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Biomedical subjects

U Lindahl

Publications and source records attributed to U Lindahl.

At least 127 records · Page 7Linked to original sources
PubMed1986

Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa.

Oligosaccharides (10-20 monosaccharide units) with high affinity for antithrombin, as well as larger high-affinity heparin fractions (having relative molecular masses between 6,000 and 21,500), all markedly accelerated the inhibition of Factor Xa by antithrombin. Moreover, all high-affinity oligosaccharides and heparins enhanced, to a similar extent, the amount of free proteolytically modified antithrombin cleaved at the reactive bond by Factor Xa. In contrast, a minimum high-affinity heparin size of approximately 18 monosaccharide units was required to significantly accelerate the inactivation of thrombin by antithrombin and to enhance the production of modified antithrombin by this enzyme. All high-affinity fractions studied had similar affinities for antithrombin, as determined by fluorescence titrations. In competition experiments, binary complexes of antithrombin with octadecasaccharide or larger high-affinity heparins, but not with smaller oligosaccharides, displaced inactivated 125I-thrombin from matrix-linked low-affinity heparin. Moreover, similar binary complexes with 3H-labeled octadecasaccharide or larger chains, but not with smaller oligosaccharides, were capable of binding to matrix-linked inactivated thrombin. These results indicate that simultaneous binding of antithrombin and thrombin to high-affinity heparin is a prerequisite to the acceleration of the antithrombin-thrombin reaction and that the minimum heparin sequence capable of binding both proteins comprises approximately 18 monosaccharide units. Similar complex formation apparently is not required for the acceleration of the antithrombin-Factor Xa reaction.

Animals↗
PubMed1986

Effect of benzyl beta-D-xyloside on the biosynthesis of chondroitin sulphate proteoglycan in cultured human monocytes.

Monocytes isolated from human blood differentiate into macrophage-like cells when maintained in vitro for 3-5 days on plastic or glass culture dishes. In the process the cells display characteristic morphological changes, and in addition, a transition in glycosaminoglycan biosynthesis, from the production of chondroitin 4-sulphate to the formation of a polysaccharide containing 20% 4,6-disulphated disaccharide units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. Cells were incubated with inorganic [35S]sulphate on day 1 or day 6 in culture, in the presence or absence of benzyl beta-D-xyloside, and labelled polysaccharide was isolated from the culture medium. In the presence of xyloside, the secretion of proteoglycans (90% galactosaminoglycan) was inhibited in a dose-dependent fashion and replaced by release of single polysaccharide chains, the size of which decreased with increasing dose of xyloside. The single polysaccharide chains produced on day 6 in the presence of 0.5 mM-xyloside showed the same proportion of disulphated disaccharide units as did the corresponding control material. Day-1 polysaccharide contained negligible amounts of this component, irrespective of the presence or absence of xyloside. It is concluded that the regulatory mechanism that induces 'oversulphation' during the differentiation process operates independently of any association between the polysaccharide chains and the core protein. Moreover, cells maintained in the presence of 0.5 mM-xyloside throughout a 6-day culture period showed the same morphological change, indicative of differentiation into macrophage-like cells, as did untreated control cells. The xyloside did not significantly affect the cytotoxicity of the monocytes, or of the differentiated macrophage-like cells, toward tumour cells.

Cell Survival↗
PubMed1986

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4.

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.

Heparin↗
PubMed1986

Structure and function of basement membrane proteoglycans.

Basement membranes contain at least three different proteoglycans. These are a large, low buoyant density heparan sulphate proteoglycan and two smaller, high density proteoglycans with either heparan sulphate or chondroitin sulphate side-chains. The large (Mr 400K-600K and small (Mr 130K) heparan sulphate proteoglycans were purified from the mouse EHS tumour. These proteoglycans are immunologically related by sharing some protein core antigenic determinants (epitopes) but do not cross-react with cell-surface heparan sulphate proteoglycans or with proteoglycans from interstitial connective tissue. This indicates that they belong to a distinct family of proteoglycans. Structural models were developed, based on electron microscopy and analytical ultracentrifugation, demonstrating that the small proteoglycan contains on average four heparan sulphate chains of about 30 nm in length, while the large proteoglycan consists of three long (about 90 nm) heparan sulphate chains connected to one end of a large core protein. Single heparan sulphate chains were isolated from the EHS tumour proteoglycans and from the corresponding proteoglycans from Reichert's membrane of the mouse embryo. The heparan sulphate from Reichert's membrane bound to antithrombin with high affinity and was found to contain the unique 3-O-sulphated glucosamine residue previously identified in the antithrombin-binding region of heparin. The EHS tumour heparan sulphate showed a higher N-/O-sulphate ratio and a lower affinity for antithrombin.

Animals↗
PubMed1986

Functional domains of rabbit thrombomodulin.

