Inhibition of rat glomerular visceral epithelial cell growth by heparin

1988 ◽  
Vol 255 (4) ◽  
pp. F781-F786 ◽  
Author(s):  
S. Adler
Keyword(s):  
Growth Inhibition ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Heparin Binding ◽  
Pentosan Polysulfate ◽  
The Media ◽  
Heparan Sulfates ◽  
Visceral Epithelial Cell

The effect of several glycosaminoglycans and sulfated polysaccharides on the growth of cultured rat glomerular visceral epithelial cells (GEC) was studied in vitro. Heparin, one preparation of heparan sulfate proteoglycan, dextran sulfate, and pentosan polysulfate significantly inhibited the growth of several GEC clones studied (36.0-77.1% inhibition at 100 micrograms/ml). Other glycosaminoglycans studied did not affect GEC growth. Growth inhibition by heparin was dose related and did not appear to reflect cytotoxicity. Heparins with high or low affinity for antithrombin inhibited growth to similar degrees. When heparin was fractionated into high- and low-anticoagulant activity fractions by physicochemical means the high activity fraction displayed significantly greater growth inhibition. The degree of growth inhibition significantly correlated with serum concentration in the media (r = 0.64; P less than 0.001). Removal of heparin binding factors from serum resulted in a loss of this correlation as well as less overall growth inhibition. These experiments suggest that interactions of GEC with heparan sulfates and other heparin-like molecules in the extracellular matrix may be important in the control of GEC growth.

Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Molecular Structures ◽  
Severe Reaction ◽  
Clinical Use ◽  
Molecular Weights ◽  
Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

INFLUENCE OF THE SULFATION PATTERN ON CERTAIN BIOLOGICAL PROPERTIES OF GALACTOSAMINOGLYCANS

1987 ◽  
Author(s):  
B Casu ◽  
L Marchese ◽  
A Naggi ◽  
G Torri ◽  
J Fareed ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Chlorosulfonic Acid ◽  
Biological Properties ◽  
Sulfated Polysaccharides ◽  
Low Molecular Weight ◽  
Antithrombotic Activity

In order to investigate the influence of charge distribution and chain length on the biological properties of sulfated polysaccharides, additional sulfate groups were introduced into the galactosaminoglycans, chondriotin sulfate and dermatan sulfate. Using a flexible method (with sulfuric acid and chlorosulfonic acid) for concurrent sulfation and controlled depolymerization, numerous products were obtained and characterized by chemical, enzymatic and nuclear magnetic resonance spectroscopic methods. The biologic actions of these products were profiled in both in vitro and in vivo assays for antithrombotic activity. Despite a weaker in vitro anticoagulant activity, low molecular weight over sulfated galactosaminoglycans produced significant dose-dependent antithrombotic actions in animal models which were similar to the actions observed with oversulfated low molecular weight heparins. These results suggest that a significant antithrombotic activity can be elicited through non-specific interactions of polysulfates with cellular and plasma components, and that clusters of sulfate groups such as the 4-6 disulfate group on D-galactosaminoglycan residues may be important for these interactions. Furthermore, these results, also suggest that supersulfation of glycosaminogly-cans results in products with biologic activity distinct from the native material.

HEPARIN DIRECTS THE INACTIVATION OF ANTITHROMBIN BY ELASTASE

1987 ◽  
Author(s):  
R E Jordan ◽  
J Kilpatrick ◽  
J Nelson ◽  
J O New gren ◽  
M A Fournel
Keyword(s):  
Alternative Explanation ◽  
Anticoagulant Activity ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Apparent Contradiction ◽  
Platelet Factor ◽  
Complete Inactivation

