A molecular mechanism for the heparan sulfate dependence of slit-robo signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.     

Heparin stimulation of plasminogen activator secretion by macrophage-like cell line RAW264.7: role of the scavenger receptor

Secretion of urokinase-type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose- and time-dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4 degrees C, indicating that heparin was not simply releasing receptor-bound uPA. Furthermore, prior removal of membrane-associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage-like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl-LDL (Falcone and Ferenc: J. Cell. Physiol., 135:387-396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]-heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]-oleate synthesis observed in these cells upon incubation with acetyl-LDL and weakly inhibited cellular binding of 125I-acetyl-LDL at 4 degrees C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte-like U937 cells which are devoid of scavenger receptor activity.     

低分子肝素的抗炎作用及机制

低分子肝素(low molecular weight heparin, LMWH)除作为抗凝血和抗血栓药在临床上广为应用外,近年来其抗炎活性也颇受重视.LMWH抗炎机制涉及炎症细胞、炎症因子和黏附分子等环节.目前对LMWH的抗炎机制研究还处在初级阶段,但是LMWH独特的性质使其有望成为有效且安全的新型抗炎药物.     

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparin's potentiation of aFGF activity observed in biological assay systems.     

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.     

Structure and activity of a unique heparin-derived hexasaccharide

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.     

Characterization of Slit protein interactions with glypican-1

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.     

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation

Heparin has a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell proliferation. To determine the minimum size of the heparin glycosaminoglycan chain essential for antiproliferative activity, porcine intestinal mucosal heparin was partially depolymerized with heparinase and fractionated to give oligosaccharides of different sizes. The structure of these oligosaccharides was fully characterized by 1D and 2D 1H NMR spectroscopy. These oligosaccharides were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells (PASMCs). The tetrasaccharide (4-mer) exhibited no heparin-like activity. Decasaccharides (10-mers) and dodecasaccharides (12-mers) displayed a reduced level of activity when compared to full-length heparin. Little effect on activity was observed in deca- and dodecasaccharides with one less 2-O-sulfo group. The 14-, 16-, and 18-mers showed comparable growth-inhibition effects on PAMSC as porcine intestinal mucosal heparin. These data suggest that a 14-mer is the minimum size of oligosaccharide that is essential for full heparin-like antiproliferative activity. Since the 14- to 18-mers have no 3-O-sulfo groups in their glucosamine residues, their full activity confirms that these 3-O-sulfonated glucosamine residues, which are required for heparin's anticoagulant activity, are not an essential requirement for antiproliferative activity.     

A botanical medicine dragon’s blood exhibited clinical antithrombosis efficacy similar to low molecular weight heparin

Deep vein thrombosis(DVT) is a common complication following traumatic fracture with a 0.5%–1% annual incidence. Low molecular weight heparin(LMWH) is the most commonly used anticoagulation drug for DVT prevention, but treatment with LMWH is invasive. Our aim is to compare the antithrombotic effect of dragon's blood, an oral botanical anticoagulant medicine approved by the Chinese FDA, with LMWH in patients undergoing hip fracture surgery and to explore the molecular mechanisms of anticoagulation treatment. Our study recruited patients and divided them into LMWH and dragon's blood treatment group. Coagulation index tests, Doppler ultrasound and m RNA sequencing were performed before and after anticoagulation therapy. There was no significant difference in postoperative DVT incidence between the two groups(23.1% versus 15.4%,P=0.694). D-dimer(D-D) and fibrinogen degradation product(FDP) showed significant reductions in both groups after anticoagulation treatments. We identified SLC4 A1, PROS1, PRKAR2 B and seven other genes as being differentially expressed during anticoagulation therapy in both groups. Genes correlated with coagulation indexes were also identified. Dragon's blood and LMWH showed similar effects on DVT and produced similar gene expression changes in patients undergoing hip fracture surgery, indicating that dragon's blood is a more convenient antithrombosis medicine(oral) than LMWH(hypodermic injection).     

Synthesis of heparin partial structures and their binding activities to platelets

A synthetic pentasaccharide corresponding to the antithrombin III-binding region in heparin was also found to bind to human platelets. To identify the platelet-binding site in the pentasaccharide which is expected to be a novel sequence in heparin responsible for its platelet-binding, five partial structures of this particular pentasaccharide were synthesized. In a competitive assay using [3H]-heparin, a trisaccharide, O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glucopyranosyl)-1--> 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranose, was concluded to be a high-affinity site for heparin's binding to platelets.     

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.     

Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with alpha and beta integrin subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.     

Heparin reduces the alpha-helical content of cobra basic phospholipase A(2) and promotes its complex formation

The interaction of phospholipase A(2) (PLA(2)) with glycosaminoglycans (GAGs) has recently attracted attention in view of its implication on inflammation and cell proliferation. By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate here that binding of cobra basic phospholipase A(2) from Naja nigricollis (N-PLA(2)) to heparin may induce a significant conformational change observed in the amide I region of the enzyme's alpha-helical and beta-sheet structure. It is observed that notable conformational change of N-PLA(2) due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by circular dichroism and optical density measurements. This correlation may be an important factor in the aggregation of N-PLA(2) and N-PLA(2)-heparin complexes. Heparin induced change in conformation of PLA(2) is suggested to be a notable link in understanding the diversity in PLA(2) activity when rendered to the extracellular matrix of cell membranes that is full of GAG molecules.     

