Heparin Fractions Research Articles

Heparin promotes the inactivation of antithrombin by neutrophil elastase.

Heparin is an acceleratory cofactor for antithrombin, a circulating inhibitor of blood coagulation enzymes. The presence of heparin on blood vessel walls is believed to contribute to the nonthrombogenic properties of those surfaces. In apparent opposition to this function, heparin was found to greatly accelerate the in vitro inactivation of antithrombin by neutrophil elastase. Inactivation rates in solution were potentiated several hundredfold by specific heparin fractions with anticoagulant activity. Although the data suggest that a heparin-antithrombin complex is essential for the inactivation by elastase to occur, the enzyme itself interacts tightly with heparin. These results suggest a mechanism which, if operating in vivo, could lead to a localized neutralization of the anticoagulant function of heparin at the endothelial surface.

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides.

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.

Inhibition of vascular smooth muscle cell proliferation in culture by pentosan polysulphate and related compounds

Pentosan polysulphate (PPS; SP 54; Hemoclar), a highly sulphated semi-synthetic polysaccharide of MW 4.7kD (1), was tested in vitro as an inhibitor of rabbit aortic smooth muscle cell (SMC) proliferation and its effects were compared with those of dextran sulphate, laminarin sulphate and heparin fractions. When added to the cell cultures simultaneously with foetal calf serum, all sulphated poly-saccharides inhibited cell growth. PPS was five fold more active than heparin and its low molecular weight fractions. A high molecular weight fraction of PPS was the most active inhibitor (IC 50: 2.7μg/ml vs 45μg/ml for PPS, and 241μg/ml for heparin). When PPS and heparin were pre-incubated with the cells for three days, then removed before foetal calf serum was added to stimulate growth, their inhibitory effects were comparable with those seen when the compounds were present throughout the growth phase, suggesting that they exert their effect by binding to the cell surface, and not by interacting with the growth factors in serum.

The disappearance of a low molecular weight heparin fraction (cy 216) differs from standard heparin in rabbits

In previous studies, we have reported that standard heparin (SH) was cleared by two mechanisms, a saturable mechanism which predominated at low doses (<100 anti-factor Xa U/kg) and a non-saturable mechanism which predominated at higher doses, when the first mechanism became saturated. In this study, we examined the importance of these two mechanisms in the disappearance of a low molecular weight heparin fraction (LMWH) (CY 216), by comparing the pharmacokinetics and the pharmacodyna-mics of a wide range of doses of SH and CY 216 (1.5 to 500 anti-factor Xa U/kg) Pharmacokinetics was measured as the disappearance of 125I-radiolabelled SH or CY 216. Pharmacodynamics was measured as the disappearance of the anti-factor Xa activity of SH and CY 216. We found that the saturable mechanism contributed little to the disappearance of CY 216 and that it was cleared predominantly by the non-saturable mechanism at all doses tested. Thus, at low doses (<100 anti-factor Xa U/kg), SH was cleared more rapidly than CY 216, whereas at higher doses, CY 216 was cleared more rapidly than SH. We conclude that the mechanism of disappearance of LMWH's differ significantly from those of SH, and that this difference may explain the apparent prolonged anticoagulant activity of LMWH's within the therapeutic range doses.

The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.

Treatment of acute pulmonary embolism by a low molecular weight heparin fraction. A preliminary study.

We evaluated the antithrombotic efficacy of the low molecular weight heparin (LMWH) fraction PK 10169 in nine consecutive patients with acute pulmonary embolism documented by pulmonary angioscan and angiography. Therapy with PK 10169 was initiated by an i.v. bolus of 0.5 mg/kg, followed by a continuous intravenous infusion during the first 10 days; the drug was then given subcutaneously twice daily during the following 15 days. The dosage of PK 10169 was adjusted by daily measurements of anti-Xa and anti-IIa activities using amidolytic methods. For a dosage ranging from 1.4 to 4.1 mg/kg per day during the i.v. period and from 0.7 to 3.5 mg/kg per day during the s.c. period, the anti-Xa activity ranged from 4 to 8.7 PK U/ml and from 4.5 to 7.2 PK U/ml respectively. Clinical improvement was observed in all the patients and was consistent with progressive reperfusion evaluated by successive angioscans. No recurrence of pulmonary embolism occurred. No deleterious hemorrhagic side-effects were observed, even in two patients at high risk of bleeding. In this pilot study, the LMWH fraction PK 10169 proved to be an effective anticoagulant therapy during the first three weeks after pulmonary embolism in man.

Potentiation by Heparin Fragments of Thrombolysis Induced with Human Tissue-Type Plasminogen Activator or Human Single-Chain Urokinase-Type Plasminogen Activator