In apparent contradiction to its anticoagulant activity, we have observed a previously undetected, and potentially opposing function for heparin: a distinct heparin-dependency for the in vitro inactivation of highly-purified human antithrombin by neutrophil elastase. Similar to its ability to accelerate antithrombin-mediated inhibition of coagulation enzymes, anticoagulantly-active heparin was also found to stimulate the rate of inactivation of antithrombin by the neutrophil enzyme.In the absence of heparin, or in the presence of the heparin antagonists platelet factor 4 or polybrene, little or no inactivation of antithrombin occurred. Catalytic amounts of heparin and elastase caused the complete inactivation of antithrombin (approximate molar ratio of 1:1:400 respectively) in 5-10 minutes. The loss of heparin binding affinity by the elastase-cleaved form of antithrombin permitted its separation from active antithrombin by heparin-agarose chromatography.The purified elastase-inactivated antithrombin was injected into rabbits for determination of its comparative clearance behavior. In contrast to intact, functional antithrombin (t 1/2 >30 hours) and the thrombin-antithrombin (T-AT) complex (t 1/2 previously shown to be minutes), elastase-inactivated antithrombin circulated for approximately 13 hours. This prolonged clearance relative to the T-AT complex may suggest an alternative explanation for the circulating, non-functional antithrombin observed in certain coagulopathic states. In summary, these results point to a potential and unexpected role for heparin in directing the inactivation of antithrombin and suggest a possible in vivo mechanism for neutralizing the usually non-thrombogenic nature of the vascular lining.

scholarly journals Sulfated polysaccharides identified as inducers of neuropilin-1 internalization and functional inhibition of VEGF165 and semaphorin3A

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4126-4136 ◽  
Author(s):  
Masashi Narazaki ◽  
Marta Segarra ◽  
Giovanna Tosato
Keyword(s):  
Cell Surface ◽  
Dextran Sulfate ◽  
Receptor Internalization ◽  
Sulfated Polysaccharides ◽  
Cell Surface Receptors ◽  
Ligand Interaction ◽  
Surface Receptors ◽  
Neuropilin 1

Abstract Neuropilin-1 (NRP1) and NRP2 are cell surface receptors shared by class 3 semaphorins and vascular endothelial growth factor (VEGF). Ligand interaction with NRPs selects the specific signal transducer, plexins for semaphorins or VEGF receptors for VEGF, and promotes NRP internalization, which effectively shuts down receptor-mediated signaling by a second ligand. Here, we show that the sulfated polysaccharides dextran sulfate and fucoidan, but not others, reduce endothelial cell-surface levels of NRP1, NRP2, and to a lesser extent VEGFR-1 and VEGFR-2, and block the binding and in vitro function of semaphorin3A and VEGF165. Administration of fucoidan to mice reduces VEGF165-induced angiogenesis and tumor neovascularization in vivo. We find that dextran sulfate and fucoidan can bridge the extracellular domain of NRP1 to that of the scavenger receptor expressed by endothelial cells I (SREC-I), and induce NRP1 and SREC-I coordinate internalization and trafficking to the lysosomes. Overexpression of SREC-I in SREC-I–negative cells specifically reduces cell-surface levels of NRP1, indicating that SREC-I mediates NRP1 internalization. These results demonstrate that engineered receptor internalization is an effective strategy for reducing levels and function of cell-surface receptors, and identify certain sulfated polysaccharides as “internalization inducers.”

scholarly journals Influence of molecular weight of chemically sulfated citrus pectin fractions on their antithrombotic and bleeding effects

2009 ◽  
Vol 101 (05) ◽  
pp. 860-866 ◽  
Author(s):  
Thales Cipriani ◽  
Ana Helena Gracher ◽  
Lauro de Souza ◽  
Roberto Fonseca ◽  
Celso Belmiro ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Venous Thrombosis ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Sulfated Polysaccharides ◽  
Antithrombotic Agent ◽  
Total Inhibition ◽  
Almost All

