Neutralization of enoxaparine-induced bleeding by protamine sulfate

It has been suggested that protamine sulfate is a poor antidote for the bleeding side-effects of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effects of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin +/- protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effects of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.     

Fucosylated chondroitin sulfate as a new oral antithrombotic agent

Fucosylated chondroitin sulfate is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of sulfated fucose branches. We have shown that intravascular injection of fucosylated chondroitin sulfate inhibits thrombus formation in a venous and an arterial shunt model in rats. Since this compound resists digestion by enzymes that cleave mammalian glycosaminoglycans, we investigated the possibility that fucosylated chondroitin sulfate might be absorbed after oral administration. In fact, after oral administration of fucosylated chondroitin sulfate to rats, we observed a dose-dependent increase in the plasma anticoagulant activity, as assessed by assays for activated partial thromboplastin time (aPTT) and thrombin time (TT) (about 3- and 5-fold, respectively) and by anti-IIa activity. Furthermore, animals receiving daily oral doses of this glycosaminoglycan showed a decrease in thrombus weight on experimental models of venous and arterial shunt thrombosis. This antithrombotic action clearly has a strong relationship with anticoagulant activity. Similar doses of heparin administered orally had no effect on the plasma anticoagulant activity or on the thrombus weight. Finally, we observed that fucosylated chondroitin sulfate given orally to rats did not modify the bleeding time. Overall, our results indicate that fucosylated chondroitin sulfate is absorbed after oral administration and could become a promising oral anticoagulant.     

Chemical Sulfonation and Anticoagulant Activity of Acharan Sulfate

Acharan sulfate is a glycosaminoglycan prepared from the giant African snail, Achatina fulica. This polysaccharide has a repeating disaccharide structure of →4)-2-deoxy-2-acetamido--D-glucopyranose (1→4)-2-sulfo--L-idopyranosyluronic acid (1→). Its structure is related to heparin and heparan sulfate but is distinctly different from all known members of these classes of glycosaminoglycans. Because of its structural similarities to heparin, chemically modified acharan sulfate was studied to understand the chemical structure effected its anticoagulant activity. After de-N-acetylation, acharan sulfate was N-sulfonated using either chlorosulfonic acid-pyridine or sulfur trioxide-trimethylamine complex. The sulfate level in these products ranged from 22 to 24%(w/w), significantly less than that of heparin at 36%. The molecular weight of both N-sulfoacharan sulfates were comparable with that of heparin. In vitro anticoagulant activity assays showed that N-sulfoacharan sulfate derivatives were moderately active for the inhibition of thrombin and neither product showed any measurable anti–factor Xa activity. The differences in the activities of N-sulfoacharan sulfates produced by these two methods are probably ascribable to a small level of concomitant O-sulfonation obtained when using chlorosulfonic acid-pyridine.     

Serpin-independent anticoagulant activity of a fucosylated chondroitin sulfate

Fucosylated chondroitin sulfate is a glycosaminoglycan from sea cucumber composed of a chondroitin sulfate-like core with branches of sulfated fucose. This glycosaminoglycan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant activity is mostly due to activating thrombin inhibition by heparin cofactor II. Here, we evaluated the anticoagulant activity of fucosylated chondroitin sulfate using antithrombin- and heparin cofactor II-free plasmas. In contrast to mammalian heparin, the invertebrate glycosaminoglycan is still able to prolong coagulation time and delay thrombin and factor Xa generation in serpin-free plasmas. These observations suggest that fucosylated chondroitin sulfate has a serpin-independent anticoagulant effect. We further investigated this effect using purified blood coagulation proteins. Clearly, fucosylated chondroitin sulfate inhibits the intrinsic tenase and prothrombinase complexes, which are critical for thrombin generation. It is possible that the invertebrate chondroitin sulfate inhibits interactions between cofactor Va and factor Xa. We also employed chemically modified polysaccharides in order to trace a structure versus activity relationship. Removal of the sulfated fucose branches, but not reduction of the glucuronic acid residues to glucose, abolished its activity. In conclusion, fucosylated chondroitin sulfate has broader effects on the coagulation system than mammalian glycosaminoglycans. In addition to its serpin-dependent inhibition of coagulation protease, it also inhibits the generation of factor Xa and thrombin by the tenase and prothrombinase complexes, respectively. In plasma systems, the serpin-independent anticoagulant effect of fucosylated chondroitin sulfate predominates over its serpin-dependent action. This glycosaminoglycan opens new avenues for the development of antithrombotic agents.     

