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J Biol Chem, Vol. 274, Issue 42, 29826-29830, October 15, 1999

Human Inhibitor Antibodies Specific for the Factor VIII A2 Domain Disrupt the Interaction between the Subunit and Factor IXa^*

Philip J. Fay^§ and Dorothea Scandella^¶

From the  Departments of Biochemistry and Biophysics and of Medicine, University of Rochester School of Medicine, Rochester, New York 14642 and the ^¶ Holland Laboratory, American Red Cross, Rockville, Maryland 20855

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Factor VIII is an essential plasma protein that, when deficient or defective, results in hemophilia A. The activated form of factor VIII, factor VIIIa, functions as a protein cofactor for the serine protease factor IXa to form an anionic phospholipid-dependent complex referred to as the intrinsic factor Xase. This complex efficiently converts zymogen factor X to the serine protease, factor Xa (for review see Ref. 1). The role of the phospholipid surface is primarily to reduce the K_m for substrate, whereas the cofactor, factor VIIIa, increases the k_cat for this reaction by several orders of magnitude (2).

Factor VIIIa is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits (3, 4). The A1 and A3-C1-C2 subunits are associated in a divalent metal ion-dependent dimer, whereas the A2 subunit is weakly associated with the dimer (K_d ~260 nM) primarily through electrostatic interaction (5, 6). The A2 subunit readily dissociates at physiologic pH and factor VIII concentration, resulting in the loss of factor VIIIa activity (5-7).

Two regions of factor VIII have been identified as interactive sites for factor IXa. A high affinity site (K_d ~14 nM) was localized to the A3 domain in and around residues 1811-1818 (8). A second, lower affinity site (K_d ~300 nM) (9) was localized to the A2 domain and is comprised of residues 558-565 (10). Recently, isolated A2 subunit was shown to stimulate the k_cat for factor IXa-catalyzed conversion of factor X by ~100-fold (9). This property appeared unique to A2 and was not observed for either the A1 or A3-C1-C2 subunits.

A significant fraction (~20%) of individuals with hemophilia A who are treated with either purified plasma-derived or recombinant factor VIII develop an immune response to factor VIII (11). These antibodies typically appear in situations where large deletions or inversions in the factor VIII gene are present (12), and some of them inhibit factor VIII activity. In rare instances individuals with normal factor VIII develop autoantibodies (13). The domain specificity of inhibitor antibodies obtained from hemophiliacs or individuals with autoantibodies identify the A2 and C2 domains as containing reactive sites in nearly 70% of inhibitor plasmas tested (14). Anti-C2 inhibitors act by preventing the interaction of factor VIII with phospholipid surfaces (15, 16). The mechanism for inhibition by anti-A2 inhibitors is poorly understood, but it may relate to affecting the transition state of intrinsic factor Xase (17). A significant fraction of anti-A2 inhibitors characterized to date show a shared epitope with mAb¹ 413, which recognizes a region comprised of residues 484-508 (18).

In this report we describe the effects of anti-A2 antibodies, either monoclonal or those derived from patients with factor VIII inhibitors, on the interaction between A2 subunit and factor IXa. Our results show that those antibodies possessing specificity to the 484-508 epitope inhibit the interaction of A2 with factor IXa, thus abrogating the A2-dependent stimulation of factor IXa activity. These results identify the molecular basis for inhibition of factor VIII activity by this class of inhibitors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Recombinant factor VIII preparations (Kogenate®) were a gift from James Brown (Bayer Corp.). Purified recombinant factor VIII was also a generous gift from Debra Pittman (Genetics Institute). The murine monoclonal antibody R8B12, which reacts with the C-terminal region of the factor VIII A2 domain (19), was prepared as described previously (4). The anti-A2 subunit mAb 413, which reacts within factor VIII residues 484-509 (18), was obtained from Leon Hoyer (American Red Cross). The synthetic peptide representing factor VIII residues 484-509 was prepared as described (15). Peptides corresponding to factor VIII residues 466-477 and 518-527 were prepared by Quality Controlled Biochemicals, Inc. Peptide concentration was determined by quantitative amino acid composition analysis. Phospholipid vesicles containing PSPCPE (20% PS, 40% PC, and 40% PE) were prepared using octyl glucoside as described previously (20). The reagents -thrombin, factor IXa_, factor X, and factor Xa (Enzyme Research Labs), Fl-FFR-factor IXa (Molecular Innovations), hirudin and phospholipids (Sigma), and the chromogenic substrate S-2765 (N--benzyloxycarbonyl-D-arginyl-glycyl-L-arginyl-p-nitroanilide-dihydrochloride, Amersham Pharmacia Biotech) were purchased from the indicated vendors.

