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The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.

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Inhaled heparin prevents antigen-induced bronchoconstriction and inhibits anti-IgE-mediated mast-cell degranulation. We hypothesized that the antiallergic action of heparin may be dependent on molecular weight and related to its nonanticoagulant properties. Therefore, in the present investigation we studied the effects of a nonanticoagulant fraction of heparin (LA-heparin) on antigen-induced bronchoconstriction, airway hyperresponsiveness (AHR), and mast-cell degranulation, and compared its antiallergic activity with that of a low molecular weight heparin (LMW-heparin, fragmin). Specific lung resistance (SRL) was measured in 15 sheep before, immediately after, and serially for as long as 2 h after airway challenge with Ascaris suum antigen, without and after pretreatment with inhaled fractionated heparins at doses of 2.5 and 5 mg/kg. Airway responsiveness was estimated before, and 2 h after antigen as the cumulative provocating dose (PD400) of carbachol in breath units, which increased SRL by 400% (one breath unit was defined as one breath of 1% carbachol solution). LA-heparin caused a dose-dependent inhibition of antigen-induced bronchoconstriction, and a 5-mg/kg nebulized dose caused a 67% inhibition of allergic bronchoconstriction, whereas a 2.5-mg/kg dose was ineffective (20% inhibition). Inhaled fragmin was more potent than LA-heparin, as shown by 84% (2.5 mg/kg) and 82% (5 mg/kg) inhibition of allergic bronchoconstriction. Fragmin (5 mg/kg) also attenuated the postantigen AHR, whereas LA-heparin was ineffective. In vitro, preincubation with both LA-heparin and fragmin inhibited the anti-IgE-induced degranulation of rat peritoneal mast-cells in a dose-dependent fashion. LA-heparin was fourfold more potent than fragmin, with IC50 of 80 and 320 microg/ml, respectively. These data suggest that: (1) fractionated heparins attenuate antigen-induced acute bronchoconstriction, (2) nonanticoagulant fractions mediate the antiallergic activity of inhaled heparin, and (3) antiallergic activity of nonanticoagulant heparin and LMW-heparin may be related to prevention of mast-cell degranulation.

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Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent elF-2 alpha protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirus VA RNA, was competed by poly(rl):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, elF-2 alpha phosphorylation was catalyzed by both heparin-and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules.

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Low-anticoagulant heparin (LA-heparin) obtained by affinity chromatography on antithrombin III Sepharose inhibits the proliferation of cultured arterial smooth muscle cells in an in vitro bioassay system as effectively as standard heparin. A growth inhibition of smooth muscle cells of about 60% is achieved when LA-heparin or heparin is added to the culture medium to a concentration of 50 micrograms/ml. In normolipemic rats LA-heparin suppresses the formation of neointimal thickenings and stenosis after balloon catheter-induced deendothelialization of the carotid artery. In terms of mass a dose of 5 mg/kg body weight/d given subcutaneously twice daily one week before and 2 weeks after balloon injury the cross sectional area of the neointima is reduced to 36% as compared with the nontreated control group (100%). This 64% reduction is statistically highly significant (p < 0.001). After treatment with 0.5 mg LA-heparin/kg/d the reduction of the neointima was 11% (p < 0.05). At a dose of 5 mg/kg body weight single or repeated administrations of LA-heparin caused only a small and transient increase in activated partial thromboplastin time values. The results show that subcutaneous administration of LA-heparin very effectively prevents smooth muscle cell proliferation and balloon catheter-induced neointimal growth. The well tolerated systemic application of this chemically non-modified LA-heparin might justify clinical trials for prevention of restenosis after percutaneous transluminal coronary angioplasty or other invasive cardiovascular interventions without complications of bleeding.

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Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity.