Thrombomodulin isolated from rabbit lung was separated by ion-exchange chromatography on DEAE-cellulose into a retarded (acidic) and a nonretarded (nonacidic) fraction. Both fractions contained the cofactor required for the activation of protein C. In addition, the acidic fraction (but not the nonacidic fraction) prevented the clotting of fibrinogen by thrombin ("direct" anticoagulant activity) and accelerated the inhibition of thrombin by antithrombin (effect corresponding to 2-10 international units of heparin per mg of protein). Both of these activities were readily neutralized by the synthetic polycation Polybrene, which did not appreciably affect protein C activation. They were also eliminated by digestion of thrombomodulin with bacterial heparinase, which, in addition, converted the acidic form of the protein C activation cofactor to a nonacidic form. Similar conversion observed during storage of thrombomodulin was attributed to endogenous proteinase activity. Density-gradient centrifugation of the acidic form of thrombomodulin in CsCl/4M guanidinium chloride failed to separate either of the direct or antithrombin-dependent anticoagulant activities from the protein C activation cofactor, which showed a buoyant density of 1.31-1.34 g/ml. The nonacidic cofactor had a lower density, 1.26-1.28 g/ml. Unreduced thrombomodulin yielded two major fractions of protein C activation cofactor on NaDodSO4/PAGE, with apparent Mr of approximately 68,000 and 57,000, respectively. The larger component contained essentially all of the direct and antithrombin-dependent anticoagulant activities. We propose that these activities as well as the negative charge and the higher buoyant density of the acidic, Mr 68,000 form of thrombomodulin are due to a heparin-like polysaccharide and, further, that this component can be separated from the major portion of the molecule, which contains the protein C activation site, through the action of a proteinase.

Animals↗
PubMed1986

Heparin and its low molecular weight derivatives: anticoagulant and antithrombotic properties.

The anticoagulant and antithrombotic effect of heparin has been known for many decades. Nevertheless, the knowledge of structure-activity relationships and its mechanisms of action has been rather limited. However, in recent years important progress has been made and our understanding of how heparin works has increased significantly. Based on this information, attempts have been made to modify heparin to get improved pharmacological properties. The present communication summarizes the recent development. It is shown that certain heparin derivatives of low molecular weight are highly interesting compounds from a clinical point of view. Biochemical and pharmacological properties of such a preparation named Fragmin are presented.

Blood Coagulation↗
PubMed1985

Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells.

Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.

Amidohydrolases↗
PubMed1985

Glycosaminoglycans in rat mucosal mast cells.

Rats were infected with the nematode Nippostrongylus brasiliensis, resulting in an approx. 5-fold increase in the number of mucosal mast cells and the histamine content of the intestinal (jejunum) wall. After injection of the infected animals with inorganic [35S]sulphate, a similar increase in the yield of labelled intestinal glycosaminoglycans was observed, compared with uninfected control rats. Autoradiography showed a highly selective labelling of the numerous mucosal mast cells and of the few connective-tissue mast cells in the subserosal region of the bowel. Analysis of the labelled polysaccharide from the infected animals showed that almost 60% of this material consisted of oversulphated galactosaminoglycan, whereas heparin-related polysaccharides accounted for only 13%. The galactosaminoglycan contained 4-monosulphated and 4,6-disulphated N-acetylgalactosamine residues in approx. 5:1 molar ratio, both being linked to D-glucuronic acid residues; the occurrence of L-iduronic acid units could not be excluded. No significant difference in structure was found between this polysaccharide and the corresponding component isolated from uninfected rats. It is concluded that the major polysaccharide produced by rat mucosal mast cells in vivo is an oversulphated galactosaminoglycan rather than heparin.

Animals↗
PubMed1984

Extension and structural variability of the antithrombin-binding sequence in heparin.

Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.

Animals↗
PubMed1984

Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates.

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.

Cells, Cultured↗
PubMed1984

High and low affinity heparin compared with unfractionated heparin as antithrombotic drugs.

A preparation of heparin was separated by affinity chromatography into two fractions: one of high ( HAH ) and the other of low (LAH) affinity to antithrombin III. These two fractions were compared with unfractionated heparin ( UFH ) by in vitro assay and their ability to impair experimental stasis thrombosis was also examined. Although the in vitro activity of HAH was double that of UFH , HAH was less effective than UFH as an antithrombotic drug; LAH was virtually inactive, both in vitro and in vivo. A mixture of 30 micrograms/kg of HAH and 50 micrograms/kg of LAH was as effective in preventing thrombosis as 80 micrograms/kg of UFH , and was more effective than 40 micrograms/kg of HAH alone, demonstrating that LAH potentiates the action of HAH in vivo.

Animals↗
PubMed1984

Low-affinity heparin potentiates the action of high-affinity heparin oligosaccharides.