Inhibitor IgG-- Inhibitor IgG and Fab fragments were prepared from patient plasma samples as described (21). Inhibitors were titered using the Bethesda assay (22).

Proteins-- The Kogenate^® concentrate was fractionated to separate factor VIII from albumin following S-Sepharose chromatography (23). Factor VIII was converted to factor VIIIa using thrombin as described (4). Purification of the A2 subunit and A1/A3-C1-C2 dimer by Mono S chromatography was as described (24). Dimer preparations were depleted of trace levels of A2 subunit using mAb R8B12 coupled to Affi-Gel (4). In some instances, proteins were concentrated using a MicroCon concentrator (Millipore, 10-kDa cut-off). Protein concentrations were determined by the Coomassie Blue dye binding method of Bradford (25).

Factor Xa Generation Assays-- The rate of conversion of factor X to factor Xa was monitored in a purified system (26). A2 subunit was reacted with factor IXa in 20 mM Hepes, pH 7.2, 100 mM NaCl, 5 mM CaCl₂, and 0.01% Tween (Buffer A) in the presence of 200 µg/ml bovine serum albumin and 10 µM PSPCPE vesicles. For reactions containing anti-A2 antibodies, A2 subunit was incubated with the indicated concentrations of the antibody for approximately 1 h before adding it to the factor Xa generation reaction. Time course reactions were initiated with the addition of factor X (300 nM). Aliquots were removed at appropriate times to assess the initial rates of product formation and were added to tubes containing EDTA (80 mM final concentration) to stop the reaction. The rates of factor Xa generation were determined by adding the chromogenic substrate S-2765 (0.46 mM final concentration). Reactions were read at 405 nm using a V_max microtiter plate reader (Molecular Devices).

Fluorescence Anisotropy-- Fluorescence anisotropy measurements were made using a SPEX Fluorolog 212 spectrometer operated in the L format. The excitation wavelength was 495 nm (5 nm band pass), and the emission wavelength was 520 nm (14.4 nm band pass). Reactions (0.2 ml) were carried out at room temperature in Buffer A containing 30 nM Fl-FFR-factor IXa, 50 µM PSPCPE vesicles, and the indicated concentrations of factor VIIIa subunits and factor X in a quartz micro cell. A2 subunit was preincubated with antibody as described above. Anisotropy measurements were made by manually rotating the polarizers and monitoring the fluorescence for 5 s at each position. Fluorescence intensity determinations (3-5) were made at each position, and the average value was obtained. Blank readings for the buffer containing phospholipid were subtracted from all determinations. Statistical analysis of the data employed a two-sided t test tested at 95% confidence ( = 0.05).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Differential Inhibition of the A2-dependent Stimulation of Factor IXa by Anti-A2 Monoclonal Antibodies-- Two monoclonal antibodies specific for distinct regions in the A2 subunit were evaluated for their ability to inhibit the A2-dependent stimulation of factor IXa-catalyzed conversion of factor X. mAb R8B12, a high affinity antibody used for immunopurification of factor VIII or A2 subunit (4), recognizes a C-terminal region of A2 contained within residues 563-740 (19). Titration of A2 subunit with this antibody prior to reaction of A2 in the factor Xa generating assay yielded ~20% inhibition of A2-dependent factor X conversion at saturating levels of antibody (Fig. 1). Conversely, mAb 413, another high affinity antibody that recognizes an epitope contained within A2 subunit residues 484-509, yielded complete inhibition of the reaction. IC₅₀ values for either antibody were similar (~40 nM) and were consistent with a high affinity interaction. However, the disparate extents of inhibition suggested that the site recognized by mAb 413 was critical for the interaction of A2 subunit with factor IXa, which results in the enhancement of k_cat.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Inhibition of A2 subunit-dependent factor Xa generation by monoclonal antibodies. A2 subunit (200 nM) was reacted with the indicated concentrations of R8B12 IgG (squares) or 413 IgG (circles) for 1 h at room temperature. The treated A2 was subsequently reacted with factor IXa (5 nM) in the presence of PSPCPE vesicles (10 µM) for 10 min prior to the addition of factor X (300 nM). Rates of factor Xa generation were determined as described under "Materials and Methods." The 100% level represented ~1 nM factor Xa generated/min/nM factor IXa.

