Low Molecular Weight Heparins: Structural Differentiation by Bidimensional Nuclear Magnetic Resonance Spectroscopy

Low Molecular Weight Heparins: Structural Differentiation by Bidimensional Nuclear Magnetic Resonance Spectroscopy Q1 Marco Guerrini,1 Sara Guglieri,1 Annamaria Naggi, Ph.D.,1 Ram Sasisekharan,2 and Giangiacomo Torri, Ph.D.Q11 ABSTRACT Individual low molecular weight heparins (LMWHs) exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy permits identification and quantification of structural peculiarities of LMWH preparations. In this article, heteronuclear single quantum coherence spectroscopy, previously used to determine variously substituted monosaccharide components of heparan sulfate (HS) and HS-like glycosaminoglycan mimics, has been applied to the structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin). Relevant residues belonging to the parent heparin, as well as minor residues generated by each depolymerization procedure, have been characterized and quantified. The use of a highsensitivity NMR spectrometer (600 MHz equipped with TCIQ2 cryoprobe) allowed the accurate quantification of residues with sensitivity better than 1 to 2%. Q2 KEYWORDS: Low molecular weight heparins (LMWHs), nuclear magnetic resonance (NMR), heteronuclear single quantum coherence (HSQC), quantitative analysis, sulfation pattern L Q3 ow molecular weight heparins (LMWHs), introduced as antithrombotic agents some 20 years ago, are now replacing heparin in the treatment of most venous thromboembolic disorders. The main advantages of LMWHs over heparin are improved bioavailability and higher anti-factor (F) Xa/anti-FIIaQ3 activity ratios, with decreased hemorrhagic risk during prolonged treatments.1 Interest in the development of generic versions of proprietary LMWHs and the recent interest in the non-anticoagulant properties of heparin and LMWHs, such as anti-inflammatory activity and inhibition of tumor cell metastasis, require a better understanding of structural requirements for these activities in both heparin and LMWHs.2 The internal structure of LMWHs ideally should match that of the parent heparin in terms of monosaccharide composition, substitution pattern, and oligosaccharide sequence. However, the depolymerization processes involved in the manufacturing of LMWHs usually involves some structural modification. Such differences are due mostly to modifications of the monosaccharidic units at the site of cleavage and are 1 guerrini@ronzoni.it. New Anticoagulants; Guest Editor, Job Harenberg, M.D. Semin Thromb Hemost 2007;33:478–487. Copyright # 2007 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584–4662. DOI 10.1055/s-2007-982078. ISSN 0094-6176. Institute for Chemical and Biochemical Research ‘‘G. Ronzoni,’’ Milan, Italy; 2Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts. Address for correspondence and reprint requests: Dr. Marco Guerrini, Institute for Chemical and Biochemical Research ‘‘G. Ronzoni,’’ G. Colombo 81, 20133 Milan, Italy. E-mail: 478 STRUCTURAL DIFFERENTIATION OF LMWHs/GUERRINI ET AL characteristic for each depolymerization procedure. The monosaccharidic sequence of LMWHs can also be different, according to the cleavage point along the heparin polysaccharide chain. For instance, the cleavage of heparin chains at the more heavily sulfated sites, rather than undersulfated sites,3 influences preservation and position of the specific pentasaccharide motif of the active site for antithrombin III (AT) along the oligosaccharidic chains. The high affinity binding of this pentasaccharide to AT activates this cofactor, with efficient inhibition of different proteases of the coagulation cascade, such as FXa and FIIa. The sequences containing the pentasaccharide, as well as the position in which it is located, may influence the biological properties of the LMWH chain. A tetradecasaccharide with the pentasaccharide sequence positioned at the nonreducing end was defined as optimal for bridging the AT/FII (thrombin) complex responsible for the AT-mediated inhibition of FIIa.4 Structural modifications at the level of pentasaccharide may reduce or cancel the affinity to AT and the corresponding antiFXa (aFXa) activity.5 Moreover, both the reducing and nonreducing extension of the pentasaccharide affects the binding properties of the oligosaccharide to AT, and consequently, its activity.6,7 Distinct pharmacological and biochemical profiles of LMWHs are claimed, resulting from differences in manufacture, and can be associated with structural modifications. For this reason it is increasingly impor- tant to gain a better understanding of their structure– activity relationship.1,8 Many efforts have been dedicated to developing analytical tools for a detailed structural characterization of preparations of LMWHs.9 Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the most important techniques for the structural analysis of glycosaminoglycans (GAGs). Monodimensional NMR spectroscopy permits structural characterization directly on unmodified GAGs, providing information on their monosaccharide composition, sulfation pattern, positional linkages, and relative amounts of iduronic acid (I) and glucuronic acid (G) residues.10,11 In these applications, the area of signals in the monodimensional proton and carbon spectra, unaffected by signal overlap, are used directly for quantification. However, the structural complexity of heparin species obtained by chemical or enzymatic treatments, including LMWHs, causes strong signal overlap in the monodimensional NMR spectra, in many cases preventing their use for quantitative analyses. The possibility of extending NMR quantitative compositional analysis methods to two-dimensional (2D) 1H–13C correlation experiments (heteronuclear single quantum coherence [HSQC]) was demonstrated recently.12 The increase in resolution gained by the 2D technique allows the quantitative evaluation of signals that overlap in the corresponding monodimensional spectrum. The bidimensional method was applied Figure 1 Scheme of depolymerization used to prepare low molecular weight heparins. Major reducing and nonreducing residues obtained are shown. 