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Heparanase activity correlates with metastatic potentials of lymphoma, melanoma and mammary adenocarcinoma cell lines. We investigated the ability of various modified species of heparin and size homogeneous oligosaccharides derived from depolymerized heparin to inhibit: a) heparanase-mediated degradation of heparan sulfate (HS) in the extracellular matrix (ECM) deposited by cultured endothelial cells, and b) lung colonization of B16-BL6 melanoma cells in C57BL mice. For this purpose, melanoma cells or conditioned medium were incubated with metabolically sulfate-labeled subendothelial ECM in the absence and presence of heparin, heparin fragment or nonanticoagulant species of heparin. Labeled HS degradation fragments released into the incubation medium were analyzed by gel filtration over Sepharose 6B. The B16-BL6 melanoma cells were also tested for lung colonization following their intravenous administration to C57BL mice, in the absence and presence of the various species of heparin. Inhibition of both heparanase and melanoma lung colonization depended on the size and degree of sulfation of the heparin molecule, the position of sulfate groups, and the occupancy of the N position of the hexosamines. Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and O positions. Low sulfate oligosaccharides were less effective heparanase inhibitors than medium and high sulfate fractions of the same size saccharide. While O-desulfation abolished the heparanase inhibiting effect of heparin. O-sulfated, N-substituted (e.g., N-acetyl or N-hexanoyl) species of heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about > or = 4,000 daltons. Potent inhibitors of heparanase activity were also efficient inhibitors of tumor invasion and lung colonization. The antimetastatic and anticoagulant activities of heparin were unrelated, as indicated by using heparin fractions with high and low affinity for antithrombin III. These heparins differ about 200-fold in their anticoagulant activity, but expressed similar high antiheparanase and antimetastatic activities. It appears that heparanase-inhibiting species of heparin interfere with the passing of tumor cells across the capillary wall, as they significantly inhibited metastasis even when injected up to 3 h after lodgment. Structural requirements for inhibition of heparanase activity and lung colonization of melanoma cells by species of heparin were different from those identified for a) release of ECM-bound basic fibroblast growth factor (bFGF), and b) stimulation of bFGF receptor binding and mitogenic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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The fibroblast growth factors (FGFs) act through high affinity tyrosine kinase receptors and, in addition, interact with lower affinity receptors that represent cell- or matrix-associated heparan sulfate proteoglycans. These lower affinity receptors modulate the biological activities of FGFs, but the mechanism by which they exert these effects is rather controversial. We have previously shown (Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. 268, 2984-2988) that heparin potentiates the mitogenic activity of acidic FGF (aFGF) but inhibits that of the keratinocyte growth factor (KGF) in cells that express the KGF receptor (KGFR). Both growth factors bind the KGFR with high affinity. To gain an insight into the mechanism by which heparin modulates the biological activity of aFGF and KGF, we studied the effect of heparin and cell-associated heparan sulfates on the binding of these two growth factors to the KGFR. To work in a well defined system, we expressed functional KGFR in L6E9 myoblasts that lack detectable high affinity binding sites for FGFs. Low concentrations of heparin inhibited the binding of KGF to the KGFR. By contrast, similar concentrations of heparin enhanced the binding of aFGF to this receptor. The effect of heparin was not unique to L6E9 cells expressing the KGFR; it was also observed in Balb/MK cells that naturally express KGFR. Treatment of cells with sodium chlorate, which blocks sulfation of proteoglycans, reduced the binding of aFGF to its low and high affinity binding sites by 95 and 80%, respectively. In contrast, the binding of KGF to its high affinity binding sites was enhanced about 2-fold. Similar results were obtained after degradation of cell-associated heparan sulfates by heparinase and heparitinase. Heparin restored the high affinity binding of aFGF to chlorate-treated cells and completely abolished the high affinity binding of KGF. Binding competition experiments suggest that aFGF and KGF bind to the same population of cell-associated heparan sulfates. In addition, KGF is apparently interacting with an as yet unidentified type of low affinity binding site that is not affected by chlorate or heparan sulfate-degrading enzymes. An important property of the FGF high affinity receptors is their ability to bind more than one ligand with high affinity. Based on the differential effect of cell-associated heparan sulfates on the binding of KGF and aFGF to the KGFR, we propose a regulatory role for cell-associated heparan sulfates as coordinators of the interaction of aFGF and KGF with the KGFR.(ABSTRACT TRUNCATED AT 400 WORDS)