Transforming growth factor-beta 1 is a heparin-binding protein: identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-beta 1.

Previous studies indicated that a major factor in heparin's ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor-beta 1 (TGF-beta 1). Heparin appeared to bind directly to TGF-beta 1 and to prevent the association of TGF-beta 1 with alpha 2-macroglobulin (alpha 2-M). The present studies indicate that 20-70% of iodinated TGF-beta 1 binds to heparin-Sepharose and the retained fraction is eluted with approximately 0.37 M NaCl. Native, unlabelled platelet TGF-beta 1, however, is completely retained by heparin-Sepharose and eluted with 0.9-1.2 M NaCl. Using synthetic peptides, the regions of TGF-beta 1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF-beta 1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin-binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF-beta 1, were found to bind to heparin-Sepharose and were eluted with 0.15 M-0.30 M NaCl. Only two of these regions were capable of blocking the binding of heparin to 125I-TGF-beta. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF-beta 1. The ability of TGF-beta 1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF-beta 1 may be a factor in some of the diverse biological actions of heparin.     

Effects of heparin and derivatives on podocytes: An in vitro functional and morphological evaluation

Unfractionated heparin (UFH) and low molecular heparin derivatives (LMWH) display numerous biological properties in addition to their anticoagulant effects. However, due to the physicochemical heterogeneity of these drugs, a better understanding concerning their effects on human cells is clearly needed. Considering that heparins are mainly excreted by the kidney, we focused our attention on the effect of UFH and LMWH on human podocytes by functional and morphological/phenotypic in vitro analyses. We demonstrated that these products differentially modulate the permeability of podocyte monolayer to albumin. The functional perturbations observed were correlated to significant cellular morphological and cytoskeletal changes, as well as a decrease in the expression of proteins involved in podocyte adherence to the extracellular matrix or intercellular interactions. This point confirms that UFH and the different LMWHs exert specific effects on podocyte permeability and underlines the need of in vitro tests to evaluate new biological nonanticoagulant properties of LMWH.     

Mathematical simulation of the blood coagulation system

The Blood coagulation system was converted to a mathematical model which was described using differential equations. The calculated output patterns of the mathematical model against various input stimulations were compared with results of in vitro assay. The simulated results of (1) Hemophilia A, (2) anticoagulation effect of antithrombin III and (3) anticoagulation effect of heparin corresponded to the results of in vitro assay and clinical reports. However, the simulated result of (4) anticoagulation effect of synthesized arginine derivative No. 805 (MD-805) did not correspond to the results of in vitro assay. Therefore, a new series simulation of MD-805 was done, supposing that MD-805 had an inhibitory activity not only on coagulation factor IIa but also VIIa. The new simulation pattern closely resembled the results of in vitro assay. From these facts, it was theoretically indicated that MD-805 also has an inhibitory activity on VIIa.     

Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties

Structural features of heparin potentially important for heparanase-inhibitoryactivity were examined by measuring the ability of heparin derivativesto affect the degradation of [³H]acetylated heparan sulphateby tumor cell heparanases. IC₅₀ values were determined usingan assay which distinguished degraded from undegraded substrateby precipitation of the latter with cetylpyridinium chloride(CPC). Removal of heparin's 2-O-sulphate and 3-O-sul-phate groupsenhanced heparanase-inhibitory activity (50%). Removal of itscarboxyl groups slightly lowered the activity (18%), while combiningthe treatments abolished the activity. At least one negativecharge on the iduronic acid/idose moiety, therefore, is necessaryfor heparanase-inhibitory activity. Replacing heparin's N-sulphategroups with N-acetyl groups reduced its activity (37%). Comparingthis heparin derivative with 2,3-O-de-sulphated heparin, theplacement of sulphate groups appears important for activitysince the two structures have similar nominal linear chargedensity. In addition, unsubstituted uronic acids are nonessentialfor inhibition since their modification (periodate-oxidation/borohydride-reduction)enhanced rather than reduced heparanase-inhibitory activity.The most effective heparanase inhibitors (2,3-O-desulphatedheparin, and [periodate-oxidized, borohydride-reduced] heparin)were tested in the chick chorioallantoic membrane (CAM) bioassayfor anti-angiogenic activity and found to be at least as efficaciousas heparin. 2,3-O-desulphated heparin also significantly decreasedthe tumor growth of a subcutaneous human pancreatic (Ca-Pan-2)adenocarcinoma in nude mice and prolonged the survival timesof C57BL/6N mice in a B16-F10 melanoma experimental lung metastasisassay. angiogenesis chemically-modified heparins endoglycosidase hepara sulphate cancer