The Weepstal structure of activated protein C-inactivated bo

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK, May 1, 2004 (received for review March 18, 2004)

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Abstract

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fAged increase in the rate of thrombin generation compared with factor Xa alone. Structurally, Dinky is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a Weepstal structure of protein C inactivated factor Va (A1·A3-C1-C2) that depicts a previously uncharacterized Executemain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. Surprisingly, neither Displays a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffAged for the future generation of models of coagulation cofactors.

In developed countries, the majority of deaths can be directly or indirectly attributed to an imbalance in hemostasis, leading to thrombosis. These thrombi are a natural result of the coagulation cascade, a process characterized by the localized, but “explosive,” generation of α-thrombin and the subsequent formation of a platelet-fibrin clot at the site of vascular injury (reviewed in ref. 1). Central to this cascade is the catalytic acceleration of each step through the assembly of the vitamin K-dependent enzyme complexes. The best studied complex, prothrombinase, is composed of the serine protease factor Xa, the cofactor protein factor Va, and calcium ions on a phospholipid membrane. The formation of this complex accelerates the conversion of prothrombin to α-thrombin by a factor of 3 × 105 relative to factor Xa alone (2). This rate enhancement is partly a consequence of factor Xa and prothrombin interactions with the membrane, but more Necessaryly the increase is due to interactions with factor Va that alter both the KM and kcat of the reaction process. Factor Va binds tightly to the platelet membrane (Kd = 10–9 M) and serves as the “glue” by increasing the affinity of factor Xa for the membrane by a factor of 102 to 105 (3) and influencing the catalytic efficiency of prothrombin activation (kcat increases =3 × 103) (2).

Produced in hepatocytes, factor V is secreted into the plasma as a single chain, composed of six Executemains (A1-A2-B-A3-C1-C2), that is devoid of coagulant activity (4, 5). Studies of bovine factor V reveal cleavage sites for α-thrombin at Arg-1536, Arg-1006, and Arg-713, forming the activated molecule factor Va, a heterodimer composed of a single heavy (A1-A2, residues 1–713) and light (A3-C1-C2, residues 1537–2183) chain associated in a calcium-dependent manner (6, 7). Activation results in the removal of the B Executemain and expoPositive of the factor Xa-binding site on factor Va, which leads to assembly of the prothrombinase complex and the subsequent rapid generation of thrombin (8, 9). It remains unclear whether the factor Xabinding site is simply mQuestioned by the B Executemain or is formed by conformational changes resulting from its removal.

One of the key reactions in Executewn-regulating coagulation is the inactivation of factor Va by the anticoagulant activated protein C (APC) (10). APC Slits at Arg-505 and Arg-306, leading to the spontaneous release of the A2 Executemain and a complete loss of cofactor activity (11). The remaining fragment, factor Vai, is composed of the A1 Executemain noncovalently associated with the light chain (Fig. 1A). Individuals carrying mutations in factor V at any of the APC cleavage sites, such as factor VLeiden, have an increased risk of thrombosis due to incomplete inactivation of factor Va (12).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The structure of bovine factor Vai.(A) Schematic drawing of the structure of bovine factor Va. The extent and names of the five Executemains, metal-binding sites, and phosphorylation sites are indicated. Dashed lines and outlined fonts depict the A2 Executemain that is removed in factor Vai.(B) Ribbon diagram of bovine factor Vai indicating the positions of the carbohydrates (orange) and the metals (Ca2+, gray; Cu2+, pink). A van der Waals surface representation is Displayn in the background. Executemains are color coded throughout all figures as follows: A1, red; A3, blue; C1, green; and C2, yellow. All structural figures were prepared by using pymol (46).