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The binding of the 165 amino-acid form of vascular endothelial growth factor (VEGF165) to the VEGF receptors of vascular endothelial cells was potentiated by heparin and heparan-sulfate, but not by other glycosaminoglycans. Heparin fragments of 16-18 sugar units inhibited the binding of 125I-VEGF165 to VEGF receptors, while fragments larger than 22 sugar units potentiated the binding. Over-sulfated heparin was a better potentiator of 125I-VEGF165 binding than native heparin. O-desulfated and N-desulfated heparins potentiated the binding to a lesser extent than native heparin. Heparin and N-desulfated heparin efficiently inhibited the binding of 125I-VEGF165 to alpha 2-macroglobulin, but surprisingly, O-desulfated heparin was an ineffective inhibitor. Since alpha 2-macroglobulin does not bind heparin, it follows that VEGF165 does not bind O-desulfated heparin efficiently. These results suggest that the mechanism by which heparin modulates the binding of VEGF165 to the VEGF receptors may require an interaction with cell surface heparin binding molecules.

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Heparanase activity correlates with the metastatic potential of lymphoma, melanoma and mammary adenocarcinoma cell lines. We investigated the ability of various modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin to inhibit (1) heparanase-mediated degradation of heparan sulfate in a naturally produced subendothelial extracellular matrix (ECM), and (2) lung colonization of B16-BL6 melanoma cells in C57BL mice. Inhibition of heparanase was best achieved by heparin species containing 16 or more sugar units and having sulfate groups at both the N and O positions. Low-sulfate oligosaccharides were less effective heparanase inhibitors than medium- and high-sulfate fractions of the same-size saccharide. While O-desulfation abolished the heparanase-inhibiting effect of heparin, O-sulfated, N-substituted (e.g. N-acetyl or N-hexanoyl) species of heparin retained high inhibitory activity. Potent inhibitors of heparanase activity were also efficient inhibitors of tumor invasion and lung colonization. Heparin fractions with high and low anticoagulant activity expressed similar high antiheparanase and antimetastatic activities. Structural requirement for the inhibition of melanoma cell heparanase and lung colonization by species of heparin were different from those identified for (1) release of ECM-bound basic fibroblast growth factor (b-FGF) and (2) stimulation of b-FGF receptor binding and mitogenic activity. These results indicate that various nonanticoagulant species of heparin and other polyanionic molecules differing in size, sulfation and substituted groups can be designed to elicit specific effects resulting in the inhibition of cell invasion in tumor metastasis and autoimmunity, or stimulation of neovascularization and wound healing.

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Heparin, with or without the addition of an adrenocorticosteroid, can inhibit normal angiogenesis in the chick embryo chorioallantoic membrane. Low- or non-sulphated heparin fragments also have anti-angiogenic effect. Attempts to define a saccharide structure responsible for the anti-angiogenic effect implicated a -[GlcA beta 1,4-GlcNAc alpha 1,4]n-sequence. This structure represents the product of the initial polymerization reaction in heparin/heparan sulphate biosynthesis. It persists in the non-sulphated regions of heparan sulphate and also occurs in the Escherichia coli K5 capsular polysaccharide. The K5 polysaccharide, fragments thereof down to octasaccharide size and analogous N-acetylated fragments of heparan sulphate, all showed anti-angiogenic activity. Hyaluronan, however, with the isomeric -[GlcA beta 1,3-GlcNAc beta 1,4]n-structure was inactive. The anti-angiogenic activity of -[GlcA beta 1,4-GlcNAc alpha 1,4]n-containing saccharides was potentiated by the presence of L-iduronic acid and one or two O-sulphate groups in the non-reducing-terminal disaccharide unit. The anti-angiogenic effect of these non- or low-sulphated saccharides was unaffected by the addition of hydrocortisone. Endothelial cell surface-bound heparan sulphate proteoglycans may represent a pool of precursors of anti-angiogenic oligosaccharides which may be of primary importance in the regulation of angiogenesis.