The effect of heparin and of two low molecular weight (low Mr) fractions of heparin on thrombolysis with recombinant human tissue-type plasminogen activator (rt-PA, Genentech Inc., So. San Francisco, CA) or human single chain urokinase-type plasminogen activator (scu-PA, Sandoz AG, Basle, Switzerland) was measured in a rabbit jugular vein thrombosis model. Four bolus injections of 200 anti-Factor Xa units/kg body weight of heparin (Liquemine, Hoffmann-La Roche, Basle, Switzerland), of 90 units/kg of CY 216 (Choay, Paris, France) or of 90 units/kg of CY 222 (Choay, Paris, France) were given intravenously, immediately after the start of the infusion of rt-PA or scu-PA and at hourly intervals during their intravenous infusion over 4 hours. The bolus injections resulted in anti-Factor Xa levels in plasma of 5.7 +/- 1.2 units/ml just before the repeat bolus injections of heparin with corresponding values of 3.9 +/- 0.2 units/ml for CY 216 and 1.6 +/- 0.2 units/ml for CY 222. Thrombolysis with 0.25 mg/kg rt-PA was 36 +/- 1 percent (n = 9) in the absence of anticoagulant, 40 +/- 1 percent (n = 7, p less than 0.05) in the presence of heparin, 49 +/- 5 percent (n = 7, p less than 0.02) with CY 216 and 62 +/- 5 percent (n = 7, p less than 0.01) with CY 222.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight heparin fractions as an alternative therapy in heparin-induced thrombocytopenia.

Eight patients with a delayed-onset heparin-induced thrombocytopenia, with thrombotic complications requiring immediate anticoagulation in 7 of them, were given low molecular weight heparin (LMWH) fractions as alternative therapy. This treatment led to normalization of platelet count within 3-5 days in 6 patients with clinical recovery in 5. In 2 patients, thrombocytopenia persisted despite LMWH therapy. In vitro platelet aggregation tests performed in all patients gave evidence of a relationship between the presence (or absence) of a LMWH-dependent platelet-aggregating factor in the patients' plasma and the persistence (or correction) of the thrombocytopenia with LMWH therapy. Although positive in vitro tests may not necessarily be associated with thrombocytopenia, in vitro testing may prove to be a useful guide before giving LMWH fractions as an alternative therapy in patients with heparin-induced thrombocytopenia requiring immediate anticoagulation.

Mechanism of inhibition of mammalian DNA topoisomerase I by heparin.

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.

Comparative Study on the Effects of a Low Molecular Weight Heparin (CY 216) and Standard Heparin in Low Dosage on Experimental Venous Thrombosis

In a venous thrombosis model in the rat obtained by ligation of the inferior vena cava, the effects of standard heparin and CY 216, a low molecular weight heparin fraction, are compared. At a dosage of less than 2 mg/kg b.w. CY 216 is an effective antithrombotic, whereas standard heparin is not.

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase

Recently, the development of low molecular weight heparin fractions and fragments. (LMHF) as potential anti-thrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anti-coagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydis-perse heparin both in vitro and in vivo . To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMMF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, CY 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal, therapy.

Are molecular weight and anti-Xa activity sufficient to predict the antithrombotic power of heparin fractions

Are molecular weight and anti-Xa activity sufficient to predict the antithrombotic power of heparin fractions

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.

Endothelial binding sites for heparin. Specificity and role in heparin neutralization

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

Heparin fractions with high and low affinities to antithrombin iii are cleared at different rates

The clearance characteristics of standard heparin (SH) and its fractions with high and low affinity to antithrombin III (HAH and LAH respectively) were studied by injecting 125I-labelled SH, HAH and LAH intravenously into the rabbits in increasing doses. Serial blood samples were then collected from an indwelling cannula for measurement of clearance, based both on radioactivity and anticoagulant activity. For equivalent weights injected, the radioactivity of HAH was cleared more slowly from the circulation than that of either SH or LAH. The radioactivity of SH, in turn, was cleared more slowly than that of LAH. The clearance of HAH, measured both by radioactivity and by anticoagulant activity were similar, whereas the clearance of the radioactivity of SH was more rapid than its anticoagulant activity (i.e. anti-factor Xa activity). These observations suggest that the radioactivity clearance curves of SH reflect a “net” estimation of the more complex clearance curves of the different heparin moieties whereas the anticoagulant clearance curves of SH reflects the clearance of the anticoagulant activity of a specific heparin fraction, in this case, the clearance of the anti-factor Xa activity of HAH. These differences suggest that the HAH:LAH ratio is, at any given time, greater in vivo than the same ratio measured in vitro.

The relative molecular mass dependence of the anti-factor Xa properties of heparin.

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.

Comparison of the haemorrhagic effects of unfractionated heparin and a low molecular weight heparin fraction (cy 216) in rabbits

Comparison of the haemorrhagic effects of unfractionated heparin and a low molecular weight heparin fraction (cy 216) in rabbits

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen.

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.

Modulation of endothelial cells growth induced by heparin

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1 % serum concentration and was abolished at 10 %. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth. In the past few years, many interactions of heparin with vascular cells have been demonstrated. Heparin binds specifically to endothelial cells (Glimelius et al, 1978; Bârzu et al, 1985a) as well as to smooth muscle cells (Castellot et al, 1985b). It inhibits migration (Majack and Clowes, 1984) and proliferation (Hoover et al, 1980) of smooth muscle cells. On the other hand, heparin stimulates the migration of capillary endothelial cells (Azizkhan, 1982), and at high concentration (90 μg/ml) potentiates the action of endothelial cell growth supplement (ECGS) on endothelial cell growth (Thornton et al, 1983). No consistent effect of heparin alone on endothelial cell proliferation has been reported so far. We therefore have investigated the effects of heparin, heparin fractions and some heparin analogues on human endothelial cell growth. Here, we show an inhibition of 3H-thymidine uptake as well as cell growth in the presence of heparins, when tested at low human serum concentration. When endothelial cells are grown in the presence of ECGS, however, heparin potentiates the growth stimulatory effect of ECGS.