Inactivation of thrombin by a fucosylated chondroitin sulfate from echinoderm

A polysaccharide extracted from the sea cucumber body wall has the same backbone structure as the mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. These branches confer high anticoagulant activity to the polysaccharide. Since the sea cucumber chondroitin sulfate has analogy in structure with mammalian glycosaminoglycans and sulfated fucans from brown algae, we compared its anticoagulant action with that of heparin and of a homopolymeric sulfated fucan with approximately the same level of sulfation as the sulfated fucose branches found in the sea cucumber polysaccharide. These various compounds differ not only in their anticoagulant potencies but also in the mechanisms of thrombin inhibition. Fucosylated chondroitin sulfate, like heparin, requires antithrombin or heparin cofactor II for thrombin inhibition. Sulfated fucans from brown algae have an antithrombin effect mediated by antithrombin and heparin cofactor II, plus a direct antithrombin effect more pronounced for some fractions. But even in the case of these two polysaccharides, we observed some differences. In contrast with heparin, total inhibition of thrombin in the presence of antithrombin is not achieved with fucosylated chondroitin sulfate, possibly reflecting a less specific interaction. Fucosylated chondroitin sulfate is able to inhibit thrombin generation after stimulation by both contact-activated and thromboplastin-activated systems. It delayed only the contact-induced thrombin generation, as expected for an anticoagulant without direct thrombin inhibition. Overall, the specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only confer high anticoagulant activity to the polysaccharide but also determine differences in the way it inhibits thrombin.     

The anticoagulant effect of heparan sulfate and dermatan sulfate

The potency of the various glycosaminoglycans (GAGs) as anticoagulants in the activated partial thromboplastin time (APTT) test system has been investigated.

Heparin, heparan sulfate (HS) and dermatan sulfate (DS) exhibit anticoagulant effect. About 70 times higher concentrations of HS and DS were required to obtain the effect of heparin. The other GAGs, including chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate and hyaluronic acid (HA) did not prolong the APTT at the concentrations used.

The influence of the GAGs on thrombin and activated factor X (Xa), and on the inactivation of these clotting enzymes by antithrombin III (At-III) has been studied, using chromogenic substrates for the assay of enzyme activity. All GAGs, except keratan sulfate, had inhibitory effect on the amidolytic activity of thrombin. However, no GAG affected the amidolytic activity of Xa.

Heparin and HS accelerate the inactivation of thrombin and Xa by At-III. About 75 times higher concentration of HS was required to obtain the effect of heparin. DS and HA had a slight accelerating effect on the inactivation of thrombin by At-III, but only at a high concentration (500 μg/ml). At concentrations where DS exhibited anticoagulant activity in the APTT test system, no accelerating effect on At-III activity could be detected. Thus, the anticoagulant effect of DS is probably not exerted via At-III, and may in this respect differ from the anticoagulant activity of heparin and HS.     

Characterization of platelet binding of heparins and other glycosaminoglycans

The binding of glycosaminoglycans to intact washed human platelets was studied. The platelet binding of a ³H-labeled unfractionated heparin was saturable and reached equilibrium in 10–15 minutes. Heparin binding was specific: a 50-fold molar excess of an equivalent unlabeled heparin displaced up to 90% of labeled heparin, while chondroitin sulfate A and hyaluronic acid minimally displaced the binding of labeled heparin. Low molecular weight heparin fragments showed intermediate efficacy in displacing the binding of unfractionated [³h]heparin. Dextran sulfate (M 8,000, sulfation 17%) was as potent as unfractionated heparin in displacing binding, while neutral dextrans were ineffective. We observed that platelet activation by the calcium ionophore A23187 increased heparin binding by 2 to 3-fold, principally by enhancement of binding capacity not binding affinity. This process of heparin binding to the platelet surface may mediate some of the reported effects of heparin on platelet behavior.     

A small fraction of dermatan sulfate with significantly increased anticoagulant activity was selected by interaction with the first complement protein

Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1-->3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution.     

Inhibition of BACE1, the β-secretase implicated in Alzheimer’s disease,by a chondroitin sulfate extract from Sardina pilchardus

The pharmaceutical and anticoagulant agent heparin,a member of the glycosaminoglycan family of carbohydrates,has previously been identified as a potent inhibitor of a key Alzheimer’s disease drug target,the primary neuronalβ-secretase,β-site amyloid precursor protein cleaving enzyme 1(BACE1).The anticoagulant activity of heparin has,however,precluded the repurposing of this widely used pharmaceutical as an Alzheimer’s disease therapeutic.Here,a glycosaminoglycan extract,composed predominantly of 4-sulfated chondroitin sulfate,has been isolated from Sardina pilchardus,which possess the ability to inhibit BACE1(IC50[half maximal inhibitory concentration]=4.8μg/mL),while displaying highly attenuated anticoagulant activities(activated partial thromboplastin time EC50[median effective concentration]=403.8μg/mL,prothrombin time EC50=1.3 mg/mL).The marine-derived,chondroitin sulfate extract destabilizes BACE1,determined via differential scanning fluorimetry(ΔTm–5°C),to a similar extent as heparin,suggesting that BACE1 inhibition by glycosaminoglycans may occur through a common mode of action,which may assist in the screening of glycan-based BACE1 inhibitors for Alzheimer’s disease.     