479 480 Q4 Q5 Q6 Q7 Q8 Q9 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 successfully to the determination of the monosaccharide composition of heparin, variously sulfated heparins, and chemically modified K5Q4 polysaccharide derivatives.12 In this study, the approach has been extended to the most common commercially available LMWHs, focusing on quantification of the reducing and nonreducing residues generated by each depolymerization method. Two LMWHs (enoxaparin and dalteparin) obtained by chemical methods and one LMWH obtained by an enzymatic method (tinzaparin) were studied (Fig. 1). STRUCTURAL FEATURES OF LMWHS The 13C and 1H spectra of each LMWHs are shown in Figs. 2 and 3, respectively. Major signals correspond to the prevalent trisulfated disaccharide repeating unit: -4)Q5-a-LQ6-IdoA2SO3-a-(1!4)-D-GlcNSO3,6SO3 (I2S-ANS,6S)Q7, typical of the parent heparin. Minor 2007 signals associated with undersulfated and oversulfated heparin sequences, as well as relevant signals associated with prevalent structural features of heparin, are shown in the spectra. Most of these additional signals are due to the presence of residues generated by the depolymerization process. The carbon spectra of dalteparin contains signals due to the presence of 2,5anhydromannitol residue (AM.ol) at the reducing end (Fig. 2). In fact, dalteparin is produced by a deamination process, in which heparin is nitrosylated at the amino group of its N-sulfoglucosamine residues. The unstable N-nitrosulfonamide residues rearrange to generate a carbocation at C2. Subsequent ring contraction and hydrolysis of the adjacent glycosidic bond generates an anhydromannose residue that is stabilized by reduction with sodium borohydride to form terminal AM.ol residues.13 Characteristic signals corresponding to C4, C2, and C5 of AM.ol are clearly observable in the dalteparin 13C spectrum at 87.9, 85.9, and 82.3 ppm, respectively14 (Fig. 2). Figure 2Q8 1H–nuclear magnetic resonance spectra of (A) enoxaparin, (B) dalteparin, and (C) tinzaparin. Spectra were recorded at 358C on a Bruker Avance 600 spectrometerQ9 equipped with TCIQ10 5-mm cryoprobe. Water signal was presaturated during relaxation delay. Recycle delay, 10 seconds; number of scans, 164. Samples were prepared by dissolution of 12 mg of heparin in 0.6 mL of deuterium oxide. Gal, galacturonic acid; G, glucuronic acid; l.r., linkage region; ANS, 2-deoxy-2-sulfoamino-D-glucose; ANS,3S, 2-deoxy-3-O-sulfo-2-sulfoamino-D-glucose; ANAc, 2-deoxy-2-acetylamino-D-glucose; ared, ???Q11; AM.ol, 2,5-anhydromannitol; I2S, 2-O-sulfo-iduronic acid; MNS, 2-deoxy-2-sulfamino-D-mannose; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose; 1,6-an.M, 2-amino-1,6-anhydro-2-deoxy-b-D-mannopyranose. Q10 Q11 STRUCTURAL DIFFERENTIATION OF LMWHs/GUERRINI ET AL Q12 Q15 Figure 3 13C–nuclear magnetic resonance spectra of (A) enoxaparin, (B) dalteparin, and (C) tinzaparin. Spectra were recorded at 400.13 MHz on a Bruker AMX400 spectrometerQ12 equipped with BB 10-mm probe at 408C. Spectra were recorded with proton decoupling during acquisition. Recycle delay, 2 seconds; number of scans, 16,000Q13. The samples were prepared by dissolution of 200 mg of low molecular weight heparin in 2.5 mL of deuterium oxide. ANS, 2-deoxy-2-sulfoamino-D-glucose; 2-O-sulfo-iduronic acid; DU2S, 2-O-sulfo-4-deoxy-a-L-threo-hex-4-enopyranosil uronic acid; AM.ol, 2,5-anhydromannitol; DU, nonsulfated uronic acid; GTANS,3S, ???Q14; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose. Enoxaparin and tinzaparin are produced by chemical b-eliminative and enzymatic methods, respectively. Both of these processes generate the 4–5Q15 unsaturated 2-O-sulfated uronic acid (DU2S) as the terminal nonreducing residue and N-sulfated, 6-O-sulfated glucosamine (ANS,6S) is prevalent at the reducing end. The proton spectrum of enoxaparin differs from the corresponding heparin spectrum more than that of tinzaparin (Fig. 3). In fact, it contains signals that are not present in the tinzaparin spectrum; in particular, a small signal at 5.83 ppm corresponding to H4 of unsaturated nonsulfated uronic acid (DU) located at the nonreducing end and a signal at 3.22 ppm in the H2 region. Additional differences between enoxaparin and tinzaparin can be observed in the carbon spectra. Two additional reducing anomeric signals at 95.6 ppm, and other two signals in the C2 region of glucosamine at 55.0 and 58.6 ppm, are present in the enoxaparin spectrum. The first signal is due to the reducing N-sulfated, 6-Osulfated mannosamine (MNS) and 2-O-sulfated iduronic acid (I2S) residues, the two others to the 2-sulfo-amino- 1,6-anhydro-2-deoxy-b-D-glucopyranose (1,6-an.A) and 2-sulfo-amino-1,6-anhydro-2-deoxy-b-D-mannopyranose (1,6-an.M) residues; these two unique bicyclic structures present at the reducing end originating from alkaline hydrolysis of the benzoyl ester of heparin.15,16 These attributes of enoxaparin are peculiar to this reaction and to other side reactions that occur when fragments so generated are submitted to additional alkaline treatment.17 Signals corresponding to the linkage region (l.r.; i.e., the tetrasaccharide corresponding to the linkage of the polysaccharide chain to the peptide of the original proteoglycan18) are more abundant in the tinzaparin spectrum than in either enoxaparin or dalteparin spectra, where these signals are almost absent (Fig. 2). Complete characterization of the structure of LMWHs is prevented by signal overlap in the monodimensional NMR spectra. In contrast, bidimensional NMR spectroscopy, particularly by using the HSQC experiment, is able to resolve and identify signals 481 Q13 Q14 482 Q16 Q18 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007 Figure 4 Ring signals of two-dimensional heteronuclear single quantum coherence (2D-HSQC) spectra of (A) enoxaparin, (B) dalteparin, and (C) tinzaparin. Anomeric region of 2D-HSQC spectra of (A0 ) enoxaparin, (B0 ) dalteparin, and (C0 ) tinzaparin. Spectra were recorded at 358C with a Bruker Avance 600 spectrometerQ16 equipped with TCIQ17 5-mm cryoprobe. Samples were prepared by dissolution of 12 mg of heparin