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Biomedical subjects

J Dawes

Publications and source records attributed to J Dawes.

At least 19 recordsLinked to original sources
PubMed1997

Production method affects the pharmacokinetic and ex vivo biological properties of low molecular weight heparins.

Low molecular weight (LMW) heparins have prolonged circulating half-lives relative to unfractionated heparin, but the rates of plasma clearance differ between different LMW preparations. To determine the impact of method of production on their pharmacokinetic and ex vivo biological properties, two LMW heparins of similar molecular weight distribution, Logiparin and Fragmin, were radiolabelled with 125I, administered intravenously with 4 mg/kg of carrier drug into rabbits, and the circulating radiolabelled material and anti-Xa activity were analysed by size exclusion chromatography and affinity for antithrombin and Polybrene. Following administration of Logiparin, the anti-Xa amidolytic activity was eliminated with the same half-life as the anti-thrombin-binding radiolabel and was not neutralised by antibody against tissue factor pathway inhibitor (TFPI). Larger molecules were cleared preferentially and were no longer detectable 8 h post injection. These findings resemble those we have previously described for Enoxaparin. After Fragmin administration the antithrombin binding radiolabel was cleared more rapidly than the anti-Xa activity, and at late times after injection a significant amount of this activity was neutralised by antibody against TFPI. Sulphated radiolabel was eliminated with a similar half-life to the anti-Xa activity and sulphated molecules > 6000 Da remained in the circulation 8 h after administration. Fragmin, unlike Logiparin and Enoxaparin, has no negatively charged sulphamino group at the reducing end of the molecule. We suggest that this minimises cellular interaction and protects the larger molecules from elimination. They remain in the circulation, contributing to anti-Xa activity by binding TFPI. Thus the method of production of LMW heparins may significantly influence their pharmacokinetic properties and circulating anticoagulant activities.

Animals↗
PubMed1996

Fibroblast growth factor and heparin protect endothelial cells from the effects of interleukin 1.

Vascular endothelium is involved in both active and passive processes in haemostasis, but inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF) have been reported to convert the comparatively inert endothelial cell to an inflammatory state. Acidic fibroblast growth factor (aFGF) in the presence of heparin has effects opposite to IL-1 on cultured human umbilical vein endothelial cells (HUVEC); therefore, we have investigated the modulation of IL-1-induced effects by the c combination of aFGF and heparin (aFGF/heparin). First passage HUVEC were cultured for 6 days in the presence of 20% human serum with and without the addition of 625 pM human recombinant aFGF (hr aFGF) and 7 microM heparin. On day 5, recombinant IL-1 beta was included for 24 h. The following day the cells were washed and measurements made of the release of prostacyclin, von Willebrand factor, plasminogen activator inhibitor type 1, and thrombospondin, both in the resting state and following stimulation for 60 min with 1 U/ml thrombin. Tissue-type plasminogen activator was assayed in HUVEC lysates. Similar experiments were performed to assess effects on the expression of vascular adhesion molecule, intracellular adhesion molecule, and E-selectin using an ELISA on cells in situ. This study indicates that aFGF/heparin in the culture medium of HUVEC abrogates the measured responses to IL-1. These data imply that routine endothelial cell culture with aFGF/heparin may cause artefacts, the effects of FGF and Il-1 may involve common pathways, and FGF/heparin may offer an approach to design therapeutics to counter the adverse effects of IL-1.

Cell Adhesion Molecules↗
PubMed1996

Long-term persistence of biological activity following administration of Enoxaparin sodium (clexane) is due to sequestration of antithrombin-binding low molecular weight fragments--comparison with unfractionated heparin.

We have previously reported (Brieger D, Dawes J. Thromb Haemost 1994; 72: 275-80) that the prolonged anti-Xa amidolytic activity following intravenous administration of the low molecular weight heparin Enoxaparin sodium is mediated by small molecules derived from the injected drug, and an antithrombin binding penta/hexasaccharide can be detected in the circulation as late as 1 week after administration. To investigate the mechanism underlying this persistence we administered 125I-labelled fractions of Enoxaparin sodium and unfractionated 125I-heparin to rabbits. Both 125I-heparin and the radiolabeled high molecular weight (> 6000 Da) Enoxaparin sodium were more effectively cleared from the circulation than the smaller components of LMW heparin. However, our data suggest that the circulating biologically active penta/hexasaccharide was not an unmodified component of the injected drug but was derived from a subpopulation of molecules of intermediate molecular weight (1800-6000 Da) which was retained in the tissues. Significant quantities of both Enoxaparin sodium and unfractionated heparin were retained in the internal organs. We propose that the sequestered subpopulations of Enoxaparin sodium and unfractionated heparin follow different catabolic routes. After administration of both unfractionated and LMW heparin additional antithrombin binding material was released into the circulation by a bolus dose of heparin. This material was not contained on circulating blood cells and was probably sequestered on the endothelium.