Inhibitor Antibodies Block the A2-dependent Stimulation of Factor IXa-- Because the epitope recognized by mAb 413 (residues 484-509) cross-reacts with antibodies obtained from patients possessing factor VIII inhibitors, the above experiment suggested that a possible mechanism for these anti-A2 inhibitors is perturbation of the interaction of the A2 subunit of factor VIIIa with factor IXa. To test this hypothesis, a panel of inhibitor antibodies showing specificity for the A2 domain (based upon inhibitor neutralization data (27)) was employed to assess the effects of these reagents on the A2-dependent enhancement of factor IXa-catalyzed conversion of factor X. The antibodies used possessed inhibitor titers ranging from 4 to 2755 Bethesda units/mg. Titration of A2 with each antibody was performed prior to addition into the factor Xa generation assay. Subsequent rates of factor Xa generation are shown in Fig. 2. All inhibitor antibodies tested resulted in a dose-dependent reduction in rate of substrate conversion. IC₅₀ values correlated inversely with the inhibitor titer (Table I). Two groupings of inhibitor response were observed. Antibodies possessing titers of >1000 Bethesda units/mg yielded IC₅₀ values in the 100 nM range, whereas antibodies possessing titers ranging from ~1 to ~10 Bethesda units/mg showed significantly weaker affinities as indicated by IC₅₀ values ranging from >1 to ~10 µM. These results indicate that anti-A2 subunit specific inhibitors alter the interaction between this factor VIIIa subunit and factor IXa and suggest that inhibitor potency is determined by affinity for the A2 subunit.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Inhibition of A2 subunit-dependent factor Xa generation by inhibitor IgG fractions. A2 subunit was treated with the indicated concentrations of inhibitor IgG and subsequently assayed in a factor Xa generation assay as described in the legend to Fig. 1. Closed circles, CC IgG; open circles, CC Fab; squares, FM IgG; inverted triangles, RJ IgG; triangles, WD IgG; diamonds, EM IgG.

In another experiment we examined the inhibitor activity of one of the more potent inhibitors, CC, with a Fab fragment derived from this IgG. The rationale for this experiment was to gain insights into inhibitor mechanism. Inhibition observed by the IgG may be steric, whereas inhibition observed with the smaller Fab fragment is consistent with blocking of a specific interactive site. The results of this analysis are also shown in Fig. 2 and indicate essentially identical inhibitory activity of the IgG and Fab fragment. This finding suggested that the A2 sequence recognized by the CC antibody may be interactive with factor IXa.