in 0.6 mL of deuterium oxide. ANS, 2-deoxy-2-sulfoamino-D-glucose; ANAc, 2-deoxy-2-acetylamino-Dglucose; aredQ18, ???; I2S, 2-O-sulfo-iduronic acid; MNS, 2-deoxy-2-sulfamino-D-mannose; ANS, 2-deoxy-2-sulfoamino-D-glucose; ANS,3S, 2-deoxy-3-O-sulfo-2-sulfoamino-D-glucose; DU2S, 2-O-sulfo-4-deoxy-a-L-threo-hex-4-enopyranosil uronic acid; G, glucuronic acid; 1,6an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose; 1,6-an.M, 2-amino-1,6-anhydro-2-deoxy-b-D-mannopyranose; l.r., linkage region; DU, nonsulfated uronic acid; Gal, galacturonic acid; AM.ol, 2,5-anhydromannitol; Xyl, ???Q19. with minimum overlap representing specific sulfation patterns or monosaccharidic sequences. Many signals belonging to the original heparin structure can be assigned by reference to previous NMR studies.10,19,20 The HSQC spectra of the anomeric and ring regions highlight structural differences among the three LMWHs (Fig. 4). Enoxaparin shows the most complex structure in both the anomeric and H2/C2 signals. As mentioned, two unique bicyclic structures, namely 1,6anhydro-glucose and 1,6-anhydro-mannose are present at the reducing ends of enoxaparin chains. In fact, treatment with base also catalyzes the C2 epimerization of the hemiacetal form of reducing terminus glucosamine N,6-disulfate (ANS,6S-ared), with partial conversion to the diastereoisomer 2-deoxy-6-O-sulfo-2-sulfamino-Dmannose (MNS,6S).22 Assignment of the corresponding NMR signals has been described recently in a NMR study of modified heparin disaccharides.16 A signal corresponding to 2-O-sulfated glucuronic acid residue (G2S), which was found in very small amounts in natural GAGs and was not detectable in Q17 Q19 spectra of non-depolymerized heparins, is clearly observable in the enoxaparin HSQC spectrum (Fig. 4A). The HSQC-TOCSYQ20 enoxaparin spectrum (not Q20 shown) reveals the correlation between H1/C1 of G2S and the corresponding H2/C2 (4.75/102.8 ppm and 4.17/82.4 ppm, respectively) in agreement with the characterization of heparin tetrasaccharide containing the same residue23 (Table 1). The enoxaparin HSQC spectrum also indicates the presence of epoxides generated by alkaline treatment.24,25 This treatment induces loss of the sulfate group at position 2 of the iduronate residue and formation of an epoxide ring between carbons 2 and 3 of this residue. In the spectrum of Fig. 4A, the two signals corresponding to the H2/C2 and H3/C3 of the epoxide group (3.74/54.2 ppm and 3.82/53.3 ppm, respectively) are highlighted.26 The presence of epoxide in the oligosaccharidic chains may explain the formation of both L-galacturonic acid (GalA) and L-IdoA residues during the depolymerization process.24 Conditions used during the alkaline treatment could cleave the epoxide ring, with prevalent formation a-L-ido STRUCTURAL DIFFERENTIATION OF LMWHs/GUERRINI ET AL 483 Table 1 (A) Proton and (B) Carbon Chemical Shifts of Reducing and Nonreducing Residues of LMWHs 1 H Chemical Shifts (ppm) A Enoxaparin H5 H6 H60 3.49 4.84 4.24 3.79 3.22 4.84 4.22 3.82 Residue H1 H2 H3 H4 ANS-red ANAc-red 5.47 5.23 MNS 5.41 3.63 1,6-an.M 5.59 1,6-an.A 5.63 DU2S 5.53 6.01 DU 5.18 5.83 I2S-ared 5.44 *G2S I2S-bred 4.75 4.99 4.17 GalA 4.69 Gnr Tinzaparin Dalteparin B 3.55 ANS-red 5.47 ANAc-red 5.23 DU2S 5.53 AM.ol6S 3.67/ 3.59 4.01 4.14 4.15 4.29 AM.ol nd 3.93 nd 4.20 4.08 6.01 13 C Chemical Shifts (ppm) Residue C1 ANS-red 93.9 ANAc-red 93.5 MNS Enoxaparin 3.54 C2 C3 C4 95.6 60.3 103.9 55.0 76.2 67.5 1,6-an.A 104.3 58.6 76.2 68.1 DU2S 100.1 108.7 DU I2S-ared 103.9 95.6 110.4 *G2S 102.8 82.4 94.8 GalA 74.5 Gnr ANS-red Q22 C6 1,6-an.M I2S-bred Q21 C5 78.1 74.8 108.7 88.0 82.4 88.7 84.3 93.9 Tinzaparin ANAc-red 93.5 Dalteparin DU2S AM.ol6S 100.1 65.4 86.0 78.2 AM.ol nd 85.5 nd *Not located at the reducing position. Chemical shifts were measured from HSQC spectra recorded at 358C on Bruker Avance 600 spectrometer. LMWHs, low molecular weight heparins; ANS, 2-deoxy-2-sulfoamino-D-glucose; ANAc, 2-deoxy-2-acetylamino-D-glucose; MNS, 2-deoxy-2sulfamino-D-mannose; red, reduced;Q21 1,6-an.M, 2-amino-1,6-anhydro-2-deoxy-b-D-mannopyranose; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-bD-glucopyranose; DU2S, 2-O-sulfo-4-deoxy-a-L-threo-hex-4-enopyranosil uronic acid; DU, nonsulfated uronic acid; I2S, 2-O-sulfo-iduronic acid; G2S, 2-O-sulfo-glucuronic acidQ22; Gal, galacturonic acid; A, ???;Q23 AM.ol, 2,5-anhydromannitol; nd, not determined; nr, nonreduced.Q24 configuration in the case of additional exposure to base or a-L-galacto configuration after inadvertent exposure to acid, perhaps following overneutralization. Signals of galacturonic acid overlap those of iduronic acid, with exclusion of H5/C5 signals, which shift to 4.69/74.5 ppm. Such signals, observable in the HSQC spectrum of enoxaparin and absent in the other two LMWHs, are compatible with this residue.24 Another minor anomeric signal at 4.99/94.8 ppm is observed in the enox- aparin preparation. Preliminary NMR studies on oligosaccharidic fractions of enoxaparin indicate a correlation of these signals with the corresponding H2/C2 at 4.25/79.1 ppm. Together with the H1-H2 proton– proton coupling constant of 8.4 Hz, these data are compatible with the presence of the expected b form of the reducing I2S residue. In addition to the presence of reducing I2S residues, signals corresponding to nonreducing glucuronic acid (G) (H3/C3 at 3.54/ Q23 Q24 484 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 2007 Table 2 Determination of Variously Substituted Monosaccharide Components (percentage) of Enoxaparin, Tinzaparin, and Dalteparin Amines Q26 ANS-I2S ANS-I ANS-G ANS,3S ANAc ANAc-ared ANS-ared ANS-bred 1,6-an.A 1,6-an.M MNS-ared AM.ol A6S Enoxaparin*44.4 8.8 14.5 4.4 10.7 0.5 8.2 1.0 2.1 2.3 3.2 0 Tinzaparin 55.7 6.9 8.2 2.0 14.6 0.4 10.5 1.6 0 0 0 0 81.9 Dalteparin 56.8 8.0 5.2 4.6 10.3 0 0 0 0 0 0 15.5 91.6 Uronic acid I2S I-A6S I-A6OH G-ANS,3S G-ANS G-ANAc G2S DU2S DU I2S-ared I2S-bred Gal-Ay Epox Enoxaparin*49.9 5.7 1.0 3.2 9.5 4.1 2.6 18.0 1.1 1.1 1.0 2.3 0.3 Tinzaparin 60.4 6.5 2.1 2.1 9.3 4.4 0.5 14.7 0 0 0 0 0 Dalteparin 75.4 9.5 0.5 4.1 6.8 3.6 0 0 0 0 0 0 0 Amount of Linkage Amount