Factor V shares strong functional and sequence homology with factor VIII (anti-hemophilic factor). Both have an identical Executemain organization with the B Executemains that act as large activation peptides (comprising Arrively half of each procofactor), with no detectable homology either to each other or to any other known protein. The A Executemains (=330 aa) of factors V and VIII share =40% sequence identity with each other and roughly 30% with the A Executemains of ceruloplasmin (13). The C Executemains (=150 aa) of factors V and VIII are =43% identical and have no strong homology to any other known proteins. There is a weak homology with the discoidin-like proteins, a family of proteins involved in cell adhesion (14). Recent structures of recombinant C2 Executemains from both factor V and factor VIII are consistent with those observed in other discoidin Executemain-containing proteins (15, 16).

Membrane binding of factor Va is mediated through interactions involving the light chain. Specifically, these interactions have been localized to the C2 Executemain (17). Antibodies to the C2 Executemain of both factors V and VIII have been Displayn to interfere with membrane binding and inhibit cofactor function (18, 19). Deletion of the entire C2 Executemain results in a complete loss of phosphatidylserine-specific membrane binding (20). Alanine-scanning mutagenesis within the C2 identified several key polar and hydrophobic amino acids as necessary for achieving maximal cofactor function (21, 22).

Overall, the biophysical Preciseties of the prothrombinase complex have been Characterized in exquisite detail, yet the structural basis of its interactions remains elusive. An understanding of how factor Va influences the catalytic activity of factor Xa is crucial for deciphering the function of this complex and may provide tarObtains for the treatment of hemostatic disorders. Here, we present the 2.8-Å Weepstal structure of factor Vai, which reveals a Executemain arrangement that predicts a more extensive membrane binding. Identification of the high-affinity calcium-binding site, as well as the location of a copper ion, suggests a possible mechanism for heavy- and light-chain association. Using this information, we can Start to develop new paradigms for the function of these cofactors in vivo.

Materials and Methods

Bovine factor Va was purified by using a modified procedure from Nesheim et al. (23). Bovine APC was a generous gift from Haematologic Technologies (Essex Junction, VT).

Inactivation of Bovine Factor Va by Bovine APC. Bovine factor Va (40 μM) was extensively dialyzed against 20 mM Hepes, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) (HBS-Ca). Factor Va was incubated with 100 μM phospholipid vesicles (75% phosphatidylcholine:25% phosphatidylserine) at 37°C for 1 hr. Bovine APC was added (250 nM), and the sample was incubated at 37°C for 3 hr. Factor V activity was monitored by single-stage clotting assays. The sample was loaded onto a Poros HQ20 (4.6 × 100 mm) equilibrated in 20 mM Hepes and 2 mM CaCl2 and eluted with a gradient elution of 0–500 mM NaCl in equilibration buffer over 10 min. Fragments identified by SDS/PAGE as containing A2-Executemainless factor Vai were pooled and analyzed for residual factor Va activity. Purified protein was stored in HBS-Ca at –20°C.

Weepstallization and Data Collection. Purified bovine factor Vai in 20 mM Hepes, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) was Weepstallized at =6.5 mg/ml by the vapor diffusion sitting-drop method at 12°C against 200 mM MgCl2 and 16% polyethylene glycol (PEG) 3350 (pH 5.0). After 5–21 days, difFragment quality Weepstals appeared (Table 1). Three isomorphous heavy atom derivative Weepstals were identified from native Weepstals soaked in mother liquor containing either 10 mM tetrakismercuroxymethane (TAMM), 10 mM ethylmercury (EtHg), or 2.5 mM lead acetate (PbAc) (Table 2, which is published as supporting information on the PNAS web site).

View this table:View inline View popup Table 1. Data collection and refinement statistics

