Unmodified Heparin Research Articles

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.

Chemoenzymatic synthesis of unmodified heparin oligosaccharides: cleavage of p-nitrophenyl glucuronide by alkaline and Smith degradation

Chemoenzymatic synthesis of unmodified heparin oligosaccharides: cleavage of p-nitrophenyl glucuronide by alkaline and Smith degradation

Chemoenzymatic synthesis of unmodified heparin oligosaccharides: cleavage of p-nitrophenyl glucuronide by alkaline and Smith degradation.

A heparin oligosaccharide having a completely natural structure was successfully synthesized through a chemoenzymatic approach using an unnatural glycosyl acceptor, p-nitrophenyl glucuronide (GlcA-pNP). The use of an inexpensive and commercially available GlcA-pNP acceptor facilitates oligosaccharide recovery and purification on C-18 resin during chemoenzymatic synthesis. Oligosaccharide chain extension and modification afforded a heptasaccharide with gluconic acid residues at its reducing and non-reducing ends. Treatment with periodate oxidation followed by Smith degradation or alkaline elimination resulted in the selective cleavage of vicinal diol-containing glucuronic acid residues affording highly sulfated heparin pentasaccharides having a completely natural structure. This methodology should facilitate the chemoenzymatic synthesis of a family of highly sulfated heparin oligosaccharides with unmodified structures for biological evaluation.

Multitasking Human Lectin Galectin-3 Interacts with Sulfated Glycosaminoglycans and Chondroitin Sulfate Proteoglycans.

Glycosaminoglycan (GAG) binding proteins (GAGBPs), including growth factors, cytokines, morphogens, and extracellular matrix proteins, interact with both free GAGs and those covalently linked to proteoglycans. Such interactions modulate a variety of cellular and extracellular events, such as cell growth, metastasis, morphogenesis, neural development, and inflammation. GAGBPs are structurally and evolutionarily unrelated proteins that typically recognize internal sequences of sulfated GAGs. GAGBPs are distinct from the other major group of glycan binding proteins, lectins. The multifunctional human galectin-3 (Gal-3) is a β-galactoside binding lectin that preferentially binds to N-acetyllactosamine moieties on glycoconjugates. Here, we demonstrate through microcalorimetric and spectroscopic data that Gal-3 possesses the characteristics of a GAGBP. Gal-3 interacts with unmodified heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). While heparin, CSA, and CSC bind with micromolar affinity, the affinity of CSPGs is nanomolar. Significantly, CSA, CSC, and a bovine CSPG were engaged in multivalent binding with Gal-3 and formed noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs was completely abolished when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was reversed by β-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Hill plot analysis of calorimetric data reveals that the binding of CSA, CSC, and a bovine CSPG to Gal-3 is associated with progressive negative cooperativity effects. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans.

Quantification of aldehyde terminated heparin by SEC-MALLS–UV for the surface functionalization of polycaprolactone biomaterials

A straight forward strategy of heparin surface grafting employs a terminal reactive-aldehyde group introduced through nitrous acid depolymerization. An advanced method that allows simultaneously monitoring of both heparin molar mass and monomer/aldehyde ratio by size exclusion chromatography, multi-angle laser light scattering and UV-absorbance (SEC-MALLS–UV) has been developed to improve upon heparin surface grafting. Advancements over older methods allow quantitative characterization by direct (aldehyde absorbance) and indirect (Schiff-based absorbance) evaluation of terminal functional aldehydes. The indirect quantitation of functional aldehydes through labeling with aniline (and the formation of a Schiff-base) allows independent quantitation of both polymer mass and terminal functional groups with the applicable UV mass extinction coefficients determined. The protocol was subsequently used to synthesize an optimized heparin-aldehyde that had minimal polydispersity (PDI<2) and high reaction yields (yield >60% by mass). The 8kDa weight averaged molar mass heparin-aldehyde was then grafted on polycaprolactone (PCL), a common implant material. This optimized heparin-aldehyde retained its antithrombin activity, assessed in freshly drawn blood or surface immobilized on PCL films. Anticoagulant activity was equal to or better than the 24kDa unmodified heparin it was fragmented from.

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase high-performance liquid chromatography (IPRP–HPLC) coupled with electrospray ionization mass spectrometry (ESI–MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains.