of Linkage Region Calculated Region Calculated by Total Degree by HSQC Method 13 of Sulfation Anti-FXa29 104 C Method (reference) Enoxaparin 0.7 0.8 2.5 Tinzaparin 3.1 2.8 2.4 90 Dalteparin 0.6 0.7 2.6 122 *Average value on three NMR measurements. y Not accurate, due to the value of the corresponding 1JC-H (see text). ANS, 2-deoxy-2-sulfoamino-D-glucose; I2S, 2-O-sulfo-iduronic acid; G, glucuronic acid; ANS,3S, 2-deoxy-3-O-sulfo-2-sulfoamino-D-glucose; ANAc, 2deoxy-2-acetylamino-D-glucose; aredQ26, ???; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose; 1,6-an.M, 2-amino-1,6-anhydro-2deoxy-b-D-mannopyranose; MNS, 2-deoxy-2-sulfamino-D-mannose; AM.ol, 2,5-anhydromannitol; A, ???;Q27 DU2S, 2-O-sulfo-4-deoxy-a-L-threohex-4-enopyranosil uronic acid; DU, nonsulfated uronic acid; Epox, ???Q28; HSQC, heteronuclear single quantum coherence; FXa, factor Xa. 78.1 ppm and H4/C4 at 3.55/74.8 ppm)27 have been detected in HSQC spectra of both enoxaparin and dalteparin. Q25 84.2 MONOSACCHARIDE COMPOSITION OF LMWHS The analytical signals were chosen from those with minimal overlap in the HSQC spectra. Signals corresponding to monosaccharides also present in the heparin structure were chosen according to a previously published method.12 Those that directly define specific residues typical for the LMWHs are shown in Table 2Q25. The prerequisite of the quantitative 2D-NMR approach is the proper selection of analytical signals from those where the effect of 1JC–H coupling is minimal and differences in relaxation effects are sufficiently small to be neglected.12 To verify whether the 1JC–H set was compatible with the average value of reducing and nonreducing residues, coupled HSQC spectra were meas- Q27 Q28 ured for enoxaparin. Results indicate that 1JC–H of both H1/C1 and H2/C2 of reducing residues do not differ significantly from those measured for the corresponding signals associated with residues within the heparin chain (Table 3). In addition, the 1JC1–H1 couplings of DU and DU2S are close to those of the other uronic acids. However, the corresponding H4/C4 signals were chosen for the integration even if the value of 1JC4–H4 differs from the average value of anomeric signals by 5 Hz. The overlap of the H1/C1 signal with part of the anomeric signals induces a larger error than that observed by integration of the better separated H4/C4 signals. The percentage of AM.ol residue was calculated from H2/C2 signals because its 1JC2–H2 value is much closer to the average of the other glucosamine residues. Given that no other signals compatible with galacturonic acid can be detected in both anomeric and H2/C2 regions of the HSQC spectrum (due to signal overlap), the H5/C5 value was used for in the calculation, even if the value of 1JC5–H5 differed slightly (by Table 3 1JC–H Coupling Constants (values in Hz) of Reducing and Nonreducing Residues of Enoxaparin and Dalteparin ANS(-I2S) ANS-(-I) ANS-ared ANAc-ared A* 1,6-an.M 1,6-an.A MNS-ared AM.ol I2S H1/C1 173 173 173 172 174 177 178 173 H4/C4 H2/C2 139 140 138 142 ** DU2S DU I2S-ared *G2S *GalA 173 165 176 148 147 151 H5/C5 G 168 172 174 166 167 151 146 1 Q29 G2S and GalA residues are located inside of the chain (*) transformation. JC-H were determined with a precision of  1 Hz by application of gradient enhanced HSQC sequence without carbon decoupling during acquisition.12 ANS, 2-deoxy-2-sulfoamino-D-glucose; I2S, 2-O-sulfo-iduronic acid; aredQ29, ???; ANAc, 2-deoxy-2-acetylamino-D-glucose; A, ???;Q30 1,6-an.M, 2amino-1,6-anhydro-2-deoxy-b-D-mannopyranose; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose; MNS, 2-deoxy-2-sulfamino-D-mannose; AM.ol, 2,5-anhydromannitol; G, glucuronic acid; DU2S, 2-O-sulfo-4-deoxy-a-L-threo-hex-4-enopyranosil uronic acid; DU, nonsulfated uronic acid. Q30 STRUCTURAL DIFFERENTIATION OF LMWHs/GUERRINI ET AL Q31 25 Hz) from the average value of the anomeric signals of uronic acid residues. The possibility of detecting and quantifying all minor signals depends on the signal-to-noise ratio obtained in the HSQC spectrum. Application of the method to heparin/heparan sulfate preparations indicated that signals corresponding to other minor components present in amounts lower than 3 to 4%, such as those belonging to the linkage region, can be detected by HSQC spectra, but not accurately quantified.12 In the present work, the spectra were recorded using a highfield NMR spectrometer (600 instead 500MHz) with a high-sensitivity probe (TCI cryoprobeQ31). This increases the sensitivity 4-fold, permitting accurate quantification of residues present in amounts below 1 to 2% (Table 4). The average contents of monosaccharide components of the three LMWHs are reported in Table 2. The complexity of the enoxaparin structure compared with the other LMWHs can be appreciated immediately: eight different reducing residues are detectable in the spectra compared with the three of tinzaparin and the single reducing unit of dalteparin. In addition to the presence of different reducing residues, only small differences were observed in the monosaccharide composition. Whereas the amount of N-sulfated glucosamine linked to glucuronic acid (ANS-G) is higher in enoxaparin, a higher ANAc content was found for tinzaparin. A slightly higher nonsulfated uronic acid content was found in enoxaparin (27%) compared with the other two LMWHs (24 to 25%). However, the total sulfation degree, calculated by adding all different sulfated monosaccharides, was almost the same (2.4/2.6) for all LMWHs, indicating that lower N-sulfation is compensated by somewhat higher O-sulfation (Table 2). Given that ANS,3S is contained in the pentasaccharidic sequence of the active site of heparin for AT, its content in LMWHs should be directly associated with the aFXa activity. However, the correlation of ANS,3S with aFXa activity is not straightfor- 485 ward, given that signals associated with ANS,3S residues were also found in heparin fractions with no affinity for AT, indicating that this residue can also be present in different sequences that do not contribute significantly to the activity.28 A more reliable correlation can be made between the