Glycosaminoglycan-Mediated Coacervation of Tropoelastin Abolishes

Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems ...
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Biomacromolecules 2008, 9, 1739–1744

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Glycosaminoglycan-Mediated Coacervation of Tropoelastin Abolishes the Critical Concentration, Accelerates Coacervate Formation, and Facilitates Spherule Fusion: Implications for Tropoelastin Microassembly Yidong Tu and Anthony S. Weiss* School of Molecular and Microbial Biosciences G08, University of Sydney, NSW 2006, Australia Received November 27, 2007; Revised Manuscript Received May 1, 2008

Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems that have been studied. To address the apparent paradox of a paucity of tropoelastin for cell surface microassembly, we examined the effects of the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfate B, on tropoelastin aggregate formation through coacervation. We found a significant effect, particularly of heparin, on the minimum or critical concentration of tropoelastin, which was required for microassembly, lowering critical concentration to a point that it was no longer detectable. The assemblies resulted in protein droplet formation that was visually indistinguishable from the spherules that typify coacervation. The spherules readily coalesced in the presence of heparin and higher concentrations of tropoelastin, resulting in an almost continuous layer of coacervated tropoelastin. Four stages of droplet behavior were observed: early droplet formation, ∼6 µm droplet formation, and fusion of droplets followed by the formation of a coalesced layer. We conclude that glycosaminoglycans in the extracellular matrix have the capacity to promote coacervation at low concentrations of tropoelastin.

1. Introduction Human tropoelastin is the soluble precursor of elastin. It is an important component of the extracellular matrix and is required for elasticity and resilience in a variety of tissues, including the lung, skin, large arteries, and bladder.1–5 Tropoelastin self-associates at the cell surface through coacervation during the initial phase of elastogenesis6 and is subsequently attached to the nascent elastic fiber and is cross-linked following oxidation by lysyl oxidases.7,8 Coacervation is characterized by the formation of spherule droplet-like aggregates that concentrate the tropoelastin. Dynamic imaging studies show that these microassembled spherical globules of tropoelastin are located on the cell surface. Subsequently, the microassembled globules associate with fibrillin-containing microfibrils after which they fuse to give a maturing elastic fiber. Microassemblies of tropoelastin therefore define the early stages of elastogenesis.9 On raising the temperature of a solution of tropoelastin to the physiological range, the solution becomes turbid because the molecules aggregate. This aggregation is promoted by interactions between multiple hydrophobic domains on the assembling proteins.10–13 This coacervation process is reversed by cooling the solution to lower temperatures. However if the coacervate is left to settle at 37 °C, it forms a lower viscoelastic phase containing highly concentrated tropoelastin while the upper layer contains the equilibrium critical concentration of tropoelastin.2,6,14 Tropoelastin monomers form 2-6 µm protein spherules during coacervation where no intermediates are detected in the progression from monomer to spherule.15,16 This process has traditionally required at least 1 mg/mL tropoelastin to serve as the critical concentration that can seed spherule formation.17 * To whom correspondence should be addressed. Tel.: +61 2 9351 3464. Fax: +61 2 9351 5858. E-mail: aweiss@ usyd.edu.au.

Glycosaminoglycans could promote the self-assembly of tropoelastin.18–20 Wu et al.21 found that heparan and chondroitin sulfates can bind tropoelastin and mediate coacervation through charge contributions by the lysine side chains on the molecules. In this study, we extend these observations by showing that tropoelastin coacervates in the presence particularly of heparin at concentrations well below the critical concentrations that are characteristic of pure tropoelastin solutions.17