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The methyl glycoside of a tetrasaccharide isolated from heparin, methyl O-(alpha-L-idopyranosyluronic acid)-(1-->4)-O-(2-acetamido-2-deoxy-alpha-D- glucopyranosyl)-(1-->4)-O-(beta-D-glucopyranosyluronic acid)-(1-->3)-O-beta-D-galactopyranoside disodium salt and a trisaccharide derivative thereof, methyl O-(alpha-L-idopyranosyluronic acid)-(1-->4)-O-(2-acetamido-2- deoxy-alpha-D-glucopyranosyl)-(1-->4)-O-beta-D-glucopyranosyluronic acid disodium salt, were synthesized using a block-type strategy. A suitable protected disaccharide block of iduronic acid and glucosamine (IdoA-GlcN) was used as a key intermediate for the syntheses and was glycosidated with a protected galactose derivative and a disaccharide block of glucuronic acid and galactose (GlcA-Gal) to give tri- and tetra-saccharide derivatives, respectively. Deprotection gave the target compounds.

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Vascular endothelial growth factor (VEGF) is a mitogen for cultured endothelial cells, and a potent angiogenic factor in vivo. Incubation of 125I-VEGF with human or bovine serum led to the formation of 125I-VEGF containing complexes that had a molecular mass greater than 300 kDa. These complexes were specifically immunoprecipitated with anti-human alpha 2-macroglobulin (alpha 2M) antibodies. Similar high molecular weight complexes were formed when 125I-VEGF was incubated with commercially available alpha 2M. The 125I-VEGF.alpha 2M complexes were resistant to boiling in the presence of SDS. The formation of 125I-VEGF.alpha 2M complexes was inhibited by iodoacetic acid, indicating that free sulfhydryl groups are required for complex assembly. Tryptic digestion of alpha 2M did not affect its VEGF binding ability. Tryptic digestion of 125I-VEGF.alpha 2M complexes on the other hand, resulted in the degradation of bound 125I-VEGF, indicating that alpha 2M does not protect bound 125I-VEGF from proteolytic digestion. The binding of 125I-VEGF to alpha 2M was partially inhibited by an excess of basic fibroblast growth factor. Other growth factors which bind to alpha 2M, such as platelet-derived growth factor and insulin, did not inhibit the binding of 125I-VEGF. The binding of VEGF to alpha 2M inhibited its receptor binding ability, indicating that alpha 2M may function as a VEGF removal and inactivation factor. Heparin and heparan sulfate, but not other glycosaminoglycans such as chondroitin sulfate, efficiently inhibited the binding of 125I-VEGF to alpha 2M. It is possible that heparin-like molecules released from extracellular matrixes could prevent the inactivation of VEGF by alpha 2M resulting in the potentiation of processes such as tumor angiogenesis.

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We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)

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Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.

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A template bleeding time study in the rat was undertaken to see if it is possible to correlate bleeding times with the molecular weight, anticoagulant activity or chemical composition of heparin or heparin-derived compounds. Heparin from porcine intestinal mucosa (PM-heparin) and from bovine lung (BL-heparin) as well as heparin fragments from these sources were compared. Heparin fragments of low anticoagulant activity were prepared by affinity chromatography on immobilized antithrombin as well as by chemical modification. A heparin fragment of high affinity for antithrombin (HA-fragment) caused a marked and dose-dependent increase in bleeding time while the corresponding heparin fragment with low affinity for antithrombin (LA-fragment) had a marginal and non-dose dependent effect on the bleeding time. Similar results were also obtained with PM-heparin with high and low affinity for antithrombin. A high anti-FXa activity was not always correlated with a marked bleeding tendency. Provided that a fragment was devoid of activated partial thromboplastin time (APTT) activity, it was not possible to provoke a bleeding time of 20 min or longer, although the compound was administered at a dose of 1,088 U/kg (anti-FXa activity). On the other hand, a N-acetylated chemically oversulphated heparin fragment, with a very low anti-FXa activity (1 U/mg) and with an APTT activity of 34 U/mg, caused a bleeding time of 20 min or longer in 70% of the animals after injection of the same number of APTT units, 1,088 U/kg. These data indicate that the APTT activity is a better and more sensitive indicator of the bleeding than is the anti-FXa activity.