Altered Fluorescence Anisotropy Following Interaction with Inhibitory IgG Molecules-- In an earlier report (9), we showed that A2 subunit resulted in a modest increase in the fluorescence anisotropy of Fl-FFR-factor IXa. Furthermore, the presence of factor X markedly increased the increment for the A2-dependent increase in anisotropy. This parameter was used as an indicator for the effects of anti-A2 antibody on the interaction between A2 subunit and factor IXa. Consistent with our earlier observation, anisotropy increases (r) of 0.013 and 0.036 were determined for the A2-dependent effect in the absence and presence of factor X, respectively (Table II). These values were somewhat lower than those determined previously and likely resulted from the presence of nonsaturating concentrations of A2 subunit. While statistical analysis revealed no significant difference in anisotropy values obtained for factor IX plus or minus A2 in the absence of factor X (p = 0.098), the inclusion of factor X magnified the A2-dependent effect so as to yield significance between values obtained in the absence and presence of A2 subunit (p = 0.002). A2 subunit was reacted with a molar excess of either mAb 413 or human inhibitor CC (IgG and Fab forms) prior to fluorescence analysis. In each situation, the antibody appeared to essentially eliminate the A2-dependent increase in fluorescence anisotropy. The r values, in the absence and presence of factor X, respectively, were 0.005 and 0.007 for mAb 413, 0.003 and 0.014 for CC IgG, and 0.001 and 0.003 for CC Fab. Statistical analysis performed for reactions run in the presence of factor X showed no significant difference between the reaction containing factor IXa alone and reactions containing factor IXa plus A2 subunit run in the presence of mAb 413 (p = 0.34), CC IgG (p = 0.087), and CC Fab (p = 0.71). These results were consistent with each antibody abrogating the interaction of A2 subunit with factor IXa.

mAb 413 was used to further determine the effect of antibody on the contribution made by A2 subunit to Fl-FFR-factor IXa anisotropy in the presence of the A1/A3-C1-C2 dimer. This analysis was restricted to the use of the 413 antibody because the human inhibitors show partial reactivity to factor VIIIa subunits other than A2 subunit (27). The A1/A3-C1-C2 dimer incrementally increases the fluorescence anisotropy of factor IXa in both the absence and presence of factor X ( Refs. 28 and 29 and Table II). Inclusion of the A2 subunit to reconstitute factor VIIIa results in maximal increases in anisotropy, with r values of 0.041 and 0.053 in the absence and presence of factor X, respectively. Similar to the results obtained using the isolated A2 subunit, preincubation of the A2 subunit with mAb 413 prior to its addition to the A1/A3-C1-C2 markedly reduced the A2-dependent effects observed in the absence (r = 0.017; p = 0.041) and presence (r = 0.012; p = 0.0043) of factor X. The anisotropy values obtained approached those determined in the absence of A2 subunit, consistent with the antibody abrogating the contribution of A2 subunit to the factor VIIIa-like effect on the factor IXa active site. Taken together, these results suggest that anti-A2 antibodies that bind to the mAb 413 epitope specifically block the interactions of factor IXa with A2 subunit, either alone or when complexed in factor VIIIa.

