in the clearance of a DMPC:cholesterol (90:10 mole %) emulsion (supplemental Figure II), demonstrating that the 4WF variant was able to interact with lipids in a cell-free context. We also prepared rHDL by cholate dialysis using POCP and the wild-type or 4WF apoAI. Both yielded a similar pattern of rHDL discs estimated by nondenaturing gels at 9.8, 12, and 17 nm, without any lipid free apoAI remaining (supplemental Figure IIIA). We tested the wild-type and 4WF rHDL, and both were equally competent to mediate ABCA1-independent cholesterol efflux from RAW264.7 cells (supplemental Figure IIIB), without ABCA1-dependent acceptor activity, as expected for fully lipidated apoAI. We examined the predicted alpha helix content of these proteins by CD and found that the rh-apoAI had 56% alpha helix, whereas the 4WL and 4WF variants both had increased estimated alpha helix contents of 68% and 71%, respectively. Thus, the loss of efflux and lipid-binding activity of the 4WL variant cannot be attributed to loss of helical content.
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Both rh-apoAI and the 4WF variant were then subjected to the MPO/Cl–/H2O2 oxidation system at increasing doses of H2O2. Figure 4B shows the result of a study representative of 4 different experiments using 2 independent preparations of each protein. As previously observed, the ABCA1-dependent cholesterol acceptor activity of wild-type apoAI was inhibited by increasing MPO induced oxidation; however, the 4WF variant maintained this activity even as an H2O2:apoAI mole ratio of 15 (Figure 4B). The cell-free lipid-binding activity of rh-apoAI 4WF was also resistant to MPO mediated inhibition, compared to rh-apoAI (Figure 4C).
MPO modification of apoAI leads to extensive cross linking resulting in dimers, multimers, and presumably intramolecular cross links as well, and we previously have shown that the MPO-mediated apoAI cross linking pattern was not altered in the variant with all 7 tyrosine residues converted to phenylalanine.9 On subjecting rh-apoAI and the 4WF variant to MPO/Cl–/H2O2 oxidation at increasing doses of H2O2 (using the identical protein products that were used for efflux in Figure 4B), we observed altered migration of these proteins in denaturing gels consistent with intermolecular cross linking (Figure 5). However, the migration patterns were different, with the 4WF variant giving a sharp predominant band at 70 kDa, whereas the wild-type protein yielded a less distinct predominant zone between 55 and 65 kDa (Figure 5). The migration of the monomer was altered for both proteins, which could be indicative of intramolecular cross links or other amino acid modifications. Although the 4WF variant is resistant to MPO-mediated loss of cholesterol acceptor activity, this variant was more susceptible to MPO-induced cross linking, particularly at low doses of H2O2 (Figure 5). We also subjected these MPO-modified proteins to structural analysis by CD (supplemental Table II), and found that both were susceptible to loss of alpha helical content, although the 4WF variant started with a higher value.
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The MPO/Cl–/H2O2 oxidation system generates HOCl,20 the active reagent of bleach, and we and others have previously demonstrated that HOCl treatment of apoAI results in loss of cholesterol acceptor and lipid-binding activity.5,6 Thus, we subjected wild-type apoAI and the 4WF variant to increasing doses of HOCl. Similar to the findings with the MPO modification system, the cholesterol acceptor activity of the 4WF variant was resistant to this treatment, whereas the efflux activity of wild-type rh-apoAI was impaired by increasing doses of HOCl (Figure 6).
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We used a quantitative mass spectrometry method, with heavy isotope internal standards, to detect total chlorotyrosine from MPO-modified rh-apoAI 4WF (H2O2:apoAI=15:1), and we detected 1.2 mole% conversion of tyrosine into chlorotyrosine, comparable to the highest levels of chlorotyrosine detected in apoAI recovered from human atheroma.4 Thus, the apoAI 4WF variant had fully functional efflux capacity (see Figure 4B) at physiological levels of tyrosine chlorination found within the highly oxidative environment of human atheroma tissues.
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Discussion |
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ApoAI is a selective target for MPO-mediated modification as demonstrated by its high chlorotyrosine content in human plasma and atheroma.4,5 Moreover, it is clear that in vitro modification of apoAI by MPO leads to reduced activity of apoAI as both a lipid-binding protein and an acceptor of cellular lipids through the ABCA1 pathway.4,5,7,6 This has led to the concept that apoAI in some subjects may be dysfunctional, and in fact the cholesterol acceptor activity of apoAI varies in different subjects and is correlated with tyrosine chlorination, which is a unique indicator of MPO-mediated modification.4,21 These basic findings have been independently observed by our group and Heinecke’s group. However, the precise mechanism by which MPO modification renders apoAI dysfunctional is controversial. We previously reported that a tyrosine-free apoAI variant was equally susceptible as wild-type apoAI to MPO-mediated loss of apoAI function, and we identified MPO-induced in vitro modifications of lysine and tryptophan residues of unknown physiological and functional consequences.9 In the current work, we identified site-specific in vivo modifications of tryptophan, methionine, and lysine in atheroma-derived apoAI and then used chemical modification and site-directed mutagenesis to determine which of these residues is associated with the MPO-mediated loss of apoAI function. We found that replacing apoAI tryptophan residues with phenylalanine led to formation of a fully functional apoAI variant with marked resistance against oxidative inactivation by pathophysiological exposure levels of MPO-generated oxidants.