2. Materials and Methods Protein Production and Reagents. Synthetic human tropoelastin (SHEL ∆26A) was prepared as previously described.22 HEPES (4-(2hydroxyethyl)-piperazine-1-ethanesulfonic acid), heparin (H3393), chondroitin sulfate B (C3788-25MG), heparan sulfate (H7640), and Tween 20 were purchased from Sigma-Aldrich. Other reagents were of analytical grade. Binding Studies between Tropoelastin Molecules Modulated by Heparin, Heparan Sulfate, and Chondroitin Sulfate B. Tropoelastin was immobilized on the surface of CM5 sensor chip at a level of 6550 response unit (RU) for analysis on a Biacore 3000 system (GE Healthcare, Inc.) as previous described.23 Heparin and chondroitin sulfate B were dissolved in running solution (0.01 M HEPES, 0.2 M NaCl, 0.005% Tween 20, pH 7; BRS) at different levels, from 0 to 250 µg/mL. Tropoelastin was prepared at a concentration of 120 µg/ mL in the same running solutions. Samples were injected over tropoelastin immobilized sensor chip surfaces at 25 °C for 3 min at flow rates of 30 µL/min. Regeneration of the immobilized tropoelastin was performed by treating the surface with 1 M NaCl, 0.05% NaOH for 120 s after 180 s dissociation. For heparan sulfate studies, tropoelastin was immobilized on the surface of a CM5 sensor chip at 7040 RU. Heparan sulfate was dissolved in BRS from 0 µg/mL to 250 µg/mL. Solution phase tropoelastin was used at 120 µg/mL. Samples were injected at 25 °C for 3 min at flow rates of 30 µL/min. Regeneration of the immobilized tropoelastin was performed by treating the surface with 1 M NaCl, 0.05% NaOH for 120 s after 180 s dissociation.

10.1021/bm7013153 CCC: $40.75  2008 American Chemical Society Published on Web 06/12/2008

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Figure 1. Binding of soluble tropoelastin to immobilized tropoelastin and its modulation by (A) heparin, (B) chondroitin sulfate B, and (C) heparan sulfate. Glycosaminoglycan concentrations are indicated beneath each column plot.

Coacervation Tests. Heparin was prepared in BRS and phosphatebuffered saline (10 mM sodium phosphate pH 7.4, 150 mM NaCl; PBS) from 0 to 250 µg/mL. Tropoelastin was prepared in the same BRS and PBS at concentrations of 30, 60, and 120 µg/mL. Each sample was transferred into a 500 µL quartz cuvette for light-scattering measurements at 300 nm in a temperature-controlled Shimadzu UV-1601 UV-visible spectrophotometer and assayed for 10 min at specific temperatures between 10-70 °C. After each coacervation time course, samples were placed at 4 °C until clarification. Chondroitin sulfate B and heparan sulfate were prepared in PBS at 5, 10, and 250 µg/mL. Tropoelastin was prepared in the same PBS at concentrations of 30, 60, and 120 µg/mL. Each sample was transferred into a 500 µL quartz cuvette for light-scattering measurements at 300 nm in UV-visible spectrophotometer and assayed for 10 min at specific temperatures between 10-60 °C. After each coacervation time course, samples were placed at 4 °C until clarification. The maximum change of absorbance at each temperature was calculated as a percentage of the maximum turbidity for each sample to generate a series of coacervation curves. Light Microscopy of Tropoelastin Coacervates. Tropoelastin was prepared in PBS at 120 µg/mL and 10 mg/mL at multiple heparin concentrations. An Axiovert 200 M MAT inverted light microscope coupled to a Carl Zeiss MicroImaging camera and software was used to capture images using a 100× oil objective under DIC phase contrast. Photographs of each sample were captured as the temperatures were raised from 0 °C. A digital thermometer was used to monitor sample temperatures. A total of 50 µL of each sample was placed between two round thin glass slides that were fixed inside a metal chamber for heat conduction and placed in a temperature controlled block.

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Figure 2. (A) Effect of 250 µg/mL heparin on tropoelastin coacervation at 37 °C in PBS. The controls without heparin contained these concentrations of tropoelastin but showed negligible coacervation. (B) Effect of 250 µg/mL heparan sulfate on tropoelastin coacervation in PBS. (C) Effect of 250 µg/mL chondroitin sulfate B on tropoelastin coacervation in PBS. The concentrations of tropoelastin are shown in µg/mL.

Figure 3. Time courses of 120 µg/mL tropoelastin coacervation at 50 °C in PBS: dotted curve, without heparin; solid curve, with 250 µg/mL heparin. Vertical lines indicate the times taken (italicized) to reach 50% coacervation.