Synthetic Peptide 484-509 Blocks the A2-dependent Effect on Factor IXa Catalysis-- To further determine the role for the A2 subunit sequence 484-509 in the interaction with factor IXa, a synthetic peptide to this region was examined for its effect on the stimulation of factor IXa activity observed in the presence of A2 subunit (Fig. 3). In this experiment, the A2-containing reaction mixture was titrated with peptide prior to the addition of factors IXa and X. Subsequent determination of rates of factor Xa generation showed that the peptide inhibited the conversion of substrate in a dose-dependent manner. The extent of inhibition for a given peptide concentration was dependent upon the concentration of A2 subunit present. A comparison of two A2 subunit concentrations (70 and 200 nM) yielded IC₅₀ values of ~90 and ~180 µM, respectively. This result suggested that peptide directly competes with A2 subunit in binding factor IXa. Peptide-dependent inhibition did not result from inhibition of the factor IXa-factor X interaction because similar levels of inhibition were observed independent of factor X concentration (data not shown). Furthermore, two peptides that flank the 484-509 sequence (466-477 and 518-527) failed to significantly inhibit the A2-dependent stimulation of factor IXa activity over the same concentration range. Taken together, these results suggest that the sequence 484-509 contributes to the interaction between A2 subunit and factor IXa.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of synthetic peptides on A2 subunit-dependent factor Xa generation. A2-dependent factor Xa generation was determined for reactions run in the presence of the indicated concentrations of peptides derived from the A2 domain sequences: 466-477 (squares), 484-509 (circles), and 518-527 (triangles). Reactions contained 5 nM factor IXa and either 200 nM (closed symbols) or 70 nM (open symbols) A2 subunit and were preformed as described in the legend to Fig. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The A2 subunit is essential for factor VIIIa activity. Functional assays have indicated that factor VIIIa can be reconstituted from A1/A3-C1-C2 dimer and A2 subunit to yield material of similar specific activity to factor VIIIa obtained following activation of the intact cofactor (4-6). Fluorescence studies have shown that whereas the A1/A3-C1-C2 dimer and A2 subunit individually increase the anisotropy of a fluorophore-labeled factor IXa active site, the synergy of all three subunits is required to yield a factor VIIIa-like effect (28, 29). The recent observation that isolated the A2 subunit directly increases k_cat for factor Xa generation (9) provides a unique functional assay to dissect the mechanisms of A2 domain-specific factor VIII inhibitors in the absence of other factor VIIIa subunits. The results from this study define a primary mechanism for inhibitor antibodies as perturbing/blocking the interaction of A2 subunit with factor IXa. The basis for this observation is (i) complete inhibition of the "cofactor" activity associated with the isolated A2 subunit and (ii) abrogation of A2-dependent increases in fluorescence anisotropy of Fl-FFR-factor IXa with either isolated A2 or the A2 subunit-dependent increment of factor VIIIa.

The association of factor VIIIa and factor IXa is complex and not fully understood. At least two subunits of the factor VIIIa heterotrimer have been implicated as possessing factor IXa interactive sites. The factor VIII light chain-derived A3-C1-C2 subunit likely possesses a high affinity site for factor IXa. The free light chain of factor VIII shows similar affinity for factor IXa (K_d ~14 nM) (30) as is observed for factor VIIIa (K_d ~2-20 nM) (31, 32). This finding suggests that little if any of the binding energy for the interaction is contributed by the factor VIII heavy chain-derived subunits, A1 and A2. This interactive site was localized to the A3 domain following studies using inhibition by a monoclonal antibody with an epitope in residues 1778-1840 (30), and it was further localized to within residues 1811-1818 based upon inhibition studies employing synthetic peptides (8). Recently it was shown that several inhibitors specific for the factor VIII light chain-derived contiguous acidic region-A3-C1 domains compete for factor IXa binding to factor VIII (33), suggesting that these molecules block the high affinity interaction of enzyme with cofactor.

Based upon the identification of an activated protein C cleavage site at Arg-562 in the A2 subunit (19) and the capacity for factor IXa to selectively protect from cleavage at this site (34), a factor IXa interactive site in the A2 subunit was postulated. Synthetic peptides spanning residues 558-565 noncompetitively inhibited factor Xase activity (10). Examination of the hemophilia A data base (35) revealed cross-reactive material-positive (CRM⁺) hemophilia resulting from mis-sense mutations at several factor VIII residues within this region. These mutations appear to alter the interaction of the A2 subunit in factor VIIIa with factor IXa (36). Taken together, these results suggest that the sequence spanning residues 558-565 in the A2 subunit represents a critical site for interaction between cofactor and enzyme.

