Heparin–protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis

Numerous extracellular proteins, growth factors, chemokines, cytokines, enzymes, lipoproteins, involved in a variety of biological processes, interact with heparin and/or heparan sulfate at the cell surface and in the extracellular matrix (ECM). The goal of this study is to investigate the relationship(s) between affinity and kinetics of heparin–protein interactions and the localization of the proteins, their intrinsic disorder and their biological roles. Most proteins bind to heparin with a higher affinity than their fragments and form more stable complexes with heparin than with heparan sulfate. Lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines) bind to heparin with very high affinity. Matrisome-associated proteins form transient complexes with heparin. However they bind to this glycosaminoglycan with a higher affinity than the proteins of the core matrisome, which contribute to ECM assembly and organization, and than the secreted proteins which are not associated with the ECM. The association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. Enzyme inhibitor activity, protein dimerization, skeletal system development and pathways in cancer are functionally associated with proteins displaying a high or very high affinity for heparin (K_D < 100 nM). Besides their use in investigating molecular recognition and functions, kinetics and affinity are essential to prioritize interactions in networks and to build network models as discussed for the interaction network established at the surface of endothelial cells by endostatin, a heparin-binding protein regulating angiogenesis.     

Heparin can activate a receptor tyrosine kinase.

Heparin, a densely sulfated glycosaminoglycan produced by mast cells, is best known for its inhibitory effects on the blood coagulation system. Heparin or heparan sulfate proteoglycans are also essential cofactors for the interaction of fibroblast growth factors (FGFs) with their receptor tyrosine kinases (FGFRs). Here we show that heparin is a growth factor-independent activating ligand for FGFR-4. Heparin stimulates FGFR-4 autophosphorylation on transfected myoblasts, fibroblasts and lymphoid cells, and is most potent on cells lacking surface heparan proteoglycan. Two functional analogs of heparin, fucoidan and dextran sulfate, are also activators of FGFR-4, while neither heparin nor its analogs can stimulate FGFR-1 in the absence of FGF. A mutation in the FGFR-4 ectodomain which impairs receptor activation by FGFs does not interfere with activation by heparin, demonstrating that receptor domains required for heparin or FGF activation are not identical. Heparin activation of FGFR-4 or of a chimeric receptor bearing FGFR-4 ectodomain and FGFR-1 cytodomain triggers downstream tyrosine phosphorylation of several signaling proteins, and induces proliferation of cells bearing the chimeric receptor. Consistent with these findings, a soluble FGFR-4 ectodomain has strong FGF-independent affinity for immobilized heparin resin, while soluble FGFR-1 requires FGF for stable heparin interaction. Heparin activation of FGFR-4 is the first example of a mammalian polysaccharide serving as a signaling ligand.     

Opposing effects of heparin with TGF-beta or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration.

The effects on vascular wound repair in vitro of aFGF and TGF-beta, growth factors having opposite influences on endothelial cell growth and angiogenesis, were studied using as a model a mechanical lesion of confluent endothelium. Modulation by heparin of the activities of these growth factors during the repair process was also examined. Whereas heparin alone inhibited repair by lowering both cell proliferation and cell migration, TGF-beta alone mainly inhibited cell proliferation. When added together, TGF-beta and heparin exerted a combined inhibitory effect resulting in a residual lesion 50% larger than in controls. aFGF alone accelerated lesion coverage and this effect was enhanced by 40% over control values when heparin was added with aFGF. This acceleration was slightly (less than 10%) but consistently diminished by TGF-beta. Cell density in confluent unwounded areas was increased by 40% in the presence of aFGF, but TGF-beta diminished cell density by 20%. A small (30%) increase in intracellular cAMP was measured whenever aFGF was present during the repair process. In comparison, intracellular cAMP inducing agents (forskolin, dbcAMP) accelerated cell migration by 20% during lesion recovery without affecting cell proliferation or density. The present results show that the inhibitory effects of TGF-beta during vascular wound repair are opposed by aFGF. Furthermore, heparin (or heparan sulfates in vivo) modulates growth factors having activating or inhibiting functions and thus plays a regulatory role during the repair process. cAMP-inducing substances other than growth factors are able to accelerate cell migration.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

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.     

Direct stenting with 3000 i.u. heparin

In order to reduce vascular complications, the authors assessed safety and feasability of a new percutaneous transluminal coronary angioplasty (PTCA) strategy consisting of direct stenting with 3000 i.u. heparin and immediate sheath removal. Predicting factors of vascular complications during PTCA include heparin dosages, sheath dwell time and use of anti-glycoprotein (GP) IIb/IIIa. A simplified PTCA with direct stenting technique may allow the use of very low doses of heparin without anti-GPIIb/IIIa in selected cases. From April 1999 to April 2000 all patients who underwent PTCA in the authors' center were screened. Exclusion criteria comprised a contraindication for direct stenting, primary PTCA for acute myocardial infarction (MI) and a TIMI (thrombolysis in myocardial infarction) grade zero flow. All other patients were included. They received 3000 i.u. heparin before direct stenting whatever their current anticoagulation and their weight. The sheath was immediately removed using manual compression. Out of 716 consecutive PTCA patients, 171 (24%) were enrolled in the study (198 sites). Complete protocol was achieved in 150 patients (88%). Activated clotting time during the procedure was 179 +/- 32 seconds. No subacute thrombosis or creatine kinase elevation was observed before discharge. Only two uncomplicated groin hematomas and two false aneurysms (one surgical repair) were noted. This study shows that direct stenting with 3000 iu heparin is safe. Immediate sheath removal can be performed with a low rate of major vascular complications.     