3. Results Heparin and Chondroitin Sulfate B Lower the Availability of Free Tropoelastin Molecules in Solution. Tropoelastin in solution associated readily with immobilized tropoelastin on the chip surface as expected for this self-associating system.24 Increasing heparin and chondroitin sulfate B concen-

Implications for Tropoelastin Microassembly

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Figure 5. Effect of heparin on the time courses of coacervation of 10 mg/mL tropoelastin at 37 °C: dotted curve, without heparin; solid curve, with 250 µg/mL heparin.

Figure 4. Tropoelastin (120 µg/mL) coacervation is modulated by GAGS in PBS. (A) Heparin at 0 µg/mL (O), 5 µg/mL (2), 10 µg/mL (0), 20 µg/mL ((), 250 µg/mL (4); (B) chondroitin sulfate B at 5 µg/mL ((), 10 µg/mL (0); (C) heparan sulfate at 5 µg/mL ((), 10 µg/mL (0).

trations were expected to encourage tropoelastin association but paradoxically less binding was detected by Biacore. Levels of binding decreased about 3-fold and 9-fold when BRS contained 10 and 20 µg/mL heparin, respectively. Binding between 120 µg/mL tropoelastin in solution and the fixed tropoelastin on the sensor chip surface was at a considerably low level when the heparin concentration was g20 µg/mL (Figure 1A), suggesting that soluble tropoelastin was unavailable due to its titration with soluble heparin. Chondroitin sulfate B elicited a similar titration response but was less effective than heparin at the same concentrations (Figure 1 B). Only 5% of the response units was seen in each case, which was mainly due to the necessarily low concentrations of protein that were injected.15,25,26

Heparan Sulfate has no Effect on the Binding of Soluble Tropoelastin Molecules to Immobilized Tropoelastin. Heparan sulfate has a structure that is closely related to that of heparin. Both types of glycosaminoglycans consist of a variably sulfated repeating disaccharide unit, where the main disaccharide units are almost the same.27 However, heparan sulfate behaved differently to heparin in this study at 25 °C as it did not alter the binding of soluble tropoelastin to the chip surface, even when the concentration was as high as 250 µg/mL (Figure 1 C). Coacervation of Tropoelastin at Low Protein Concentrations in the Presence of Heparin, Heparan Sulfate, and Chondroitin Sulfate B. Time courses of coacervation for tropoelastin at 37 °C in PBS at 30, 60, and 120 µg/mL indicated that tropoelastin did not coacervate. This was anticipated because the critical concentration for coacervation would be above 1 mg/mL under these conditions.17 However, the addition of 250 µg/mL heparin to the same tropoelastin solutions caused an immediate coacervation with a substantial increase in the maximum change in turbidity. Samples containing 120 µg/mL tropoelastin and 250 µg/mL heparin became opaque, giving 1.3 maximum absorbance, while samples containing 30 µg/mL tropoelastin plus 250 µg/mL heparin gave 0.41 maximum absorbance (Figure 2A). Similarly, enhanced coacervation occurred when tropoelastin and heparin were prepared in BRS. The same concentration of heparan sulfate had a remarkably similar effect to heparin. The 30, 60, and 120 µg/mL tropoelastin solutions showed maximum changes in turbidity that were almost identical (Figure 2B). Chondroitin sulfate B had less of an effect than heparin and heparan sulfate but still facilitated coacervation (Figure 2C). At 50 °C, 120 µg/mL tropoelastin solutions reached 50% coacervation more than 8 times faster in the presence of 250 µg/mL heparin (Figure 3). Tropoelastin Coacervation is Modulated by Heparin, Heparan Sulfate, and Chondroitin Sulfate B. Heparin was prepared in PBS and BRS at concentrations ranging from 0 to 250 µg/mL, while tropoelastin was kept constant at 120 µg/ mL. Coacervation time courses were performed over 10 to 70 °C. The maximum change of absorbance at each temperature was recorded and used to express a percentage of the maximum turbidity for each sample, in order to generate a series of coacervation curves. Similar effects were seen in both solutions, where heparin substantially reduced the temperature required for coacervation. Higher concentrations of heparin facilitated coacervation by shifting the entire set of coacervation curves to lower temperatures (Figure 4A). To further explore these effects over time, heparan sulfate and chondroitin sulfate B were prepared in PBS at 5 µg/mL and 10 µg/mL, while tropoelastin was kept constant at 120 µg/ mL. Coacervation time courses were performed over 10 to