SummaryEvaluated were the anticoagulant and antithrombotic activities, and bleeding effect of two chemically sulfated polysaccharides, obtained from citric pectin, with different average molar masses. Both low-molecular-weight (Pec-LWS, 3,600 g/mol) and high-molecular-weight sulfated pectins (Pec-HWS, 12,000 g/mol) had essentially the same structure, consisting of a (1→4)-linked α-D-GalpA chain with almost all its HO-2 and HO-3 groups substituted by sulfate. Both polysaccharides had anticoagulant activity in vitro, although Pec-HWS was a more potent anti-thrombotic agent in vivo, giving rise to total inhibition of venous thrombosis at a dose of 3.5 mg/kg body weight. Surprisingly, in contrast with heparin, Pec-HWS and Pec-LWS are able to directly inhibit α-thrombin and factor Xa by a mechanism independent of antithrombin (AT) and/or heparin co-factor II (HCII). Moreover, Pec-HWS provided a lower risk of bleeding than heparin at a dose of 100% effectiveness against venous thrombosis, indicating it to be a promising antithrombotic agent.

Studies into the Mechanism by Which Glycosaminoglycans Potentiate Protein C Activation by Factor Xa.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1724-1724
Author(s):  
Simon J. McRae ◽  
Alan R. Stafford ◽  
James C. Fredenburgh ◽  
Jeffrey I. Weitz
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Protein C ◽  
Dextran Sulfate ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Sulfated Polysaccharides ◽  
Heparin Binding ◽  
Kd Values ◽  
Mean Molecular Weight

Abstract Previous studies have demonstrated that protein C (PC) can be activated by factor Xa (fXa) in a reaction that requires Ca2+ and negatively-charged phospholipid. Sulfated polysaccharides, such as heparin or dextran sulfate, have been shown to accelerate this reaction, although their mechanism of action remains elusive. To further explore this phenomenon, we first examined the effect of glycosaminoglycans of varying degrees of sulfation on the kinetics of PC activation by fXa in the presence of Ca2+ and phosphatidylcholine-phosphatidylserine vesicles (75%/25% w/w). Heparin increased the rate PC activation in a concentration-dependent and saturable fashion producing a 4-fold increase in catalytic efficiency (kcat/Km of 105 M−1 min−1) by reducing the Km for the reaction. In contrast N-desulfated heparin had no effect on the rate of this reaction, whereas dextran sulfate, which is more sulfated than heparin, increased the catalytic efficiency 21-fold. These data suggest that the capacity of glycosaminoglycans to catalyze PC activation by fXa is dependent on their degree of sulfation. The extent of sulfation is more important than chain length because hypersulfated low-molecular-weight heparin (HSLMWH) and dextran sulfate, both of which have a mean molecular weight of 5000, increased the catalytic efficiency 16- and 21-fold respectively. In contrast, enoxaparin, which also has a mean molecular weight of about 5000, had little effect. The capacity of heparin to enhance PC activation by fXa is similar in the presence of factor Va as it is in its absence, suggesting that heparin can accelerate this reaction even when fXa is incorporated within the prothrombinase complex. To begin to explore the mechanism by which these glycosaminoglycans enhance PC activation by fXa, we measured their affinities for PC and fXa, both of which have heparin-binding domains, in the presence of Ca2+. This was performed by monitoring changes in extrinsic fluorescence of fluorescein-labeled fXa or PC after addition of glycosaminoglycan. Heparin binds PC with similar affinity in the absence or presence of negatively-charged phospholipid (Kd values of 1.9 and 1.0 mM, respectively). In contrast, heparin binds fXa with 86-fold higher affinity in the presence of phospholipid vesicles than in its absence (Kd values of.007 and 0.61 mM, respectively). These findings suggest that fXa binding to phospholipid exposes a high-affinity heparin-binding site. In the absence of phospholipid, more sulfated glycosaminoglycans (dextran sulfate and HSLMWH) bind fXa with 2- to 3-fold higher affinity than heparin. These compounds exhibit a smaller increase in affinity for PC. These observations suggest that the capacity of glycosaminoglycans to enhance PC activation is dependent on the extent of sulfation, a feature that determines their affinity for fXa. How glycosaminoglycan binding to fXa modulates this reaction is uncertain, but it is more likely to reflect conformational changes in the enzyme than bridging of the enzyme to the substrate.