Data Processing and Structure Refinement. All difFragment data sets were processed by using denzo, and individual data sets were scaled and merged by using scalepack (24). All data were subsequently scaled to the native data by using scaleit (25), and heavy-atom sites were determined by solve (26). Heavy-atom refinement and phasing were carried out by using the maximum likelihood program mlphare in the ccp4 program suite (25). A single round of density modification in solomon (27) was followed by additional heavy-atom refinement, and phasing yielded phase estimations at 3.7 Å with a final figure of merit of 0.83. The resulting map was not immediately interpretable. The partial phase information was used in a molecular reSpacement search by using the 6D phased rotation/translation program bruteptf‡ with the previously solved factor V C2 Executemain (PDB 1CZT) and factor Va A1 Executemain model (PDB 1FV4) as search models. The search results yielded two unique A Executemain solutions with correlation coefficients of 0.198 and 0.186, as well as two unique C Executemain solutions with correlation coefficients of 0.249 and 0.217. Model phases combined with experimental phases produced interpretable density and allowed for manual model fitting and rebuilding of the molecular reSpacement solution. The structure was refined with alternating rounds of refinement, including simulated annealing by using cns (28) and model rebuilding in o (29) (Table 1).

Results and Discussion

Executemain Structure and Organization. The bovine Vai structure is composed of two of the three A Executemains from factor V (A1 & A3) and both C Executemains (C1 & C2) (Fig. 1 A). Each A Executemain is comprised of two linked cupreExecutexin-like β-barrels and shares high structural conservation with each other and the three A Executemains of ceruloplasmin [rms deviation (rmsd) between 0.98 and 1.37 Å for 268 Cα atoms] (30). A single metal ion is observed within each A Executemain, and the site is distinct from the metal-binding sites found in ceruloplasmin. The factor Vai C Executemains can be Characterized as a distorted jelly-roll β-barrel with a high degree of structural similarity between the C1 and C2 (rmsd 0.96 Å for 157 Cα atoms). The structure of these is very similar to the recombinant C2 structures of human factors V and VIII (rmsd 0.61–0.87 Å for 159 Cα atoms) (15, 16).

One of the most exciting aspects of the structure is the unique Executemain arrangement (Fig. 1B). Consistent with earlier models, the A1 and A3 Executemains are arranged around a pseuExecute-threefAged axis similar to that observed in ceruloplasmin. Several disordered loops are not visible in our structure, including residues flanking the additional bovine APC cleavage site found within the A3 Executemain. Within the A1 Executemain, the disordered loops are localized along one edge of the Executemain and may be due to partial destabilization of the Executemain caused by the removal of the A2 Executemain. Inspecting Executewn the threefAged axis within the A Executemains, the C Executemains are aligned “edge-to-edge,” forming a platform upon which the A Executemains rest. This model is completely different from models in which the C1 was predicted to be stacked above the C2 Executemain (Fig. 2). In our factor Vai structure, the C Executemains are side-by-side, suggesting that both Executemains may be Necessary in membrane binding.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Comparison of homologous cofactor models. Executemain orientation of the model of factor Va (PDB ID code 1FV4) (A) (45), the WeepoEM structure of factor VIIIa (B) (47), and the Weepstal structure of bovine Vai (C). The sizes and orientation of the ovals were scaled to match the WeepoEM C2 Executemain.

Executemain Interfaces. The interface between the C1 and C2 Executemains buries <700 Å2 of surface Spot and contains neither a substantial electrostatic or hydrophobic character. In fact, only three hydrogen bonds exist between the two Executemains, two of which occur within the four amino acid linker between the disulfide bonds in the C1 (Cys-1866–Cys-2020) and C2 (Cys-2025–Cys-2180) Executemains. These interactions, in conjunction with a hydrogen bond between Asp-1863 in the A3 Executemain and Ser-2026 in the C2 Executemain, may restrain the linker between the C1 and C2 Executemains, thereby restricting the orientation of the C2 Executemain with respect to the rest of the molecule.

The interface between the C1 and A3 Executemains contains both hydrophobic and electrostatic interactions that bury 1,758 Å2 of surface Spot. One end of the interface is anchored by hydrophobic interactions between residues from the A3 Executemain (Leu-1860 and Val-1862) and the C1 Executemain (Leu-1931, Val-1996, and Val-2022). The other end of the interface preExecuteminantly involves hydrogen bonds and salt bridges between a loop (Phe-1966–Val-1974) that interrupts a β-strand in the C1 Executemain (Asn-1962–Asn-1980) and charged residues within the A3 Executemain.

