Heparan Sulfate Mimetics Differentially Affect Homologous Chemokines and Attenuate Cancer Development.

Achieving selective inhibition of chemokine activity by structurally well-defined heparan sulfate (HS) or HS mimetic molecules can provide important insights into their roles in individual physiological and pathological cellular processes. Here, we report a novel tailor-made HS mimetic, which furnishes an exclusive iduronic acid (IdoA) scaffold with different sulfation patterns and oligosaccharide chain lengths as potential ligands to target chemokines. Notably, highly sulfated-IdoA tetrasaccharide (I-45) exhibited strong binding to CCL2 chemokine thereby blocking CCL2/CCR2-mediated in vitro cancer cell invasion and metastasis. Taken together, IdoA-based HS mimetics offer an alternative HS substrate to generate selective and efficient inhibitors for chemokines and pave the way to a wide range of new therapeutic applications in cancer biology and immunology.

Conformational Modulation of Iduronic Acid-Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs.

1 H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

A non-anticoagulant heparin-like snail glycosaminoglycan promotes healing of diabetic wound.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

Synthesis of Heparan Sulfate- and Dermatan Sulfate-Related Oligosaccharides via Iterative Chemoselective Glycosylation Exploiting Conformationally Disarmed [2.2.2] l-Iduronic Lactone Thioglycosides.

Heparan sulfate (HS) and dermatan sulfate (DS) are l-iduronic acid containing glycosaminoglycans (GAGs) which are implicated in a number of biological processes and conditions including cancer and viral infection. Chemical synthesis of HS and DS is required to generate structurally defined oligosaccharides for a biological study. Herein, we present a new synthetic approach to HS and DS oligosaccharides using chemoselective glycosylation which relies on a disarmed [2.2.2] l-ido lactone motif. The strategy provides a general approach for iterative-reducing end chain extension, using only shelf-stable thioglycoside building blocks, exploiting a conformational switch to control reactivity, and thus requires no anomeric manipulation steps between glycosylations.

Susceptibility of enoxaparin reducing end amino sugars to periodate oxidation.

There is a growing interest on glycol-split low-molecular weight heparins (gs-LMWHs), obtained by periodate oxidation of LMWHs, optionally followed by borohydride reduction, as potential anticancer and anti-inflammatory drugs. However, their structural characterization is still a challenging task, mainly because of the high microheterogeneity of the starting material. In addition, susceptibility to oxidation of some end-groups of LMWHs induces additional heterogeneity, making analysis of gs-LMWHs more complex. In our previous study we showed that 1,6-anhydro-d-mannosamine N-sulfate was affected by periodate, while its epimer 1,6-anhydro-d-glucosamine N-sulfate was resistant. In order to understand the apparently anomalous behavior of terminal 1,6-anhydro-d-mannosamine N-sulfate residues, in the present work we have studied by NMR spectroscopy and LC/MS the behavior of the reducing end amino sugar residues of the tetrasaccharides, isolated from the LMWH enoxaparin, in the presence of periodate. Their molecular mechanics conformational characterization has been also performed. We have shown that the C(2)-C(3) bond of the 1,6-anhydro-d-mannosamine residue can be split by periodate despite the N-substitution. Moreover, we have found that both terminal d-mannosamine N-sulfate and d-glucosamine N-sulfate, lacking the 1,6-anhydro-bridge, can be also oxidized by periodate but with a significantly lower rate. The present results suggest that the cis-e-/a-position of OH and NHSO3(-) groups of N-sulfated 1,6-anhydro-d-mannosamine is not the only factor that makes these end residues susceptible to the oxidation. The 1,6-anhydro-bridge that 'blocks' the ring conformation appears another crucial factor for oxidation to occur. Moreover, we have shown that controlling the reaction time could permit to selectively split non-sulfated iduronic acids of enoxaparin chains without oxidizing terminal amino sugar residues, a finding that may be useful to obtain more structurally homogeneous gs-LMHWs.

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments.

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

Interfering with UDP-GlcNAc metabolism and heparan sulfate expression using a sugar analogue reduces angiogenesis.

Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analogue of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analogue was activated intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited up to ∼96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analogue, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors such as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Microinjection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All of these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis, which hampers pro-angiogenic signaling and neo-vessel formation.