How to handle low-molecular-weight heparins in patients with decreased renal function: an open issue

Chronic renal failure is a leading cause of morbidity and mortality in Western countries [1]. The prevalence of patients with end stage renal disease (ESRD) is growing constantly, and ever more frequently require physicians of several specialties to manage these patients with reduced renal function. An example of this event is the prevention and treatment of venous thromboembolism (VTE), acute deep vein thrombosis (DVT) or pulmonary embolism (PE) with heparins. Heparins are the most effective treatment for the thromboembolic disorders, but their management requires careful usage in patients with reduced glomerular filtration rate (GFR). Heparin is an indirect thrombin inhibitor that forms complexes with antithrombin (AT, formerly known as AT III), and converts this circulating cofactor from a slow to a rapid inactivator of thrombin, factor Xa, and to a lesser extent, factors XIIa, XIa, and IXa [2, 3]. The binding of heparin to the heparin binding site on AT produces a conformational change in AT that accelerates the inactivating process by 1,000to 4,000-fold [4]. The type of unfractionated (UF) heparin in current clinical use is polydispersed unmodified heparin, with molecular weights ranging from 3,000 to 30,000 Da and a mean molecular weight of approximately 15,000. Low molecular weight (LMW) derivatives of commercial heparin have been obtained. They have a molecular weight range from 2,000 to 9,000 Da, and a mean molecular weight of 4,000–5,000 [5]. Like UF heparins, LMW heparins inactivate factor Xa, but have a lesser effect on thrombin, because most of the molecules do not contain enough saccharide units to bind to form the quaternary complex in which thrombin and AT are bound simultaneously [5]. As a result, LMW heparins do not prolong the aPTT. LMW heparins are at least as effective as unfractionated heparin for the treatment and prevention of venous thromboembolism [5]. LMW heparins also have a number of advantages over UF heparins [6]. They have greater bioavailability when given by subcutaneous injection, and their anticoagulant effect is longer than UF heparins because of reduced binding to macrophages and endothelial cells, permitting once or twice daily administration. The anticoagulant response (anti-Xa activity) to LMW heparin is highly correlated with body weight, permitting administration of a fixed dose. Nevertheless, the dose may have to be adjusted for patients who are extremely obese or have renal failure [2]. LMW heparins are much less likely to induce immune-mediated thrombocytopenia than UF heparins (0 vs. 2.7% in one study) [7] and they do not increase osteoclast number and activity as much as UF heparins, thus producing less bone loss [8, 9]. All these features make LMW heparin suitable for safe administration in the outpatient setting and particularly in renal patients. Two Cochrane reviews have explored the benefits of LMW heparin over UF heparin, and conclude that in patients with acute VTE, fixed-dose subcutaneous LMW heparin is more effective than adjusted-dose of UF heparin for reducing the incidence of symptomatic recurrent VTE, major hemorrhage, and all-cause mortality [10]. Moreover, home management using LMW heparin is cost effective, G. S. Netti L. Gesualdo (&) Department of Biomedical Sciences, Section of Nephrology, University of Foggia, Viale Luigi Pinto, 1, 71100 Foggia, Italy e-mail: l.gesualdo@unifg.it

Antiangiogenic and Apoptotic Properties of a Novel Amphiphilic Folate-Heparin-Lithocholate Derivative Having Cellular Internality for Cancer Therapy

Anitangiogenic and apoptotic properties of a novel chemically modified heparin derivative with low anticoagulant activity were evaluated on the experimental in vitro and in vivo model. Heparin-lithocholate conjugate (HL) was initially synthesized by covalently bonding lithocholate to heparin. Folate-HL conjugate (FHL) was further synthesized by conjugating folate to HL. Antiangiogenic and apoptotic abilities of HL and FHL were characterized in vitro and in vivo experimentations. Compared to unmodified heparin, both HL and FHL represented a low anticoagulant activity (38 and 28%, respectively). HL and FHL maintained antiangiogenic activity even further modification from the results of Matrigel plugs assay. FHL specifically induced apoptosis on KB cells having highly expressed folate receptor after cellular internalization. Both administered HL and FHL had similar antiangiogenic activity and inhibitory effect on tumor growth in vivo although FHL induced higher apoptosis on tumor tissues. In vivo tumor growth inhibition was possibly due to the decrease of vessel density and apoptotic cell death, although antiangiogenic effect of FHL seemed more actively affected on growth inhibition than apoptotic potential in vivo system. Thus, Low anticoagulant FHL having antiangiogenic and apoptotic properties would provide benefits for the development of a new class of anticancer agent.