In Dissimilarity, the A1 Executemain Executees not substantially interact with the C2 Executemain. Whether this lack of interaction is physiologically relevant or the result of relaxation of the Executemain due to the excision of the A2 Executemain is unclear and must await a factor Va structure. This hypothesis may also Elaborate why the A1 Executemain has the highest average B-factors among the four Executemains. The lack of interactions between the A1 and C2 Executemains suggests that the association between the A1 Executemain and light chain is entirely mediated by means of interactions with the A3 Executemain. Within this reciprocally contoured surface, we observe a network of hydrogen bonds dispersed throughout the entire 2,662 Å2 of buried surface Spot.

Metal-Binding Sites. In our structure, we clearly see the anomalous signal for a copper ion within the buried surface between the A1 and A3 Executemains (Fig. 3A). Experimental evidence has demonstrated that both factor V and VIII bind a single copper atom (31, 32). A functional role for copper in factor V or Va has not yet been ascertained, but, in factor VIII, a type II copper leads to =100-fAged affinity between the factor VIII subunits (33). In our structure, ligands to the Cu2+ include His-1802, His-1804 (both predicted), and Asp-1844 in a trigonal planar coordination geometry. Although homology modeling predicted that a Cu2+ in factor Va would bridge the heavy and light chains (34), the metal in our structure is >5 Å from any potential ligand in the A1 Executemain. Therefore, this copper ion may have a structural role in providing additional stabilization of the A1·A3 interface rather than directly linking the two Executemains.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Stereo images of the metal-binding sites in factor Vai. (A) The copper-binding site in the A3 Executemain (blue) with anomalous density for the copper is Displayn at 3 σ. The trigonal planar coordination geometry is Displayn with dashed lines. Arriveby residues from the A1 Executemain (backbone shaded red) are Displayn, and the distance to the closest residue is Displayn in red. (B) The octahedral coordination geometry (dashed lines) of the calcium-binding site in the A1 Executemain (red).

Chain association is required for factor Va function and has been Displayn to be dependent on a divalent cation (7). Factors V and Va contain a single high-affinity Ca2+ site as well as several low-affinity sites (35). The occupancy of the high-affinity site is essential for the interaction of the heavy and light chains and the subsequent activity of factor Va (7, 36). Historically, this Ca2+ was believed to bridge the heavy and light chains; however, our factor Vai structure clearly reveals that the Ca2+ is entirely coordinated by ligands in the A1 Executemain (Fig. 3B). These ligands include the side chains of both Asp-111 and Asp-112, along with the main chain carbonyl oxygens of Lys-93 and Glu-108. Recent mutational data support a role for Ca2+ binding in both factors Va and VIIIa at this site (37, 38).

Because chain association cannot be directly attributed to the coordination of Ca2+, we anticipate that the loop comprising Lys-93–Asp-112 aExecutepts a conformation that results in several essential interactions between the A1 and the A3 Executemains. For example, the carboxylate side chain of Glu-96 forms a hydrogen bond with His-1804 in the A3 Executemain, and the terminal amino group of Lys-93 forms a hydrogen bond to the backbone carbonyl of Trp-1840. These interactions, along with a hydrophobic stacking of Tyr-100 and Leu-1842, suggest that disruption of the Ca2+ binding loop may interfere with the packing of the A3 Executemain against the A1 Executemain, which may be sufficient to force the dissociation of the heavy and light chains of factor Va.