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Figure 6. Phase contrast light microscopy of tropoelastin solutions. (A) 120 µg/mL tropoelastin at 30 °C, (B) 120 µg/mL tropoelastin with 20 µg/mL heparin at 30 °C, (C) 10 mg/mL tropoelastin at 30 °C, (D) 10 mg/mL tropoelastin with 20 µg/mL heparin at 30 °C, (E) 10 mg/mL tropoelastin with 250 µg/mL heparin at 30 °C, (F) same as E but focused on the surface of the slide, (G) 10 mg/mL tropoelastin heated rapidly to above 40 °C. The scale bar represents 10 µm in each panel.

Implications for Tropoelastin Microassembly

60 °C. The maximum change of the absorbance at each temperature was recorded and used to express a percentage of the maximum turbidity for each sample to generate a series of coacervation curves. Chondroitin sulfate B had a similar but weaker effect compare to heparin (Figure 4B). Heparan sulfate also could help tropoelastin to coacervate at a concentration as low as 120 µg/mL. However, the coacervation started at a much higher temperature than the experiments that were modulated by heparin and chondroitin sulfate B, and it was also much close to physiological temperature (Figure 4C). The coacervation curve for 10 mg/mL tropoelastin with 250 µg/mL heparin showed an accelerated start and a less pronounced decrease at the end of the experiment than in the absence of heparin (Figure 5). Spherules Appear at Low Concentrations of Tropoelastin during Coacervation in the Presence of Heparin as Evidenced by DIC Microscopy. At 30 °C, 120 µg/mL tropoelastin solutions remained clear (Figure 6A) but in the presence of 20 µg/mL heparin, the tropoelastin solutions showed some ∼6 µm spherules (Figure 6B). In the presence of heparin, small spherules started to appear at 10 °C then coalesced to form bigger droplets at 20 °C, where the diameter of the droplets did not change appreciably between 25 and 40 °C. Spherules Readily Coalesce in the Presence of Heparin at Higher Concentrations of Tropoelastin. Tropoelastin (10 mg/mL) formed spherules that increased in size with increasing temperature, then stabilized at ∼6 µm when the temperature was higher than 30 °C (Figure 6C). A blend of 10 mg/mL tropoelastin and 20 µg/mL heparin showed similar behavior (Figure 6D) where droplets were observed at lower temperatures. At the higher concentration of 250 µg/mL heparin with 10 mg/ mL tropoelastin, there was a mixture of spherules (Figure 6E) and fusing spherules (Figure 6F). An accumulation of tropoelastin spherules on the glass slide led to increasing amounts of fusion until an almost continuous layer of fused spherules formed on the glass slide. In the absence of heparin, when tropoelastin solutions were rapidly (e20 s) shifted from ∼0 to >40 °C, they quickly formed very large (>10 µm) droplets (Figure 6G), but these were unstable and rapidly transitioned to ∼6 µm droplets. The ∼6 µm droplets appear to represent amassed tropoelastin whose collective size is due to a balance of associative and dissociative forces under these conditions.

4. Discussion This study demonstrates that glycosaminoglycans can effectively lower the critical concentration for tropoelastin coacervation to a level where it is no longer detected. This effect was particularly noticeable for heparin, which encouraged the formation of tropoelastin microassemblies, as evidenced by the lower temperature requirement and its ability to kinetically assist by accelerating the process. These effects were more significant than those obtained for chondroitin sulfate B, in agreement with the observations of Mecham and co-workers who showed that cell-surface interactions with tropoelastin were predominantly facilitated through heparan sulfate and to a lesser extent by chondroitin sulfate B.18 Heparin is an accepted chemical model for heparan sulfate’s sulfation that forms the principal recognition mode for heparan sulfate-binding proteins.19 In support of this model, heparan sulfate had a very similar effect to heparin on effecting the coacervation of low concentrations of tropoelastin at 37 °C. Heparin has the highest negative charge density of any known biological molecule. These negative charges can neutralize the