Iduronic acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells.

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.

Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family.

Ulvans are complex sulfated polysaccharides found in the cell walls of green algae belonging to the genus Ulva. These polysaccharides are composed of disaccharide repetition moieties made up of sulfated rhamnose linked to either glucuronic acid, iduronic acid, or xylose. Two ulvan lyases of 30 and 46 kDa were purified from the culture supernatant of Persicivirga ulvanivorans. Based on peptide sequencing, the gene encoding the 46-kDa ulvan lyase was cloned. Sequence analysis revealed that the protein is modular and possesses a catalytic module similar to that of the 30-kDa ulvan lyase along with a module of unknown function. The ulvan-degrading function of the gene was confirmed by expression of the catalytic module in a heterologous system. The gene encoding the catalytic module has no sequence homolog in sequence databases and is likely to be the first member of a novel polysaccharide lyase family. Analysis of degradation products showed that both the 30- and 46-kDa ulvan lyases are endolytic and cleave the glycosidic bond between the sulfated rhamnose and a glucuronic or iduronic acid.

Noncovalent interactions in the gas-phase conformers of anionic iduronate (methyl 2-o-sulfo-α-L-iduronate): variation of subconformer versus ring conformer energetics for a prototypical anionic monosaccharide studied using computational methods.

The conformational preferences of the prototypical anionic monosaccharide (methyl 2-O-sulfo-α-L-iduronate) have been studied at various computational levels to investigate the energetic variation of 17 subconformers associated with the (4)C(1), (2)S(0), (5)S(1), and (1)C(4) ring conformers. These calculations include the first fully optimized MP2 calculations that have been performed for an anionic sugar system, and therefore allow an assessment of the performance of a group of DFT functionals (B3LYP, PW91PW91, and M05-2X) for treating the noncovalent dispersion and anomeric effects that are present in this system. We find that the recently developed M05-2X functional of Truhlar and co-workers [Y. Zhao, N. E. Schultz, D. G. Truhlar, J. Chem. Theory Comput., 2006, 2, 364] reproduces the MP2 results most closely, thus indicating that it may well be suitable for computational studies of larger ionic saccharides. Most importantly, the results presented indicate that it is crucial to consider the subconformers (which correspond to rearrangements of the sugar-ring side-chains) of the main ring-conformers in order to obtain a reliable overview of the potential energy surface of such systems. We find that the lowest isolated (gas-phase) conformer corresponds to a (4)C(1) chair conformer, which displays a pair of strong C(3)-OH···SO(3)(-) and OMe···SO(3)(-) electrostatic hydrogen-bonding interactions, coupled with a looser C(4)-OH···SO(3)(-) interaction. Overall, the relative energies of the subconformers appear to be straightforwardly related to the number of hydrogen-bonding interactions that each conformer displays among its pendant functional groups.

Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density.

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.

Gas-phase and solution conformations of the alpha-L-iduronic acid structural unit of heparin.

The IdoA2S structural unit of heparin (subunit G) may oscillate among the three conformations (4C1, 1C4, and 2So). Only the twisted boat conformation allowed the biologically active pentasaccharide unit of heparin (DEFGH) to bind to antithrombin. Our work reports, in detail, the results of systematic large-scale theoretical investigations of the three basic conformations (4C1, 1C4, and 2So) of the IdoA2S structural unit of heparin, its anionic forms, and its sodium salt using the B3LYP/6-311++G(d, p) and B3LYP/6-31+G(d) model chemistries. According to our calculations, the most stable structure of these molecules corresponds to the 2So skew-boat conformation. This form is also the most stable in a water solution. The 2So conformation of neutral molecules is not maintained in the anionic species. With anions, both 1C4 and 4C1 conformations are present. The relative stability of individual species of the substituted iduronic acid affects extra stabilization by means of intramolecular hydrogen bonds. The calculated macroscopic pKa of 1,4-DiOMe IdoA2S are as follows: pKa = 0.25 for the terminal C(2)-OSO3H group, pKa = 3.67 for the terminal C(5)-CO2H group, and pKa = 14.00 for the C(3)-OH hydroxyl group. The computed Gibbs interaction energies, DeltaGdegrees , for the reaction 1,4-DiOMe IdoA2S(2-) + 2Na+ <==> 1,4-DiOMe IdoA2SNa2 (4C1, 1C4, and 2So conformations) are negative and span a rather small energy interval (from -1244 to -1290 kJ mol(-1)).

Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: an effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide.

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.

Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n.

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.

Short heparin sequences spaced by glycol-split uronate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors.

Fibroblast Growth Factor-2 (FGF2) is a major inducer of neovascularization (angiogenesis). Heparin activates FGF2 by favoring formation of ternary complexes with its cellular receptors (FGFRs). Controlled 2-O-desulfation followed by exhaustive periodate oxidation/borohydride reduction has been used to generate sulfation gaps within the prevalent heparin sequences, building-up arrays of pentasulfated trisaccharides (PST, consisting of a 2-O-sulfated iduronic acid flanked by two N,6-disulfated glucosamines) spaced by reduced, glycol-split uronic acid (sU) residues. The structure of the prevalent sequences of the novel heparin derivative has been confirmed by mono- and two-dimensional NMR analysis. NMR spin-lattice relaxation times (T2) and nuclear Overhauser effects suggest that the sU residues act as flexible joints between the PST sequences and cause a marked distortion of the chain conformation of heparin required for formation of ternary complexes. Since the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, the heparin derivative has no anticoagulant activity. However, it fully retains the FGF2-binding ability of the original heparin, as shown by its capacity to protect FGF2 from trypsin cleavage and to prevent the formation of heparan sulfate proteoglycan (HSPG)/FGF2/FGFR1 ternary complexes. However, when compared to heparin it showed a reduced capacity to induce FGF2 dimerization and to favor the interaction of [125I]FGF2 with FGFR1 in HSPG-deficient, FGFR1-transfected CHO cells. Accordingly, it was more effective than heparin in inhibiting the mitogenic activity exerted by FGF2 in cultured endothelial cells. Finally, it inhibited angiogenesis in a chick embrio chorioallantoic membrane (CAM) assay in which heparin is inactive.

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans.

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts.

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]-PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti-proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti-proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.

Neuropilin-1 is a placenta growth factor-2 receptor.

Placenta growth factor (PlGF) belongs to the family of vascular endothelial growth factors (VEGFs). It binds to the flt-1 VEGF receptor but not to the KDR/flk-1 receptor which is thought to mediate most of the angiogenic and proliferative effects of VEGF. Three PlGF isoforms are produced by alternative splicing. PlGF-1 and PlGF-3 differ from PlGF-2 since they lack the exon 6 encoded peptide which bestows upon PlGF-2 its heparin binding properties. Cross-linking experiments revealed that 125I-PlGF-2 binds to two endothelial cell surface receptors in a heparin dependent fashion. The binding of 125I-PlGF-2 to these receptors was inhibited by an excess of PlGF-2 and by the 165-amino acid form of VEGF (VEGF165), but not at all by VEGF121 and very marginally if at all by PlGF-1. The apparent molecular weight and the binding characteristics of these receptors correspond to those of the recently identified VEGF165 specific receptor neuropilin-1, and we therefore conclude that neuropilin-1 is a receptor for PlGF-2. The binding of 125I-PlGF-2 as well as the binding of 125I-VEGF165 to these receptors was inhibited by a synthetic peptide derived from exon 6 of PlGF. Furthermore, the binding of 125I-PlGF-2, but not that of 125I-VEGF165, was also inhibited by a synthetic peptide derived from exon 7 of PlGF. These observations indicate that the peptides encoded by these exons probably participate in the formation of the domain which mediates the binding of PlGF-2 to these receptors. We have also determined, using chemically modified heparin species, that the presence of sulfate moieties on the glucosamine-O-6 and on the iduronic acid-O-2 groups of heparin was required for the potentiation of 125I-PlGF-2 binding to these receptors. To determine if PlGF-2 is able to induce biological responses that are not induced by PlGF-1, we compared the effects of PlGF-1 and PlGF-2 on the migration and proliferation of endothelial cells. Both PlGF forms induced migration of endothelial cells. However, there was no quantitative difference between the response to PlGF-2 and the response to PlGF-1. Furthermore, neither PlGF-1 nor PlGF-2 had any effect upon the proliferation of the endothelial cells.