N- and 6-O-Sulfated Heparan Sulfates Mediate Internalization of Coxsackievirus B3 Variant PD into CHO-K1 Cells

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO(3)(6OSO(3))](n), but not basic fibroblast growth factor binding to [HexUA-GlcNSO(3)-HexUA-GlcNSO(3)-IdUA(2OSO(3))](n), was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO(3)(6OSO(3))](n) is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.

Diversity-oriented chemical modification of heparin: Identification of charge-reduced N-acyl heparin derivatives having increased selectivity for heparin-binding proteins

The diversity-oriented chemical modification of heparin is shown to afford charge-reduced heparin derivatives that possess increased selectivity for binding heparin-binding proteins. Variable N-desulfonation of heparin was employed to afford heparin fractions possessing varied levels of free amine. These N-desulfonated heparin fractions were selectively N-acylated with structurally diverse carboxylic acids using a parallel synthesis protocol to generate a library of 133 heparin-derived structures. Screening library members to compare affinity for heparin-binding proteins revealed unique heparin-derived structures possessing increased affinity and selectivity for individual heparin-binding proteins. Moreover, N-sulfo groups in heparin previously shown to be required for heparin to bind specific proteins have been replaced with structurally diverse non-anionic moieties to afford identification of charge-reduced heparin derivatives that bind these proteins with equivalent or increased affinity compared to unmodified heparin. The methods described here outline a process that we feel will be applicable to the systematic chemical modification of natural polyanionic polysaccharides and the preparation of synthetic oligosaccharides to identify charge-reduced high affinity ligands for heparin-binding proteins.

Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin, respectively

Pasteurella multocida is a pathogenic Gram-negative bacterial species that infects a wide variety of animals and humans. A notable morphological feature of many isolates is the extracellular capsule. The ability to remove the capsule by treatment with certain glycosidases has been utilized to discern various capsular types called A, D and F. Based on this preliminary evidence, these microbes have capsules made of glycosaminoglycans, linear polysaccharides composed of repeating disaccharide units containing an amino sugar. Glycosaminoglycans are also abundant components of the vertebrate extracellular matrix. It has been shown previously that the major Type A capsular material was hyaluronan (hyaluronic acid). We report that the Type D polymer is an unmodified heparin ( N-acetylheparosan) with a →4)-β- d-Glc p-UA-(1→4)-α- d-Glc p-NAc-(1→ repeating unit and the Type F polymer is an unmodified chondroitin with a →4)-β- d-Glc p-UA-(1→3)-β- d-Gal p-NAc-(1→ repeating unit. The monosaccharide compositions, disaccharide profiles, and 1H NMR analyses are consistent with these identifications. The molecular size of the Pasteurella polymers is approximately 100–300 kDa as determined by gel electrophoresis and multi-angle laser light scattering; this size is much greater than the 10–30 kDa size of the analogous polymers isolated from animal tissues. The glycosaminoglycan capsular polymers are relatively non-immunogenic virulence factors that enhance microbial pathogenicity.

Local delivery of basic fibroblast growth factor (bFGF) using adsorbed silyl-heparin, benzyl-bis(dimethylsilylmethyl)oxycarbamoyl-heparin.

A growth factor delivery system was developed that is based on the use of silyl-heparin, a chemically modified analogue of heparin. The silyl-heparin was adsorbed onto surfaces by hydrophobic interaction via the prosthetic unit and can then be used as a solid-phase adsorbent for bFGF. All the coating steps were performed by adsorption, a process that allowed preparation of surfaces by immersion or "dip-coating". In this study a series of silyl-heparins were synthesized and each of the analogues found to function similar to unmodified heparin relative to their binding of antithrombin III and also the binding of bFGF. The silyl-heparins were found to be adsorbed onto a wide variety of substrates including polystyrene and lactide:glycolide copolymer. Enzyme-linked immumosorbant assay (ELISA) was used to establish that bFGF was readily bound to surface adsorbed silyl-heparin, and that the amount bound was directly related to amount offered for binding. Once adsorbed the silyl-heparin/FGF was able to induce capillary tube formation of endothelial cells and to increase the growth of endothelial cells. When coated onto suture material and implanted in muscle, the FGF/silyl-heparin coating caused an increased density of mesenchymal cells in the area of the implant. This coating method could prove to be useful in a number of tissue engineering applications for the local delivery of FGF and other growth factors.