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positive charges brought by lysine and arginine on tropoelastin molecules, which makes it easier for tropoelastin molecules to interact.11,21 Calculations reveal there were approximately equal numbers of positive charges on tropoelastin as there were negative charges on heparin, which is consistent with a cooperative interaction (data not shown). We would not expect salt to have a similar effect as heparin because salt alone would not prevent tropoelastin:tropoelastin charge repulsion at lysines or arginines. Tests repeated in BRS gave similar results to those conducted in PBS. Again chondroitin sulfate B had a weaker effect than heparin. Heparan sulfate also helped coacervation at 120 µg/mL, but the coacervation start temperature was closer to 37 °C. This is why there was no effect of heparan sulfate at 25 °C in Figure 1C. Light microscopy of coacervation showed that heparin promoted the formation of tropoelastin droplets by achieving the effect at temperatures lower than in the absence of heparin. There were four distinct stages of assembly during coacervation. These stages were (1) smaller droplet formation, (2) ∼6 µm droplet formation, (3) fusion of droplets, and then (4) the formation of a coalesced layer. Initially, tropoelastin monomers formed tropoelastin droplets that were smaller than 6 µm, and then these small droplets appeared to recruit tropoelastin from solution to grow into larger, terminal (∼6 µm) droplets similar to those seen in classical coacervates. The droplets were unable to further coalesce in suspension. However, these ∼6 µm droplets showed they have the capacity to merge after settling from suspension onto the glass slide. Heparin decreased the time taken to form the ∼6 µm droplets. These droplets and those in the absence of heparin eventually merged after settling on the glass slide to form amorphously shaped protein aggregates. More tropoelastin droplets fused over time, until a layer of coalesced tropoelastin was formed. These findings may be related to in vivo elastin assembly, where there is minimal tropoelastin available at the cell surface at any one time that could participate in the formation of elastic fibers. The presence of biologically available glycosaminoglycans could aid this process by assisting in the initial step of coacervation, even at very low protein concentrations.

5. Conclusion Heparin, heparan sulfate, and chondroitin sulfate B abolished the critical concentration of coacervation for tropoelastin. Multiple stages of coacervation were identified. Tropoelastin assembly in the presence of heparin resulted in the formation of protein droplets. These droplets eventually merged to give an almost continuous layer of coacervated tropoelastin.

References and Notes (1) Mithieux, S. M.; Weiss, A. S. Elastin. AdV. Protein Chem. 2005, 70, 437–61. (2) Vrhovski, B.; Weiss, A. S. Biochemistry of tropoelastin. Eur. J. Biochem. 1998, 258 (1), 1–18. (3) Pasquali-Ronchetti, I.; Baccarani-Contri, M. Elastic fiber during development and aging. Microsc. Res. Tech. 1997, 38 (4), 428–35. (4) Pasquali-Ronchetti, I.; Fornieri, C.; Baccarani-Contri, M.; Quaglino, D. Ultrastructure of elastin. Ciba Found Symp. 1995, 192, 31–42; discussion 42-50. (5) Keeley, F. W.; Bellingham, C. M.; Woodhouse, K. A. Elastin as a self-organizing biomaterial: Use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and selfassembly of elastin. Philos. Trans. R. Soc.London, Ser. B 2002, 357 (1418), 185–9. (6) Urry, D. W. Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces. J. Protein Chem. 1988, 7 (2), 81–114.