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Heparin was previously reported to potentiate the mitogenic activity of endothelial cell mitogens in a crude extract of bovine hypothalami (Thornton, S. C., Mueller, S. N., and Levine, E. M. (1983) Science 222, 623-625). We and others (Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484) have reported that the growth stimulatory effects of acidic fibroblast growth factor (aFGF) are potentiated in a similar manner. We have used these observations as the basis of an assay to characterize the importance of size, sulfation, and anticoagulant activity of heparin in mediating this effect. Partial nitrous acid depolymerization of heparin from porcine intestinal mucosa resulted in a mixture of heparin fragments, containing oligosaccharides ranging from disaccharides to polysaccharides of about 40 monosaccharides in length. This mixture was fractionated by ion exchange chromatography and gel permeation chromatography to obtain size-homogeneous oligosaccharides with different degrees of sulfation. Assay of these heparin-derived saccharides in the presence of a suboptimal concentration of aFGF revealed that a minimum chain length and a certain degree of sulfation is required in order to potentiate the action of aFGF. Low sulfate oligosaccharides (4-16 units) were unable to potentiate aFGF, whereas medium sulfate fractions of octadecasaccharides and larger were able to moderately potentiate aFGF. The potentiation of aFGF by the high sulfate fraction correlated with the saccharide size: 12 or more monosaccharide units were necessary to achieve potentiation equivalent to whole heparin, octa- and decasaccharides were mildly stimulatory, and hexasaccharides were without effect. In the absence of aFGF, intact heparin as well as all the oligosaccharides examined, inhibited the proliferation of capillary endothelial cells to approximately the same degree, between 20 and 50% inhibition. When a tetradecasaccharide was separated into a binding and a nonbinding fraction on matrix-bound antithrombin III, no difference was seen for these fractions in the endothelial cell proliferation assay. These results indicate that both size and sulfation of a heparin-derived oligosaccharide contribute to its ability to interact with aFGF and/or endothelial cells and that this interaction is independent of anticoagulant activity. In addition, our findings suggest that the inhibitory and potentiating effects of heparin on capillary endothelial cells have different structural requirements.

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Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

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The absorption of trans-4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid, Cyklokapron) administered as the prodrug trans-4-(aminomethylcyclohexanecarboxylate hydrochloride (Kabi 2161) was investigated in 3 healthy volunteers. Kabi 2161 was given orally in doses of 1, 2, 3 and 3.5 mmol, respectively, and as a reference a clinical dose of 1.5 g tranexamic acid (9.6 mmol) was administered. At 3 mmol of Kabi 2161 the same maximum plasma concentration of tranexamic acid was obtained as with the reference drug but with Kabi 2161 it appeared earlier. The recovery of tranexamic acid in the urine 0-48 h after administration of Kabi 2161 was 84.7, 82.4, 89.4 and 97.5%, resp., of the increasing doses. For the tranexamic acid 37.0% could be recovered. A similar result was seen in the areas under the plasma concentration-time curves normalized for dose. With Kabi 2161, 13.1, 19.6, 14.4 and 14.3 mg.h/l.mmol were found compared to 8.0 mg.h/l.mmol with tranexamic acid. From these results it was concluded that Kabi 2161 markedly increased the bioavailability of tranexamic acid in man.

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Derivatives of the antifibrinolytic drug tranexamic acid [trans-4-(aminomethyl)cyclohexanecarboxylic acid] containing one or two tranexamic acid moieties were synthesized. Most of the derivatives have good stability in acidic and neutral solutions but are easily hydrolyzed in plasma. By measuring the amount of tranexamic acid excreted in the urine after an oral dose, relative absorptions of a number of derivatives in the rat were estimated. Most of the derivatives showed greater absorption than tranexamic acid itself. 1-[(Ethoxycarbonyl)oxy]ethyl trans-4-(amino-methyl)cyclohexanecarboxylate hydrochloride was chosen for studies in man.

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