amount of glucuronic acid linked to ANS,3S (G-ANS,3S) and the aFXa activity, given that this disaccharide has been detected only in active sequences (Table 2). In fact, it has been demonstrated that heparinases I and II cleave the pentasaccharide between ANS,3S and I2S residues, leaving the trisaccharide ANAc,6S-G-ANS,3S unit at the reducing end. Because significant aFXa activity is retained by oligosaccharides having such trisaccharide sequences at the reducing end, the G-ANS,3S disaccharide content can be considered proportional to the aFXa activities of LMWHs, as shown in Table 2.30 As discussed, analysis of 13C spectra indicates that all LMWHs contain a small amount of linkage region. Comparable results were obtained by HSQC (average value of the integral of the corresponding four anomeric signals) and 13C spectra integration methods, indicating a decrease in the content of linkage region in both enoxaparin and dalteparin compared with the average value for pig mucosal heparin.31 In contrast, the linkage region content of tinzaparin is about twice as high as that of pig mucosal heparin, clearly indicating that heparinase does not cleave the linkage region. Another parameter that can be derived from the NMR data are the number-average mean molecular weight (Mn), which can be evaluated by integration of reducing peaks with respect to the total anomeric signals in the carbon spectrum.32 The total amount of reducing residues, obtained by integration of the appropriate signals in HSQC spectra, can be correlated to the average length of the chains and the Mn value by estimation of the average molecular weight of the disaccharide. The calculated Mn (3200, 4700, and 4000 d for enoxaparin, tinzaparin, and dalteparin, respectively) are substantially in agreement with those calculated by applying the HP-SEC/TDAQ35 method,33 optimized Q35 Table 4 Monosaccharide Components Average Contents (percentage), Standard Deviation (SD), and Coefficient of Variation (CV) for Enoxaparin, Determined by Three Measurements of the HSQC Spectrum Q32 Q34 Amine ANS-I2S ANS-I ANS-G ANS,3S ANAc Average 44.4 10.7 8.8 14.5 4.4 ANS-ared ANAc-ared ANS-bred 1,6-an.A 1,6-an.M MNS ared A6S 8.2 0.5 1.0 2.1 2.3 3.2 SD 0.49 0.21 0.23 0.16 0.02 0.05 0.05 0.06 0.05 0.05 0.09 0.10 CV % 1.1 2.4 1.6 3.6 0.2 0.6 9.7 5.7 2.1 2.3 2.8 0.1 Uronic acid I2S I-A6S I-A6OH G-ANS,3S G-ANS G-ANAc G2S DU2S DU I2S-ared I2S-bred Average 5.7 1.0 3.2 9.5 4.1 2.6 18.0 1.1 1.1 1.0 49.9 84.2 Gal-A* Epox 2.3 0.3 SD 0.28 0.04 0.01 0.04 0.09 0.06 0.04 0.40 0.03 0.10 0.06 0.03 0.01 CV % 0.5 0.7 1.1 1.2 1.0 1.5 1.5 2.3 2.9 9.3 6.3 1.3 4.2 ANS, 2-deoxy-2-sulfoamino-D-glucose; I2S, 2-O-sulfo-iduronic acid; G, glucuronic acid; ANS,3S, 2-deoxy-3-O-sulfo-2-sulfoamino-D-glucose; ANAc, 2deoxy-2-acetylamino-D-glucose; aredQ32, ???; 1,6-an.A, 2-amino-1,6-anhydro-2-deoxy-b-D-glucopyranose; 1,6-an.M, 2-amino-1,6-anhydro-2deoxy-b-D-mannopyranose; MNS, 2-deoxy-2-sulfamino-D-mannose; A, ???;Q33 DU2S, 2-O-sulfo-4-deoxy-a-L-threo-hex-4-enopyranosil uronic acid; DU, nonsulfated uronic acid; Epox, ???Q34. Q33 486 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 33, NUMBER 5 for the analysis of LMWHs (3600, 5500, and 4600 d; manuscript in preparation). CONCLUSION Knowledge of the monosaccharidic composition of heparin and LMWHs is essential for deriving structure– activity relationships. The use of high-sensitivity NMR instruments permits a detailed analysis of the structural peculiarities of LMWHs, also permitting the quantification, with an acceptable error, of minor monosaccharide components. Combination of the present NMR method with disaccharide analysis performed by highperformance liquid chromatography or capillary electrophoresis, so far, is the best way to characterize heparin and heparin-like species. In addition to the structural differences typical of each manufacturing process, variation in the heparin monosaccharidic composition among the three analyzed samples was found. Such differences may also depend on structural differences among the parent heparins. Given that different pharmacokinetic profiles of LMWHs may be essentially due to their different molecular weight and polydispersity, a more exhaustive study should involve isolation of size-equivalent fractions for each LMWH. ACKNOWLEDGMENTS We thank Professor B. Casu (G. Ronzoni Institute) for useful discussions, and LEO-Pharma (Denmark) and Sanofi-Aventis (France and Italy) for providing tinzaparin and enoxaparin samples, respectively. This study was supported by National Institutes of Health grant 1R01-HL080278-01. ABBREVIATIONS 1,6-an.A 2-amino-1,6-anhydro-2-deoxy-b-Dglucopyranose 1,6-an.M 2-amino-1,6-anhydro-2-deoxy-b-Dmannopyranose 2D-NMR two-dimensional nuclear magnetic resonance aFXa anti-factor Xa AM.ol 2,5-anhydromannitol ANAc 2-deoxy-2-acetylamino-D-glucose 2-deoxy-2-sulfoamino-D-glucose ANS 2-deoxy-3-O-sulfo-2-sulfoamino-DANS,3S glucose ANS,6S 2-deoxy-6-O-sulfo-2-sulfoamino-Dglucose AT antithrombin III G glucuronic acid GAGs glycosaminoglycans GalA a-L-galacturonic acid HS heparan sulfate 2007 HSQC I I2S l.r. MNS DU2S heteronuclear single quantum coherence a-L-iduronic acid 2-O-sulfo-iduronic acid linkage region 2-deoxy-2-sulfamino-D-mannose 2-O-sulfo-4-deoxy-a-L-threo-hex-4enopyranosil uronic acid REFERENCES 1. Hoppensteadt D, Iqbal O, Fareed J. Basic and clinical differences of heparin and low molecular weight heparin treatment. In: Garg HG, Linhardt RJ, Hales CA, eds. Chemistry and Biology of Heparin and Heparan Sulfate. Elsevier Ltd; 2006:583–606Q36 2. Lever R, Page CP. Novel drug development opportunities for heparin. Nat Rev Drug Discov 2002;1:140–148 3. Casu B. Structure and active domains of heparin. In: Garg HG, Linhardt RJ, Hales CA, eds. Chemistry and Biology of Heparin and Heparan Sulfate. Elsevier Ltd; 2006:1–28Q37 4. de Kort M, Buijsman RC, van Boeckel CAA. Synthetic heparin derivatives as new anticoagulant drugs. Drug Discov Today 2005;10:769–779 5. van Boeckel CAA, Petiou M. The unique antithrombin III binding domain of heparin: a lead to new synthetic antithrombotics. Angew Chem Int Ed Engl 1993;32:1671– 1690Q38 6. Belzac KJ, Dafforn TR, Petitou