Anticoagulant properties of extracellular slime substance produced by Staphylococcus epidermidis

Slime produced by S. epidermidis strain KH 11 was extracted with phenol-saline. The saline phase was fractionated on a DEAE-Sepharose CL-6B column. The crude slime solution and its phenol-saline fraction were found to possess anticoagulant properties. They inhibited the coagulation of human plasma by thrombin, prolonged the activated partial thromboplastin time, but did not change the rate of plasma coagulation by reptilase. The anticoagulant effect of slime could be neutralized by rather high concentrations of protamine sulphate. In the presence of plasma, the staphylococcal slime also inhibited in a concentration dependent fashion the amidolytic activity of thrombin and factor Xa against synthetic chromogenic substrates. Both antithrombin III (AT III) and other plasma component(s), presumably heparin cofactor II, were required for the full expression of the slime effect. The anticoagulant action of slime was markedly less AT III dependent than that of heparin. The activity was resistant to heating (100 degrees C, 30 min). Slime and its fractions were stronger inhibitors of thrombin than of factor Xa. Fraction IV separated by DEAE-Sepharose chromatography and particularly rich in galactose and glucuronic acid had the highest inhibitory potency. It is conceivable that slime component(s) similar to glycosaminoglycans from other sources can carry the anticoagulant activity.     

Dual effects of sulfated D-galactans from the red algae Botryocladia occidentalis preventing thrombosis and inducing platelet aggregation.

Sulfated D-galactans occur on the red algae Botryocladia occidentalis as three fractions that differ in their sulfate content. Fractions F2 and F3 are potent anticoagulants. Like heparin, they enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. The inhibition potency increases simultaneously with the sulfate content of the fractions. The antithrombotic activity of these sulfated D-galactans was investigated on an experimental thrombosis model in which thrombus formation was induced by a combination of stasis and hypercoagulability. In contrast with heparin. the sulfated D-galactans showed a dual dose-response curve preventing thrombosis at doses up to approximately 0.5 mg/ kg body weight but losing the effect at higher doses. This unexpected behavior is probably due to a combined action of the sulfated D-galactan as anticoagulant and also as a strong inducer of platelet aggregation. In platelet-depleted animals the antithrombotic activity at higher dose of sulfated D-galactan is restored and almost total inhibition of thrombus formation is achieved. The sulfated D-galactan has no hemorrhagic effect even at high doses, possibly as a consequence of its effect on platelet aggregation. At comparable dose heparin has an intense bleeding effect. These results indicate that new polysaccharides, with well-defined structures, can help to distinguish events, such as antithrombotic and anticoagulant activities, bleeding and platelet-aggregating effects, which are obscure when induced simultaneously by a single compound.     

Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro - modulatory action of glycosaminoglycans

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC₅₀ 255 ng/ml) was more pronounced than that of factor Xa (IC₅₀ 684 ng/ ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 μg/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3–40 μg/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40–50 % was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.     

The interaction of platelets with a tritium-labelled heparin

Platelets were found to take up tritium-labelled heparin either when platelet-rich plasma (PRP) was treated with the labelled heparin or if platelets, free of plasma proteins, obtained from chromatography of PRP on Sepharose 2B were treated with the tagged heparin. Sodium dodecyl sulfate (SDS) prevented uptake of heparin by platelets. Platelets were found to take up only “free” heparin but not heparin complexed with antithrombin III. 20% of the heparin was released by complete aggregation of the platelets. Treatment of ³H-heparinized platelets with a low dose of sodium dodecyl sulfate which did not disrupt the platelets liberated 40%. The heparin released in both cases had anticoagulant activity. Treatment with the low dose SDS followed by aggregation and then higher doses of detergents freed almost all of the heparin. It is concluded that uptake of heparin by platelets may involve a combination of several constituents: 1) platelet factor 4, 2) surface membranes, and 3) other granules or cytoplasmic components. Inasmuch as platelets do not take up heparin combined with antithrombin III, the physiologic significance of the complexing of heparin by platelets has not been settled.     

Treatment of established thrombotic events in patients with cancer

Acutely ill medical patients with cancer and cancer patients requiring Anticoagulants remain the cornerstone of therapy for venous thromboembolism. In patients with cancer, monotherapy with low molecular weight heparin (LMWH) is preferred over initial therapy with heparin followed by long-term treatment with vitamin K antagonists (VKA) because it is more efficacious and does not interfere with chemotherapy. The shorter duration of action of LMWHs compared with VKAs also offers greater flexibility to accommodate invasive procedures and thrombocytopenia. Newer oral antithrombotic agents may further simplify treatment of cancer-associated thrombosis because they are given at fixed doses and do not require laboratory monitoring of their anticoagulant effect. However, there are very limited data and experience with these agents in patients with cancer and some of these drugs do interact with a number of chemotherapeutic agents. Other unanswered clinical questions include the optimal duration of anticoagulant therapy, which anticoagulant to use after 6 months of treatment, how to treat patients with recurrent thrombosis or patients with a high risk for bleeding. Formal studies are needed to address these unmet clinical needs.     

Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route.

Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desulphation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.     

Complement inhibitory and anticoagulant activities of fractionated heparins

Almost monodisperse heparin fractions (Mw/Mn less than 1.1) were obtained by gel filtration of a commercial heparin. These fractions were assayed for anticoagulant activity (thrombin times and APTT), chromogenic anti-factor Xa activity, inhibitory activity for the human classical complement pathway, carboxyl group content and total sulfate content. Linear relationships were observed between the molecular weight of the heparin fractions and the anti-coagulant activities as determined by thrombin time- and APTT-assay and the classical complement pathway inhibitory activity. On the other hand a hyperbolic-like relationship was observed between the molecular weight of the heparin fractions and the chromogenic anti-factor Xa activity. The heparin fractions did not show significant differences with respect to the carboxyl group and total sulfate content. Low- and high affinity heparin fractions were obtained by affinity chromatography using immobilized AT III. High- and low-affinity fractions greatly differed not only with respect to their APTT activity, but also where their complement-inhibitory activities were concerned. The latter in contrast to literature data available. These differences could not be explained by the observed differences in molecular weight of high and low affinity heparin respectively.     

The nontoxic mushroom Auricularia auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin

An acidic polysaccharide with anticoagulant activity was isolated from the edible mushroom Auricularia auricula using water, alkali or acid extracts. The alkali extract showed the highest anticoagulant activity and was thereby further purified using gel filtration chromatography. Specific anticoagulant activity of the purified polysaccharide was 2 IU/mg and its average mass was approximately 160 kDa. The polysaccharide from this species of mushroom contains mainly mannose, glucose, glucuronic acid and xylose but no sulfate esters. Its anticoagulant activity was due to catalysis of thrombin inhibition by antithrombin but not by heparin cofactor II. Inhibition of Factor Xa by antithrombin was not catalyzed by the polysaccharide. The glucuronic acid residues were essential for the anticoagulant action of the mushroom polysaccharide since the activity disappeared after reduction of its carboxyl groups. In ex vivo tests using rats orally fed with the polysaccharide, we observed an inhibitory effect on platelet aggregation as observed with aspirin, a well-known antiplatelet agent. The polysaccharides from these mushrooms may constitute a new source of compounds with action on coagulation, platelet aggregation and, perhaps, on thrombosis.     

Low molecular weight heparins: are they superior to unfractionated heparins to prevent and to treat deep vein thrombosis?

In many countries, low molecular weight heparins (LMWHs) have replaced unfractionated heparin (UH) for prevention and treatment of venous thromboembolism. The present paper reviews the possible advantages of LMWHs over UH. In spite of their lower molecular weight distribution, LMWHs are functionally more heterogeneous than UH. Their anti-Xa/anti-IIa ratio varies significantly, and the injection of the same dose generates different anti-Xa activities and activated partial thromboplastin time (APTT) prolongations. Their pharmacodynamic properties account for their more convenient use in comparison with UH; however, there is a risk of accumulation in case of renal insufficiency. Even if they are less anticoagulant on the basis of the APTT prolongation, they are not less prohemorrhagic than UH. LMWHs are probably less immunogenic and probably induce less osteoporosis. Several meta-analyses published between 1992 and 1999 indicate that LMWHs are as efficient as UH in preventing postoperative deep vein thrombosis (DVT) in general surgery and more efficient than UH in preventing DVT in orthopedic surgery and treating established DVT.     

Fractionated tritium-labelled heparin studied in vitro and in vivo

Tritium-labelled commercially prepared porcine mucosal heparin was fractionated by chromatography on Sephadex G-200. The fractions obtained were studied for sulfate content, metachromasia, and ability to complex with antithrombin to produce anticoagulant activity. When added to platelet poor plasma and rechromatographed most of the higher molecular weight fractions were found between the globulin and albumin protein peaks combined with antithrombin.The fractions were grouped into three pools, which were injected into rats and their anticoagulant activity, tissue distribution and urinary excretion were studied in comparison with the whole heparin.The higher molecular weight material had the greater metachromasia and higher sulfate content. It yielded a higher and longer lasting anticoagulant activity than the whole heparin. When injected into rats about 40% of the material appeared free (uncomplexed with protein) in the urine in three hours although the major portion was excreted in the first half hour. Large amounts were also found in the liver and kidneys.