Using model peptides, Heinecke and colleagues reported that a lysine downstream of a tyrosine in a YXXK peptide motif can increase tyrosine chlorination by MPO or HOCl.22 Shao et al then used site directed mutagenesis of apoAI lysine 195 to arginine, and found that this led to decreases in susceptibility to MPO or HOCl-mediated tyrosine 192 chlorination.15 They also suggested that altering glutamate 198 to methionine, creating a YXXMXXK motif, inhibited MPO-mediated chlorination of tyrosine 192, consistent with protein bound methionine acting as a scavenger for MPO generated reactive halogenating species.15 This was supported also by substitution of methionine 112 to lysine (going from a MXXY to a KXXY motif), which increased tyrosine 115 chlorination by MPO.15 They also demonstrated that MPO treatment of apoAI led to the modification of its 3 methionine residues to methionine sulfoxide, and that this could be reversed by adding the enzyme methionine sulfoxide reductase.15 However, Shao et al did not directly test whether methionine substitution altered the sensitivity of apoAI to the MPO-mediated loss of function, but they did find that treatment of MPO-modified apoAI with the enzyme methionine sulfide reductase could partially restore the cholesterol acceptor activity of apoAI.15 In our studies, we directly observed that apoAI methionine residues play a protective scavenging role, as we found a markedly increased susceptibility of the methionine substituted 3MV apoAI variant to low doses of H2O2 in the complete MPO chlorination system. Thus, our site-directed substitution and cholesterol efflux data clearly show that neither methionine nor tyrosine serve as the oxidant sensitive residue involved in MPO-dependent apoAI inactivation. We also performed chemical modification of apoAI lysine residues, which failed to alter the sensitivity of apoAI to MPO-mediated loss of function.
We substituted all 4 apoAI tryptophan residues with either leucine or phenylalanine. The apoAI 4WL variant lost its lipid binding and cholesterol-accepting activities, whereas the 4WF variant retained these activities. Because tryptophan and phenylalanine are the most hydrophobic residues on the Wimley and White scale,23 our results imply that highly hydrophobic and bulky residues are required at the tryptophan positions on the nonpolar face of apoAI for its lipid binding and accepting functions. The global replacement of tryptophan with phenylalanine, which is far less susceptible to oxidative modification by MPO, created an apoAI that was clearly resistant to the MPO-mediated loss of function, but still susceptible to other modifications that lead to cross linking. In regard to the MPO-mediated cross linking of apoAI, in the current work we observed an altered cross linking pattern comparing the 4WF variant with wild-type apoAI, whereas in our prior study we did not observe an alteration of the cross linking pattern comparing the tyrosine-free 7YF variant.9 These combined data suggest that: (1) tryptophan residues either contribute directly to the cross links observed in wild-type apoAI, or that tryptophan substitution alters the tertiary structure and this alters the preferred sites of cross linking; and (2) tyrosine residues do not directly participate in the cross links observed in wild-type apoAI.
We conclude that tryptophan oxidation, which we observed in apoAI isolated from human atheroma, is likely to be the causative alteration that results in the production of dysfunctional apoAI. Although we cannot exclude the possibility that the oxidation resistance of the 4WF variant may be attributable to an altered tertiary structure of this variant leading to protection of some other sensitive residue. All of our in vitro MPO treatments were performed with lipid-free apoAI; similar to pre-β particles formed in vivo during lipoprotein remodeling. It is these lipid-free and lipid-poor apoAI particles that are capable to participate in ABCA1-mediated lipid efflux and thus play an important and physiological role in reverse cholesterol transport.24,25,26,27 We speculate that the 4WF apoAI variant would be a better therapeutic reagent, compared to wild-type apoAI or apoAI Milano,28 to promote the regression of plaques, a location where the levels of MPO-generated oxidants as well as modified apoAI are high.
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Acknowledgments |
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Sources of Funding
This work was supported by National Institutes of Health Grants HL66082 (J.D.S.), P50 HL077107 (S.L.H. and J.D.S.), and PO1 HL076491 (S.L.H.). D.-Q.P. was the recipient of an American Heart Association Fellowship Award (0525386B).
Disclosures
Dr. Hazen is named as co-inventor on pending and issued patents filed by the Cleveland Clinic that relate the use of biomarkers in inflammatory and cardiovascular disease. Dr. Hazen reports he is the scientific founder of PrognostiX Inc; has received speaking honoraria from Pfizer, AstraZeneca, Merck, Merck Schering Plough, BioSite, Lilly, Wyeth and Abbott; and has received research grant support from Abbott Diagnostics, Pfizer, Merck, PrognostiX Inc, Hawaii Biotech, ArgiNOx, Sanofi, and Takeda; and has received consulting fees from Abbott Diagnostics, Pfizer, PrognostiX Inc, Wyeth, BioPhysical, and AstraZeneca.
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Footnotes |
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D.-Q.P. and G.B. contributed equally to this study.
Original received March 17, 2008; final version accepted July 29, 2008.
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References |
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