M, et al. The effect of a reducing-end extension on pentasaccharide binding by antithrombin. J Biol Chem 2002;275:8733–8741Q39 7. Guerrini M, Guglieri S, Beccati D, et al. Conformational transitions induced in heparin octasaccharides by binding with antithrombin III. Biochem J 2006;399:191–198 8. Hirsh J, Warkentin T, Raschke R, et al. Heparin and low molecular weight heparin. Mechanism of action, pharmacokinetics, dosing consideration, monitoring safety and efficacy. Chest 1998;114:489S–501S 9. Capila I, Guany NS, Shriver Z, Venkataraman G. Methods for structural analysis of heparin and heparin sulfate. In: Garg HG, Linhardt RJ, Hales CA, eds. Chemistry and Biology of Heparin and Heparan Sulfate. Elsevier Ltd; 2006Q40 10. Guerrini M, Bisio A, Torri G. Combined quantitative 1H and 13 C-NMR spectroscopy for characterization of heparin preparations. Semin Thromb Hemost 2001;27:100–123Q41 11. Casu B, Guerrini M, Naggi A, et al. Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy. Arzneimittelforchung/ Drug Res 1996;46:472–477Q42 12. Guerrini M, Naggi A, Guglieri S, et al. Complex glycosaminoglycans: profiling substitution patterns by twodimensional nuclear magnetic resonance spectroscopy. Anal Biochem 2005;337:35–47 13. Lormeau J-C, Petitou M, Choaj J. Oligosaccharides having anti Xa activity and pharmaceutical compositions containing them. US patent #RE 35770. 1998 14. Huckerby TN, Sanderson PN, Nieduszynski ANMR. Studies of the disulphated disaccharide obtained by degradation of bovine lung heparin with nitrous acid. Carbohydr Res 1985;138:199–206 15. Mourier P, Viskov CUS. Patent 2005/0119477 A1; Chem Abstr 142:89363 Q36 Q37 Q38 Q39 Q40 Q41 Q42 STRUCTURAL DIFFERENTIATION OF LMWHs/GUERRINI ET AL Q43 Q44 Q45 Q46 Q47 Q48 16. Mascellani G, Guerrini M, Torri G, et al. Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydrohydro-b-D-glucosamine or N-sulfated 1,6-an b-D-mannosamine residues. Carbohydr Res 2007;342:835–842Q43 17. Černy I, Buděšinsky M, Trnka T, Černy M. Preparation of 2-amino-1,6-anhydro-2,3-dideoxy-b-D-arabino-hexopyranose. 1H- and 13C-n.m.r. spectra of deoxy derivatives of 2-amino-1,6-anhydro-2-deoxy-D-glucose and 2-amino-1,6anhydro-2-deoxy-D-mannose. Carbohydr Res 1984;130: 103–114Q44 18. Robinson HC, Horner AA, Hook A, et al. A proteoglycan form of heparin and its degradation to single-chain molecules. J Biol Chem 1978;253:6687–6693 19. Chuang WL, McAllister H, Rabenstein D. Hexasaccharides from the istamine-modified depolymerization of porcine intestinal mucosal heparin. Carbohydr Res 2002;337:935–945 20. Yates EA, Santini F, Guerrini M, et al. 1H and 13C NMR spectral assignment of the major sequences of twelve systematically modified heparin derivatives. Carbohydr Res 1996;294:15–27 21. Černy I, Buděšinsky M, Trnka T, Černy M. Carbohydr Res 1984;130:115–124Q45Q46Q47 22. Yamada S, Watanabe M, Sugahara K. Conversion of N-sulfated glucosamine to N-sulfated mannosamine in an unsaturated heparin disaccharide by non-enzymatic, base-catalyzed C-2 epimerization during enzymatic oligosaccharide preparation. Carbohydr Res 1998;309:261–268 23. Yamada S, Murakami T, Tsuda H, et al. Isolation of the porcine heparin tetrasaccharides with glucuronate 2-Osulfate. J Biol Chem 1995;270:8696–8705 24. Rej RN, Perlin AS. Base-catalyzed conversion of the a-L-iduronic acid 2-sulfate unit of heparin into a unit of a-L-galacturonic acid, and related reactions. Carbohydr Res 1990;200:437–447Q48 25. Mourier PAJ, Viskov C. Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases. Anal Biochem 2004;332:299–313 26. Hricovini M, Guerrini M, Torri G, et al. Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr Res 1995;277:11–23 27. Cipolla L, Nicotra F, Lay L, et al. Synthesis of the disaccharides methyl 4-O-(20 /30 -O-sulfo-beta-D-glucopyranosyluronic-acid)-2-amino-2-deoxy-alpha-D glucopyranoside disodium salts, related to heparin biosynthesis. Glycoconj J 1996;13:995–1003 28. Casu B, Torri G. Structural characterization of low molecular weight heparins. Semin Thromb Hemost 1999;25(suppl 3): 17–25 29. Fareed J, Ma Q, Florian M, et al. Differentiation of lowmolecular-weight heparins: impact on the future of the management of thrombosis. Semin Thromb Hemost 2004; 30(suppl 1):89–104 30. Shriver Z, Sundaram M, Venkataraman G, et al. Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin. Proc Natl Acad Sci USA 2000;97:10365– 10370 31. Iacomini M, Casu B, Guerrini M, et al. Linkage region sequences of heparins and heparan sulfates. Detection and quantification by NMR spectroscopy. Anal Biochem 1999; 274:50–58 32. Desai UR, Linhardt RJ. Molecular weight of heparin using 13 C nuclear magnetic resonance spectroscopy. J Pharm Sci 1995;84:212–215 33. Bertini S, Bisio A, Torri G, et al. Molecular weight determination of heparin and dermatansulfate by size exclusion chromatography with a triple detector array. Biomacromolecules 2005;6:168–173 487 Author Query Form (STH/01307) Special Instructions: Author please write responses to queries directly on proofs and then return back. Q1: AU: Please supply professional degrees for all authors, if applicable. Q2: AU: Please define. Q3: AU: Correct with "factor" as inserted throughout instead of "anti-Xa and anti-IIa"? Throughout text, "factor" was changed to "F" in combination with a specific Roman numeral for consistency with journal style. Q4: AU: Please verify the use of italics for K5. Q5: AU: Please verify the use of an unpaired right parenthesis. Q6: AU: Per standard chemical nomenclature for carbohydrates, OK with "D" and "L" as changed throughout to small capital letters to indicate absolute configuration? Q7: AU: Please verify all chemical compound names throughout text for accuracy and consistency. All chemical compound acronyms/abbreviations should be defined at first mention in text and within each figure and table, if applicable. Q8: AU: In Fig 2, 3, and 4 legends, and all table footnotes, please verify all definitions of chemical compounds and ensure all applicable items in art/table are defined. Q9: AU: Please supply city/country or city/state location (if in U.S.) for manufacturer. Q10: AU: Please define TCI. Q11: AU: Please define ?red. Q12: AU: Please supply city/country or city/state location (if in U.S.) for manufacturer. Q13: AU: As meant? Q14: AU: Please define. Q15: AU: Please clarify: Does this refer to a specific number (four to five) of residues or to part of the chemical name? Q16: AU: Please supply city/country or city/state location (if in U.S.) for manufacturer. Q17: AU: Please define TCI. Q18: AU: Please define ared. Q19: AU: Please define. Q20: AU: Please define TOCSY. Q21: AU: Correct as defined? Q22: AU: Correct as defined? Q23: AU: Please define. Q24: AU: Correct as defined? Q25: AU: Original Table 4 was cited out of order. Tables were renumbered accordingly. Q26: AU: Please define ared. Q27: AU: Please define. Q28: AU: Please define. Q29: AU: Please define ared. Q30: AU: Please define. Q31: AU: Please define TCI. If it is a trade name of a product, please supply manufacturer’s name and city/state (or city/country if not in U.S.) location. Q32: AU: Please define ared. Q33: AU: Please define. Q34: AU: Please define. Q35: AU: Please define. Q36: AU: Please supply city/country or city/state (if U.S.) location for publisher. Q37: AU: Please supply city/country or city/state (if U.S.) location for publisher. Q38: Medline indexes "Angew Chem Int Ed Engl" but cannot find a listing for the reference 5 "van Boeckel, Petiou, 1993". Please check the reference for accuracy. Q39: Medline indexes "J Biol Chem" but cannot find a listing for the reference 6 "Belzac, Dafforn, Petitou, et al, 2002". Please check the reference for accuracy. Q40: AU: Please supply city/country or city/state (if U.S.) location for publisher and page range. Q41: Medline indexes "Semin Thromb Hemost" but cannot find a listing for the reference 10 "Guerrini, Bisio, Torri, 2001". Please check the reference for accuracy. Q42: Medline cannot find the journal "Arzneimittelforchung/Drug Res" (in reference 11 "Casu, Guerrini, Naggi, et al, 1996"). Please check the journal name. Q43: AU: Reference was updated per PubMed. Please verify. Q44: Medline indexes "Carbohydr Res" but cannot find a listing for the reference 17 "Černy, Buděinsky, Trnka, Černy, 1984". Please check the reference for accuracy. Q45: The reference 21 "Černy, Buděinsky, Trnka, Černy, 1984" is not cited in the text. Please add an in-text citation or delete the reference. Q46: Medline reports the first author "Černy I" is not correct in the reference 21 "Černy, Buděinsky, Trnka, Černy, 1984". Q47: The article title is missing from this reference. Please provide one. (in reference 21 "?erny, Bud??insky, Trnka, ?erny, 1984"). Q48: Medline indexes "Carbohydr Res" but cannot find a listing for the reference 24 "Rej, Perlin, 1990". Please check the reference for accuracy. Instructions to Contributors Dear Contributor: Enclosed in this document please find the page proofs, copyright transfer agreement (CTA), and offprint order form for your article in the Seminars in Thrombosis and Hemostasis, Volume 33, Number 5, 2007. Please print this document and complete and return the CTA and offprint form, along with corrected proofs, within 72 hours (3 business days). 1) Please read proofs carefully for typographical and factual errors only; mark corrections in the margins of the proofs in pen. Answer (on the proofs) all editor’s queries indicated in the margins of the proofs. Check references for accuracy. Please check on the bottom of the 1st page of your article that your titles and affiliations are correct. Avoid elective changes, as these are costly and time consuming and will be made at the publisher’s discretion. 2) Please pay particular attention to the proper placement of figures, tables, and legends. Provide copies of any formal letters of permission that you have obtained. 3) Please return the corrected proofs, signed copyright transfer agreement, and your offprint order form, with color prints of figures, if you received any. 4) As a contributor to this journal you will receive one copy of the journal, at no charge. • If you wish to order offprints or e-prints, please circle the quantity required (left column) and the number of pages in your article. If you wish to order additional copies of the journal please enter the number of copies on the indicated line. • If you do not want to order offprints or journals simply put a slash through the form, but please return the form. Please send all materials back via overnight mail, within 72 hours of receipt, to: Xenia Golovchenko Production editor Thieme Medical Publishers 77 Gregory Blvd. Norwalk, CT 06855 Tel: 845-548-8127 Fax: +1 (203) 857 4996 E-mail: xeniag@optonline.net Please do not return your materials to the editor, or the compositor. Please note: Due to a tight schedule, if the publisher does not receive the return of your article within 7 business days of the mail date (from the compositor), the publisher reserves the right to proceed with publication without author changes. Such proofs will be proofread by the editor and the publisher. Thank you for your contribution to this journal. Xenia Golovchenko, Production Editor, Journal Production Department Thieme Medical Publishers, Inc. Thieme Medical Publishers, Inc. (the “Publisher”) will be pleased to publish your article (the “Work”) entitled _____________________________ in the Seminars in Thrombosis and Hemostasis, Volume 33, Number 5, 2007. The undersigned Author(s) hereby assigns to the Publisher all rights to the Work of any kind, including those rights protected by the United States Copyright laws. The Author(s) will be given permission by the Publisher, upon written request, to use all or part of the Work for scholarly or academic purposes, provided lawful copyright notice is given. If the Work, subsequent to publication, cannot be reproduced and delivered to the Author(s) by the publisher within 60 days of a written request, the Author(s) is given permission to reprint the Work without further request. The Publisher may grant third parties permission to reproduce all or part of the Work. The Author(s) will be notified as a matter of courtesy, not as a matter of contract. Lawful notice of copyright always will be