Previous studies have shown that high-affinity (HA) heparin oligosaccharides, with molecular weights of 3,000-5,000, were less effective than unfractionated heparin in preventing serum-induced venous thrombosis in rabbits, using a Wessler stasis model. In the present study, a larger high-affinity fragment (M.Wt. 6,000-6,500) was also found to be less effective than unfractionated heparin as an antithrombotic agent. However, addition of 80 micrograms/kg low affinity (LA) heparin to 80 micrograms/kg of this HA fragment significantly potentiated its antithrombotic activity, and the antithrombotic action of the mixture was equivalent to that of unfractionated heparin. Significant potentiation of antithrombotic activity was also observed on the addition of LA heparin to a HA decasaccharide (M.Wt. 3,000-3,500) with anticoagulant activity only against Factor Xa. The LA heparin content of low molecular weight heparin fractions appears to be an important determinant of their antithrombotic activity.

Affinity Labels↗
PubMed1984

Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4.

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.

Anticoagulants↗
PubMed1984

Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase.

The substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase from a mouse mastocytoma was examined to determine the effects of N-acetyl and O-sulfate groups on substrate recognition by the enzyme. [5-3H]Glucuronosyl-labeled heparosan N-sulfate was prepared enzymatically and was modified chemically by partial N-desulfation and N-acetylation. After enzymatic release of tritium, the location of remaining label was determined by deaminative cleavage and analysis of resulting di-, tetra-, and higher oligosaccharides. This analysis indicated that a D-glucuronosyl residue is recognized as a substrate if it is linked at C-1 to an N-acetylated glucosamine residue and at C-4 to an N-sulfated unit. However, the reverse structure, in which the D-glucuronosyl moiety is bound at C-1 to an N-sulfated residue and at C-4 to N-acetylated glucosamine, is not a substrate. Similar studies with O-sulfated heparin intermediates showed that O-sulfate groups either at C-2 of the L-iduronosyl moieties or at C-6 of vicinal D-glucosaminyl moieties prevent 5-epimerization. These findings were confirmed by studies of the reverse reaction, in which tritium was incorporated from 3H2O into partially O-desulfated heparin and the location of incorporated radioactivity was determined. These and more direct experiments corroborated the previous conclusion that the L-iduronosyl moieties are formed after N-sulfation but before O-sulfation. Assessment of the influence of substrate size on the reaction further showed that a large substrate is preferred; an octasaccharide released tritium at a rate approximately 10% of that observed for the parent polysaccharide, and some release occurred also with smaller oligosaccharides.

Animals↗
PubMed1983

The antithrombin-binding sequence in heparin. Identification of an essential 6-O-sulfate group.

An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin, was previously found to have an L-iduronosyl-N-acetylglucosaminyl-6-O-sulfate nonreducing terminal disaccharide unit. After digestion of this octasaccharide with alpha-L-iduronidase and N-acetylglucosamine-6-sulfate sulfatase, two fractions, with high and low affinity for antithrombin, respectively, were isolated by affinity chromatography on antithrombin-Sepharose. Structural analysis showed that the high affinity fraction contained intact octasaccharide, whereas the low affinity fraction consisted of the expected heptasaccharide, lacking a 6-sulfate group on the terminal N-acetylglucosamine residue. Digestion of the octasaccharide with alpha-L-iduronidase only yielded heptasaccharide which was identical with the low affinity species except for the presence of this 6-sulfate group. This less degraded heptasaccharide retained high affinity for antithrombin. It is concluded that the 6-sulfate group on the N-acetylglucosamine residue is of critical importance to the interaction between heparin and antithrombin.

Animals↗
PubMed1983

Changes in glycosaminoglycan biosynthesis during differentiation in vitro of human monocytes.

Monocytes isolated from human blood were maintained in vitro on plastic culture dishes. After 3-4 days, adherent cells displayed morphological changes previously attributed to differentiation of the cells into histiocytes. 35S-labelled glycosaminoglycans were isolated after incubation of the cells with inorganic [35S]sulphate. Polysaccharide recovered from the culture medium after labelling from day 0 to day 2 or from day 5 to day 7 in vitro was approximately 90% galactosaminoglycan (resistant to deamination by HNO2), irrespective of labelling period. Whereas day-0-2 material was extensively degraded to disaccharide on incubation with the bacterial eliminase chondroitinase AC, a significant portion, about 30%, of the day-5-7 material resisted degradation under the same conditions. The resistant portion was readily depolymerized by treatment with chondroitinase ABC and may be dermatan sulphate. Paper electrophoresis and paper chromatography of the disaccharides obtained by eliminase digestion identified the day-0-2 labelled galactosaminoglycan as chondroitin 4-sulphate. In contrast, the corresponding day-5-7 material yielded approximately 20% disulphated disaccharide, both on digestion with chondroitinase AC and on subsequent enzymic degradation of the chondroitinase AC-resistant fraction. Further treatment of the disulphated disaccharide with chondro-4-sulphatase and chondro-6-sulphatase indicated that both sulphate groups were located on the N-acetylgalactosamine residue. In accordance with these findings, the day-5-7 polysaccharide showed a higher negative charge density than the day-0-2 material on ion-exchange chromatography. It is concluded that the novel properties acquired by the monocyte during prolonged culturing on plastic include the ability to synthesize glycosaminoglycan(s) containing 4,6-disulphated N-acetylgalactosamine units.

Carbohydrates↗