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 147-155
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkaduisz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Biologically Active ◽  
Matrix Proteins ◽  
Hematopoietic Progenitor Cell ◽  
Heparin Binding ◽  
Heparan Sulfates ◽  
Kinetics Of ◽  
Better Than

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

Heparin-Associated Thrombocytopenia: The Antibody Is Not Heparin Specific

1992 ◽  
Vol 67 (05) ◽  
pp. 545-549 ◽  
Author(s):  
A Greinacher ◽  
I Michels ◽  
C Mueller-Eckhardt
Keyword(s):  
Platelet Activation ◽  
Dextran Sulfate ◽  
Dermatan Sulfate ◽  
Solid Phase ◽  
Binding Capacity ◽  
Low Grade ◽  
Type Ii ◽  
Pentosan Polysulfate ◽  
Heparin Administration

SummaryIn this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration. As Org 10172 contains a small amount of a LMW heparin-like substance (3%) this heparinoid should not be used in HAT patients without prior in vitro testing. We conclude that heparin-associated thrombocytopenia is not caused by a heparin-specific antibody and that a major factor contributing to the pathomechanism is the high grade of sulfation present in a variety of polysulfated oligosaccharides.

Sulfated Polysaccharide Anticoagulants Suppress Natural Killer Cell Activity In Vitro

1995 ◽  
Vol 74 (04) ◽  
pp. 0998-1002 ◽  
Author(s):  
Sabine Johann ◽  
Christine Zoller ◽  
Sylvia Haas ◽  
Günther Blümel ◽  
Martin Lipp ◽  
...  
Keyword(s):  
Natural Killer Cell ◽  
Natural Killer ◽  
Cell Function ◽  
Anticoagulant Activity ◽  
Natural Killer Cell Activity ◽  
Killer Cell ◽  
Cell Activity ◽  
Sulfated Polysaccharides ◽  
Killer Cell Activity

SummaryIn contrast to the well studied anticoagulant activity of sulfated polysaccharides (SPS), little is known about their influence on immune competent cells. Using two naturally derived SPSs (unfractionated heparin and low molecular weight heparin), one semi-synthetic SPS (pentosanpolysulfate), and one synthetic SPS (lactobionic acid) as well as the polypeptide hirudin we investigated the effect of these drugs on natural killer cell activity in vitro. We demonstrate that all SPSs tested significantly suppress the activity of natural killer cells at clinically relevant dosages. At all concentrations and at all effector:target ratios tested pentosanpolysulfate was the most potent natural killer cell inhibitor. In contrast, hirudin had no effect on natural killer cell function. Furthermore, scanning electron microscopy revealed that reduced natural killer cell activity is paralleled by decreased lymphocyte cell size and altered cell surface structures. Our results indicate that defined therapeutically applied SPSs can interfere with the tumor cell killing process.

Unbalanced Effects of Dermatan Sulfates with Different Sulfation Patterns on Coagulation, Thrombosis and Bleeding

2001 ◽  
Vol 86 (11) ◽  
pp. 1215-1220 ◽  
Author(s):  
C. P. Vicente ◽  
P. Zancan ◽  
L. L. Peixoto ◽  
R. Alves-Sá ◽  
F. S. Araújo ◽  
...  
Keyword(s):  
Dermatan Sulfate ◽  
Thrombus Formation ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Heparin Cofactor Ii ◽  
Iduronic Acid ◽  
Bleeding Effect ◽  
Mammalian Counterpart

SummaryWe compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated α-L-iduronic acid and 4-O-sulfated N-acetyl-β-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated α-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-β-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.

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