given. Check appropriate box below and affix signature. [ ] I Sign for and accept responsibility for transferring copyright of this article to Thieme Medical Publishers, Inc. on behalf of any and all authors. Author’s full name, degrees, professional title, affiliation, and complete addre ss: __________________________________ ____________________________ Author’s printed name, degrees Professional title Complete professional address ______________________________ ___________________ Author’s signature Date [ ] I prepared this article as part of my official duties as an employee of the United States Federal Government. Therefore, I am unable to transfer rights to Thieme Medical Publishers, Inc. ______________________________ _________________ Author’s signature Date Order Form for Offprints and additional copies of the Seminars in Thrombosis and Hemostasis (Effective October 2005) Please circle the cost of the quantity/page count you require (orders must be in increments of 100) Quantity 100 200 300 400 500 1000 1 to 4 $198 $277 $356 $396 $446 $792 Pages in Article/Cost 5 to 8 9 to 12 $317 $440 $444 $615 $570 $791 $634 $879 $713 $989 $1,267 $1,758 Volume/Issue #: 13 to 16 $578 $809 $1,041 $1,156 $1,301 $2,313 17 to 20 $693 $970 $1,247 $1,386 $1,559 $2,772 Page Range (of your article): Article Title: MC/Visa/AmEx No: Exp. Date: Signature: Name: Address: City/State/Zip/Country: Corresponding author will receive one complimentary copy of the issue in which the manuscript is published. Number of additional copies of the journal, at the discounted rate of $20.00 each: Notes 1. The above costs are valid only for orders received before publication of the issue. Please return the completed form, even if your institution intends to send a Purchase Order (the P.O. may sometimes be supplied after the issue has been printed). 2. Orders from outside the U.S. must be accompanied by payment. 3. A shipping charge will be added to the above costs. 4. Reprints are printed on the same coated paper as the journal and side-stapled. 5. For larger quantities or late orders, please contact reprints dept. Phone: +1(212) 584-4662 Fax: +1(212) 947-1112 E-mail: reprints@thieme.com As an added benefit to all contributing authors, a discount is offered on all Thieme books. See below for details or go to www.thieme.com As a T hie m e a ut hor you are entitled to a 2 5 % disc ount for new books and a 3 5 % disc ount for forthcoming books. We selected two books that might be of interest for you: ne w ! 25% fort hc om ing! 35% Thurlbeck's Pathology of the Lung Vascular Diagnosis with Ultrasound 3 Edition Cerebral and Peripheral Vessels rd Andrew M. Churg, M.D. Ph. D. Professor of Pathology, University of British Columbia; Pathologist, Vancouver Hospital & Health Sciences Center, Vancouver, BC, Canada Thurlbeck's cornerstone textbook and reference on pulmonary pathology returns in a brand new edition! Updated with the latest advances in the field, you will save time with all-inclusive coverage of neoplastic, non-neoplastic, infectious, occupational/environmental, and developmental pathologies in one book, learn how molecular biology provides a greater understanding of lung development, gain new insights into the diagnosis of neoplastic and non-neoplastic lung disease, find pertinent information on clinical features, epidemiology, and pathogenetic mechanisms of lung disease and much more! Comprehensive in its scope and authoritative in its scholarship, Thurlbeck's Pathology of the Lung is a virtual one-volume encyclopedia written by a ''who's who'' list of specialists. It is the one text that no pathologist, pulmonologist, or resident in either specialty can afford to be without. $187.46 $249.95 2005, app.1032 pp., app.1064 illus., hardcover ISBN 1-58890-288-9 Michael Hennerici, M.D. Professor and Chairman, Department of Neurology, University of Heidelberg, Mannheim, Germany Covering the entire venous and body circulation as examined by vascular ultrasound, this unique text/atlas is invaluable for diagnosing arterial and venous disease. It includes comprehensive chapters on vascular ultrasonography in the arteries and veins of the cerebral circulation and the peripheral upper and lower limb circulation, systematic coverage of all available ultrasound technologies, including continuous and pulsed-wave Doppler mode, b-mode, and conventional and color-coded duplex analysis in frequency and amplitude power modes, anatomy and physiology, normal and abnormal findings, test accuracy and sensitivity, pitfalls, and comparison with other diagnostic tests in each vascular region and special, difficult-to-interpret cases discussed in a separate section 2006, 336 pp., 530 illus., hardcover, ISBN 1-58890-144-0 If you want to view more Thieme books, fell free to visit $149.95 $97.47 T hie m e Book s Thieme Author order form For faster service, call TOLL-FREE 1-800-782-3488 or fax this order form to 212-947-1112 Quantity ISBN (last 4-digits only) Author/Title Price Subtotal: Shipping & Handling (Add $7.50 for the first book and $1.00 for each additional book): NY and PA residents add applicable sales tax: TOTAL: Enclosed is my check for $____________________ Charge my: AMEX MasterCard VISA Discover Card#____________________________________________________________________Exp.____________________________________________________ First Name_____________________________________ MI ________________________Last Name_______________________________________________ Address__________________________________________________________________________________________________________________________ City/State/Zip______________________________________________________________________________________________________________________ Telephone________________________________________________________________FAX_____________________________________________________ e-mail____________________________________________________________________________________________________________________________ Signature_________________________________________________________________________________________________________________________ EM1- 05