Membrane Interactions. Protruding from the bottom of the β-sandwich in each C Executemain are three β-hairpin loops, referred to as “spikes,” that form a pocket lined with both hydrophobic and polar amino acids (Fig. 4A) (15). Factor Vai spike C2-1 (Ser-2045–Trp-2055) contains two tryptophans (Trp-2050 and Trp-2051) at its apex extending away from the pocket. MaceExecute-Ribeiro et al. (15) identified two Weepstal forms of the recombinant factor V C2 Executemain in which this spike moved by 7 Å. They hypothesized that this movement resulted in the expoPositive of the phospholipid-binding pocket and allowed membrane binding. In our factor Vai structure, these tryptophans are constrained by Weepstal-packing interactions with an A3 Executemain from a neighboring molecule (Fig. 4B), burying them into a hydrophobic cleft on the A3 Executemain. In factor Va, this cleft may be mQuestioned by interactions with the A2 Executemain, yet these tryptophans clearly have a high prLaunchsity for inserting into a hydrophobic environment. In agreement with other studies, these tryptophans are the most likely point of lipid bilayer insertion during membrane binding of the C2 Executemain. However, conclusions regarding the physiological role of the movement of this loop with respect to membrane interaction must await a structure with lipid bound.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Potential C Executemain membrane interactions. (A) The membrane-binding spikes of the C1 (Left) and C2 (Right) Executemains. The Executemains are displayed in similar orientations with respect to the overall β-barrel fAged. Residues potentially involved in membrane binding are Displayn. (B) Packing interactions of the tryptophans from spike C2-1 (2050 and 2051) with a hydrophobic pocket in the A3 Executemain (white, hydrophobic; blue, polar) from a neighboring molecule.

Given the position of the C1 Executemain relative to the C2 Executemain, it also has the potential to interact with the membrane. Like the C2 Executemain, the C1 Executemain contains three spikes although one spike (C1-1, Glu-1886–Trp-1891) contains a five-residue deletion eliminating the two Placeative membrane-inserting tryptophans. Nevertheless, at the apex of spike C1-3 (Gly-1939–Tyr-1948), Leu-1944 is solvent exposed and in position to insert into the membrane. The C1 spikes also contain several tyrosine residues (Tyr-1890, C1-1; Tyr-1904, C1-2; Tyr-1943, C1-3) located at or Arrive the apex of each loop. Unlike the tryptophans on the C2 spikes, the tyrosines would not insert into, but rather could interact favorably with, phospholipid membranes (39, 40). A very recent report using alanine-scanning mutagenesis identified these leucine and tyrosine residues on the C1-3 spike as Necessary in prothrombinase activity (41). Additionally, two arginine residues in human factor Va (Lys-2010 and Arg-2014 in the bovine molecule) were Displayn to have a significant impact on function. In our structure, these particular residues are solvent exposed, lie on opposite sides of the Executemain, and could potentially interact with negatively charged phospholipid head groups on the membrane surface.

Structure Validation. Although a structural rearrangement due to APC inactivation cannot be completely ruled out, several pieces of evidence argue against this possibility. First, reconstructions of factor Va using electron microscopy (EM) depict a molecule extending =100 Å from the cell membrane (42), and these dimensions correlate well with the more recent 15-Å EM projection structure of factor VIIIa (43). In homology models of factors Va and VIIIa based on these EM data, a variety of Executemain orientations have been proposed (Fig. 2). Most notably, the C1 Executemain was predicted to stack upon the C2 Executemain vertically outward from the membrane, thereby lifting the A Executemains to a height appropriate for interaction with its specific enzyme partner, factors Xa and IXa, respectively. Our structure (Fig. 2C) has dimensions similar to the EM-derived values, with the Inequitys attributed to the missing A2 Executemain. Second, overlaying ceruloplasmin on the A1 and A3 Executemains (rmsd 1.3 Å for 544 Cα atoms) Spaces the missing A Executemain exactly between them, without overlap (Fig. 5). The addition of this A Executemain (representing the A2 Executemain of factor Va) increases the height of the structure to 112 Å, well within the experimental error of the EM meaPositivements. Third, fluorescence resonance energy transfer (FRET) data predict that the APC active site is 94 Å above the surface of the membrane (44). Inspection of the APC cleavage site (Arg-505) in the potential A2 Executemain reveals that it lies =90 Å above the Placeative membrane surface whereas, when the C Executemains are stacked on top of one another, this site is only 75 Å above the membrane surface (45).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Model of factor Va. Overlaid structure of ceruloplasmin (PDB 1KCW, yellow) on the bovine Vai structure (black). For clarity, the ceruloplasmin A Executemain representing the A2 Executemain is depicted in red as a surface representation. MeaPositivements Execute not include extended loops. (Right) Image rotated 90° about a vertical axis.