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(7) Kagan, H. M.; Sullivan, K. A. Lysyl oxidase: Preparation and role in elastin biosynthesis. Methods Enzymol. 1982, 82, 637–50. (8) Kagan, H. M.; Cai, P. Isolation of active site peptides of lysyl oxidase. Methods Enzymol. 1995, 258, 122–32. (9) Kozel, B. A.; Rongish, B. J.; Czirok, A.; Zach, J.; Little, C. D.; Davis, E. C.; Knutsen, R. H.; Wagenseil, J. E.; Levy, M. A.; Mecham, R. P. Elastic fiber formation: A dynamic view of extracellular matrix assembly using timer reporters. J. Cell. Physiol. 2006, 207 (1), 87– 96. (10) Urry, D. W.; Starcher, B.; Partridge, S. M. Coacervation of solubilized elastin effects a notable conformational change. Nature 1969, 222 (5195), 795–6. (11) Vrhovski, B.; Jensen, S.; Weiss, A. S. Coacervation characteristics of recombinant human tropoelastin. Eur. J. Biochem. 1997, 250 (1), 92–8. (12) Toonkool, P.; Jensen, S. A.; Maxwell, A. L.; Weiss, A. S. Hydrophobic domains of human tropoelastin interact in a context-dependent manner. J. Biol. Chem. 2001, 276 (48), 44575–80. (13) Cox, B. A.; Starcher, B. C.; Urry, D. W. Communication: Coacervation of tropoelastin results in fiber formation. J. Biol. Chem. 1974, 249 (3), 997–8. (14) Urry, D. W.; Long, M. M. On the conformation, coacervation and function of polymeric models of elastin. AdV. Exp. Med. Biol. 1977, 79, 685–714. (15) Clarke, A. W.; Wise, S. G.; Cain, S. A.; Kielty, C. M.; Weiss, A. S. Coacervation is promoted by molecular interactions between the PF2 segment of fibrillin-1 and the domain 4 region of tropoelastin. Biochemistry 2005, 44 (30), 10271–81. (16) Clarke, A. W.; Arnspang, E. C.; Mithieux, S. M.; Korkmaz, E.; Braet, F.; Weiss, A. S. Tropoelastin massively associates during coacervation to form quantized protein spheres. Biochemistry 2006, 45 (33), 9989– 96. (17) Toonkool, P.; Regan, D. G.; Kuchel, P. W.; Morris, M. B.; Weiss, A. S. Thermodynamic and hydrodynamic properties of human tropoelastin. Analytical ultracentrifuge and pulsed field-gradient spinecho NMR studies. J. Biol. Chem. 2001, 276 (30), 28042–50. (18) Broekelmann, T. J.; Kozel, B. A.; Ishibashi, H.; Werneck, C. C.; Keeley, F. W.; Zhang, L.; Mecham, R. P. Tropoelastin interacts with

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cell-surface glycosaminoglycans via its COOH-terminal domain. J. Biol. Chem. 2005, 280 (49), 40939–47. Cain, S. A.; Baldock, C.; Gallagher, J.; Morgan, A.; Bax, D. V.; Weiss, A. S.; Shuttleworth, C. A.; Kielty, C. M. Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly. J. Biol. Chem. 2005, 280 (34), 30526–37. Gheduzzi, D.; Guerra, D.; Bochicchio, B.; Pepe, A.; Tamburro, A. M.; Quaglino, D.; Mithieux, S.; Weiss, A. S.; Pasquali Ronchetti, I. Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers. Matrix Biol. 2005, 24 (1), 15–25. Wu, W. J.; Vrhovski, B.; Weiss, A. S. Glycosaminoglycans mediate the coacervation of human tropoelastin through dominant charge interactions involving lysine side chains. J. Biol. Chem. 1999, 274 (31), 21719–24. Martin, S. L.; Vrhovski, B.; Weiss, A. S. Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin. Gene 1995, 154 (2), 159–66. Keane, F. M.; Clarke, A. W.; Foster, T. J.; Weiss, A. S. The N-terminal A domain of Staphylococcus aureus fibronectin-binding protein A binds to tropoelastin. Biochemistry 2007, 46 (24), 7226–32. Tu, Y.; Mithieux, S. M.; Clarke, A. W.; Weiss, A. S., Tropoelastin initially assembles through head-to-tail interactions as a prelude to coacervation, 2008, manuscript in preparation. Varadarajan, R.; Sharma, D.; Chakraborty, K.; Patel, M.; Citron, M.; Sinha, P.; Yadav, R.; Rashid, U.; Kennedy, S.; Eckert, D.; Geleziunas, R.; Bramhill, D.; Schleif, W.; Liang, X.; Shiver, J. Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120M9: Implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design. J. Virol. 2005, 79 (3), 1713–23. Du, L.; Zhang, Z.; Luo, X.; Chen, K.; Shen, X.; Jiang, H. Binding investigation of human 5-lipoxygenase with its inhibitors by SPR technology correlating with molecular docking simulation. J. Biochem. (Tokyo) 2006, 139 (4), 715–23. Gallagher, J. T.; Walker, A. Molecular distinctions between heparan sulphate and heparin: Analysis of sulphation patterns indicates heparan sulphate and heparin are separate families of N-sulphated polysaccharides. Biochem. J. 1985, 230 (3), 665–674.

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