Derivatized dextrans mimic heparin as stabilizers, potentiators, and protectors of acidic or basic FGF.

Acidic and basic fibroblast growth factors (aFGF and bFGF) belong to a family of structurally related polypeptides characterized by a high affinity for heparin. a and bFGF display mitogenic activity for many cell types. Biological activity is strongly potentiated by heparin which stabilizes their molecular conformation by preventing physicochemical or enzymatic degradation. In our previous study we have shown that a water-soluble derivatized dextran named DDE, containing 82.2% methyl carboxylic acid groups, 6.1% benzylamide, and 5.6% sulfonate with a specific anticoagulant activity equivalent to heparin of 0.5 IU/mg could potentiate the mitogenic activity of aFGF on CCL39 cells. Optimal concentrations for maximal potentiation of 400 micrograms/ml and 20 micrograms/ml were obtained respectively for DDE and heparin. In the present report, we have uncovered the fact that several carboxymethyl benzylamide sulfonate dextrans differing in degree and positioning of the substituent groups can mimic heparin in regard to the protection, stabilization, and potentiating effects with aFGF or bFGF. Our data establishes that the dextran derivatives studied can act as potentiating agents for FGFs. Native dextran (DDA) had no effect. Dextran derivatives can also protect aFGF and bFGF from heat as well as from pH denaturation, and against trypsic and chymotrypsic degradation. The dextran derivative DDI (82% methylcarboxylic acid, 23% benzylamide, 13% sulfonate) was studied in greater detail and exhibited a greater protection for bFGF and a lesser protecting effect for aFGF than heparin. Derivatized dextrans which have very weak anticoagulant activity are of great interest as alternatives to heparin for use as stabilizers, potentiators, protectants, and slow-release matrices for FGFs in pharmaceutical formulations.     

Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.

The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.     

Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine-mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes.     

Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focusing.

The mechanism of complexation of pI range 3.5--5 Ampholine to heparin in isoelectric focusing has been explored by the dye-binding technique at different pH values in solution. There is no significant interaction between heparin and Ampholine at pH 6.7. Weak, or selective, binding occurs at pH 5.1, and very strong interaction at pH 3.5. In the latter system, the Ampholine components appear to behave as polycations due to their ordered sequence of positive charges, each two methylene groups apart, which favors a strong binding to polyanions. In addition, there appear to be variable stoichiometries for the strong binding between heparin and Ampholine, depending on their relative amounts. It is proposed that at a low ratio of heparin to Ampholine (Ampholine excess), aggregation is perpendicular to the heparin chain, with the end ammonium charge of each Ampholine molecule neutralizing one negative charge along the heparin molecule; at higher ratios (heparin excess), the bound Ampholine segment is aligned parallel to the heparin molecule, so that on the average one Ampholine component neutralizes approx. three negative charges. The banding of heparin in isoelectric focusing in the pH range 3.0--4.5 can be explained by aggregation of the various components on heparin in amounts dependent upon the net charge on the Ampholine species at the given pH, and upon the changing stoichiometries as a function of the variation in ratio of heparin to Ampholine along the pH gradient. Binding of Ampholine to polygalacturonate was also demonstrated in excess Ampholine in a pH range dependent on the degree of protonation of the carboxyl groups of this acidic polysaccharide as well as on the net positive charge of the Ampholine. The aggregation seen at pH 4.2--4.5 led to the prediction and subsequent demonstration that polygalacturonate would also exhibit binding upon isoelectric focusing. This supports the hypothesis that aggregation of Ampholine on polyanions having sufficient charge density is a general phenomenon which can lead to spurious banding of certain polymers at appropriate pH ranges in isoelectric focusing. On the basis of their behavior in isoelectric focusing at pH 3.0--4.5, strength of aggregation of the polyanions studied appears to be heparin A = heparin B greather than polyglutamate greater than carboxyl-reduced heparin B greater than polygalacturonic acid.     

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparin's efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin-binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF-2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF-2 binding to endothelial cells and stripped pre-bound FGF-2 from cells. SOS also regulated FGF-2 stimulated proliferation. Further, SOS facilitated FGF-2 diffusion through Descemet's membrane, a heparan sulfate-rich basement membrane from the cornea, suggesting a possible role in FGF-2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study.     

Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.     

Heparin augments osteoclast resorption-stimulating activity in serum

Increased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long-term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet-poor plasma-derived serum, heparin, in the range 25-100 micrograms/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption-stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor-beta, platelet-derived growth factors, insulin-like growth factors I or II, acidic or basic fibroblast growth factors, fibronectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin-binding factor. The ability of glycosaminoglycans (GAGs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin-2-sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor involved in the local and systemic regulation of osteoclastic bone resorption.     

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.     

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.     

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Complexation with heparin prevents adhesion between fibrin-coated surfaces.

Heparin, in Langmuirian fashion, binds stoichiometrically with high affinity, Kd approximately 100 nM, to both fibrinogen and fibrin adsorbed as monomolecular films to lecithin-coated, microscopic, polystyrene-divinylbenzene beads. Complex formation inhibits aggregation of fibrin-coated beads, and it also results in dissociation of preformed aggregates of fibrin-coated beads. These phenomena are not caused by desorption of fibrin(ogen), indirect inhibition of thrombin activity, or mere electrostatic repulsion of charged particles. Instead, these data are consistent with the proposal that the complexed heparin interferes directly with dimer formation between fibrin molecules adsorbed to colliding beads. We describe these phenomena and their application to the development of sensitive analytical methods for quantitating heparin. Based on these observations, we also propose a role for endogenous heparin in the physiologic regulation of fibrin-mediated adhesion of surfaces.