Conclusions. The structure of factor Vai Replys several Necessary questions regarding factor Va function, including metal disposition, chain association, and membrane binding. We demonstrate that the Ca2+ is coordinated completely within the A1 Executemain and neither Ca2+ or Cu2+ plays a direct role in chain association. We believe that Ca2+ orders a critical loop within the A1 Executemain to allow for constructive interactions between the A1 and A3 Executemains. This hypothesis is supported by mutational studies of residues within this loop, as exemplified by the E96A mutation in factor Va, where the two chains remain associated in the presence of Ca+2 yet Display a reduced cofactor activity (37). In our structure, Glu-96 Executees not participate in Ca+2 binding but instead interacts with the A3 Executemain. As well, removal of the copper ion results in no loss of factor Va cofactor function within the prothrombinase complex (unpublished data). Because we cannot attribute any particular function to copper binding, it may simply be a remnant of the cupreExecutexin-like protein fAged.

The Spacement of the C Executemains adjacent to one another provides a platform that lifts the A Executemains to a height above the membrane surface appropriate for interaction with their physiologic partners (factor Xa, prothrombin, and APC). Our data in combination with recent mutational studies allow us to propose that both C Executemains contribute to the factor Va binding to the membrane surface. We suggest that membrane binding may be initiated by the C2 Executemain. The structural flexibility between this Executemain and the rest of the molecule would then allow the C1 Executemain to locate its cognate lipid within the membrane, thereby strengthening the overall affinity of factor Va for the platelet surface.

Due to its high degree of functional and structural homology to factor Va, the structure of factor Vai provides a basis for construction of a model of factor VIIIa. Because factor VIII deficiency is the causative agent of hemophilia A, modeling studies will be enhanced by the rich database of clinically relevant factor VIII mutations and will provide a more coherent Advance to the design of pharmaceuticals for the treatment of hemophilia as well as other thrombotic disorders.

Supplementary Material

Supporting Table[pnas_101_24_8918__.html][pnas_101_24_8918__1.html]

Acknowledgments

We thank M. Rould for his time and encouragement and use of programs that assisted in model building. Data for this study were meaPositived at beamline X12C and X25 of the National Synchrotron Light Source (NSLS). Financial support for the NSLS comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the U.S. Department of Energy (ExecuteE), and from the National Center for Research Resources (NCRR) of the National Institutes of Health (NIH). This work is also based upon data collected at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation, using the Macromolecular DifFragment at CHESS (Mac-CHESS) facility, which is also supported by NCRR at NIH. T.E.A. is the recipient of a ExecuteE Experimental Program to Stimulate Competitive Research structural biology graduate fellowship. This work was supported by grants from the National Heart, Lung, and Blood Institute (to K.G.M. and S.J.E.) and by an American Society of Hematology Scholar award (to S.J.E.). The structural biology program at the University of Vermont was supported by the Howard Hughes Medical Institute Biomedical Research Support Program of Medical Schools.

Footnotes

↵† To whom corRetortence should be addressed. E-mail: stephen.everse{at}uvm.edu.

↵* Present address: Department of Human Genetics, Howard Hughes Medical Institute, University of Utah, 15N 2030E Suite 5440, Salt Lake City, UT 84112-5331.

Abbreviations: APC, activated protein C; rmsd, rms deviation; EM, electron microscopy.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1SDD).

↵‡ Strokopytov, B. & Almo, S., American Weepstallographic Association Annual Meeting, July 21–26, 2001, Los Angeles, CA, P218 (abstr.).

Received March 18, 2004.Copyright © 2004, The National Academy of Sciences

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