Inhibitor alloantibodies generated in response to therapeutic infusions of factor VIII and mAb 413 bind an epitope within residues 484-508 of the factor VIII A2 domain (18). Because of this cross-reactivity, mAb 413 has been used to model mechanisms of inhibition by this class of molecules. Based on kinetic data showing that mAb 413 is a noncompetitive inhibitor of factor Xase and on steady-state fluorescence analyses, Lollar et al. (17) concluded that the anti-A2 antibodies inhibit factor VIIIa by blocking conversion of the intrinsic factor Xase/factor X complex to the transition state rather than by blocking formation of the enzyme/substrate complex. Anisotropy studies presented in that report showed little or no change in the factor VIIIa-dependent anisotropy of fluorescein-labeled factor IXa in response to mAb 413. However, a major decrease in anisotropy (r ~0.05) was observed in the presence of factor X when the antibody was bound to factor VIIIa. Similarly, we observed an equivalent decrease in anisotropy for A1/A3-C1-C2 dimer plus A2 subunit in the presence of factor X (r = 0.320) when mAb 413 was bound to A2 (r = 0.279). The latter value is significantly greater than that for factor IXa plus factor X (r = 0.229), indicating a contribution of the A1/A3-C1-C2 dimer to the orientation of active site with substrate that is independent of the A2 subunit. However, in the presence of isolated A2 subunit, there is a large incremental increase in anisotropy in the presence of factor X compared with the absence of substrate (Ref. 9 and this study). Thus, one explanation for the results obtained by Lollar and co-workers (17) is that mAb 413 eliminates the A2-dependent contribution to anisotropy observed in the presence of factor VIIIa.

Results from the present study implicate residues 484-509 as contributing to the interaction of A2 subunit with factor IXa. Complete inhibition observed for both CC IgG and the derived Fab fragment support the importance of this epitope in the intermolecular interaction. Conversely, mAb R8B12, which recognizes a C-terminal epitope, yielded only modest (~20%) inhibition of A2-dependent activity. This loss of activity may reflect partial steric interference in forming the A2 subunit-factor IXa complex. Inhibition studies using the 484-509 peptide yield IC₅₀ values (~100-200 µM) similar to the K_I determined for the 558-565 peptide (~100 µM) (10). Thus, both regions may contribute to an extended factor IXa-interactive surface on the A2 subunit. This notion is supported by the homology model of the factor VIII A domains (37), which shows both of the above sequences to be in close spatial proximity and on the same face of the A2 domain.

In summary, inhibitor antibodies that cross-react with the epitope of mAb 413 appear to inhibit the interactions of A2 subunit with factor IXa, which contributes to the factor VIIIa-dependent k_cat effect. This conclusion is based in part upon a factor Xa generation assay in which the A2 subunit is the sole factor VIIIa component present. Furthermore, use of this assay offers a means for direct identification of functionally active, anti-A2 inhibitor molecules.

	ACKNOWLEDGEMENTS

We thank Debra Pittman of Genetics Institute and James Brown of Bayer Corp. for the gifts of recombinant factor VIII, Tammy Linder for assistance with statistical analysis, and Leon Hoyer of the American Red Cross for the mAb 413.

	FOOTNOTES

^* This work was supported by grants HL 30616 and HL 38199 (to P. J. F.) and HL 55273 (to D. S.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Vascular Medicine Unit, University of Rochester Medical Center, 601 Elmwood Ave., P. O. Box 610, Rochester, NY 14642. Tel.: 716-275-6576; Fax: 716-473-4314; E-mail: Philip_Fay@urmc.rochester.edu.

	ABBREVIATIONS

The abbreviations used are: Fl-FFR-factor IXa, factor IXa modified in its active site with fluorescein-Phe-Phe-Arg chloromethyl ketone; mAb, monoclonal antibody; PSPCPE, phosphotidylserine-phosphotidylcholine-phosphotidylethanolamine; S-2765, N--benzoyloxycarbonyl-D-arginyl-glycyl-L-arginyl-p-nitroanilide dichloride.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fay, P. J. Articles by Scandella, D. Search for Related Content PubMed PubMed Citation Articles by Fay, P. J. Articles by Scandella, D. Social Bookmarking                      What's this?