Heparin and heparan sulfate bind interleukin-10 and modulate its activity

AbstractGlycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-β, IL-7, and interferon-γ. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin–agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, Kd, of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10–induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC50 values of 100 to 500 μg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC50 of 2000 to 5000 μg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10– induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10–mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity.

Heparin and heparan sulfate bind interleukin-10 and modulate its activity

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-β, IL-7, and interferon-γ. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin–agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, Kd, of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10–induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC50 values of 100 to 500 μg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC50 of 2000 to 5000 μg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10– induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10–mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity.

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [ 125I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α vβ 3 or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

AbstractStem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1α (MIP-1α), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1α and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Antithrombotic agents stimulate the synthesis and modify the sulfation pattern of a heparan sulfate proteoglycan from endothelial cells

Low molecular weight heparins, namely CY 216 and CY 222 (Sanofi/Choay); OP 622 and OP 386 (Opocrin); PK 10169 (Pharmuka); an oligosaccharide prepared from heparin by heparitinase II digestion; chemically sulfated glycosaminoglycans and polysaccharide namely Suleparoid (Syntex), Aprosulate (Luitpold-Werk); chemically modified glycosaminoglycans GAGPS and MPS (Luitpold-Werk) as well as unmodified heparin stimulate two to three fold the synthesis of a heparan sulfate with antithrombotic activity secreted by endothelial cells in culture. The stimulation is concentration dependent and specific for the endothelial cell. The [ 35S]-heparan sulfate synthesized in the presence of heparin and/ or the tested antithrombotic agents has shown a high degree of sulfation of the iduronic acid residues as revealed by the analyses of the disaccharide products formed from the heparan sulfate by the action of bacterial heparitinases. The features of the above compounds in common with heparin are their polymeric nature and a high charge density, as well as their pharmacological activities as potent antithrombotic agents “in vivo”. These combined observations reinforce the proposition that the antithrombotic activity of heparin, low molecular weight heparins and the chemically modified polysaccharides could be related to the increased production of this peculiar heparan sulfate by endothelial cells.

Immobilization of a lysine-terminated heparin to polyvinyl alcohol.

Lysine terminated heparin, prepared by the nitrous acid partial depolymerization and reductive amination of heparin, failed to increase the active heparin content of a heparin-polyvinyl alcohol (heparin-PVA) hydrogel relative to the unmodified commercial heparin. The depolymerization of heparin resulted in a loss of biological activity which outweighed the increase in the terminal amine groups (produced by reductive amination), that were used for glutaraldehyde immobilization to the PVA. The loss in anti-thrombin activity (thrombin time or chromogenic substrate) paralleled the increase in anhydromannose end groups due to depolymerization making it necessary to optimize the loss of activity against the increase in terminal amine groups after amination. For example, depolymerization at a high sodium nitrite concentration (81 g/l) at pH4 and 25 degrees C for 20 min, resulted in a loss of 22-40% of the biological activity but achieved an anhydromannose content of 600 nmoles/mg (approximately 7 cleavage sites/molecule). After the anhydromannose groups were reductively aminated by lysine, the anhydromannose content was reduced to 190 nmol/mg indicating a terminal lysine content of 410 nmol/mg. This resulted in an increase in heparin content of the final hydrogel by 53% on mass terms. However, given the reduction in biological activity, it was not surprising that the modified heparin-PVA hydrogel coated on a polyethylene tube was no better than the hydrogel with unmodified heparin in inactivating thrombin in a flow circuit. These results point out the need for care in interpreting heparin immobilization results and for new strategies to increase the active heparin content of this hydrogel.

Structural determinants of the capacity of heparin to inhibit collagen synthesis in 21-day fetal rat calvariae

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700-5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665-3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.