Animals↗
PubMed1996

Low molecular weight heparin is responsible for the anti-Xa activity of Desmin 370.

Dermatan sulphate does not catalyse the inactivation of factor Xa. However, the low molecular weight (LMW) dermatan sulphate Desmin 370 has been shown to generate circulating anti-Xa activity following administration to humans. Using a single batch of Desmin 370, we measured 3 U/mg of anti-Xa activity by amidolytic assay in vitro. The material responsible for this activity had a lower molecular weight range (6000 and 1800 Da) than Desmin 370 and was more highly sulphated than the bulk of the drug. Heparinase digestion of Desmin 370 eliminated 90% of the in vitro anti-Xa activity without significantly interfering with its ability to potentiate inactivation of thrombin by HCII, suggesting that the anti-Xa activity is not due to dermatan sulphate and is probably heparin. When 125I-labelled Desmin 370 together with 40 mg/kg carrier drug was administered intravenously to a rabbit, anti-Xa activity was readily detectable in the plasma for up to 10 h and had a longer half-life than the sulphated radiolabel. Most of this anticoagulant activity was recovered from the plasma by Polybrene affinity chromatography and was probably a sulphated glycosaminoglycan. Administration of the heparinase-digested drug to a rabbit resulted in 70% less anti-Xa activity than the undigested drug. We conclude that Desmin 370 contains detectable quantities of biologically active low molecular weight heparin, which is responsible for persistent anti-Xa activity following intravenous administration.

Animals↗
PubMed1995

Fibrinogen inhibits the heparin cofactor II-mediated antithrombin activity of dermatan sulfate.

Dermatan sulfate is a naturally occurring antithrombotic glycosaminoglycan. The antithrombin activity of several dermatan sulfate preparations has been measured in whole human plasma and found to be -55% of that in purified systems. Kinetic studies under pseudo-first-order conditions indicated that the reduction in antithrombin activity of dermatan sulfate in plasma compared with that in buffer was due to noncompetitive inhibition with respect to dermatan sulfate. Analysis of the protein profile bound to immobilized dermatan sulphate showed that on a molar basis, histidine-rich glycoprotein and apolipoprotein E were the most abundant proteins specifically bound, together with significant amounts of fibrinogen and vitronectin. Addition of these proteins to the purified system showed that only fibrinogen inhibited the antithrombin activity of dermatan sulfate and that it did so in a concentration-dependent manner over the physiologic range of plasma fibrinogen levels. These results indicate that the anticoagulant activity of dermatan sulfate may be modulated in human plasma by fibrinogen.

Animals↗
PubMed1995

Acidic and basic fibroblast growth factors have comparable effects on the haemostatic function of vascular endothelium.

Acidic and basic fibroblast growth factor (aFGF and bFGF respectively) are closely related mitogens (55% homology) of the heparin binding growth factor family. Reports of the relative potency of these growth factors and the ability of heparin to potentiate the activity of bFGF are conflicting. We have examined the effect of heparin and human recombinant aFGF and bFGF on basal and thrombin challenged release of metabolites from cultured human umbilical vein endothelial cells (HUVEC). Culture supernatant was assayed for thrombospondin, prostacyclin and PAI-1 and cell lysates were analysed for t-PA. aFGF and bFGF were equipotent in regulating ther release of all metabolites studied, except thrombin stimulated release of PGI2 where bFGF was more potent than aFGF in the absence of heparin. Heparin potentiated the mitogenic and metabolic effects of both bFGF and aFGF. However, heparin was not essential for the expression of the biological activity of FGF.

Animals↗
PubMed1995

Augmentation of vascular endothelial barrier function by heparin and low molecular weight heparin.

Glycosaminoglycans (GAGs) are an important component of endothelial barrier function. Early passage human umbilical vein endothelial cells were grown to confluence on transparent micropore filters and barrier function assessed as transendothelial electrical resistance (TEER) and permeability to albumin and sucrose. Unfractionated heparin and the LMW heparin Clexane decreased endothelial permeability to both sucrose and albumin and increased TEER. Chondroitin 6-sulphate also augmented barrier function, but other GAGs had no effect. Interleukin-1 increased permeability to albumin and sucrose and decreased TEER. Although heparin attenuated the effect of IL-1 on TEER and sucrose permeability, it could not restore the barrier to albumin transfer. Denuded endothelial matrix presented a negligible barrier, which was not enhanced by heparin. When sulphation of endogenous GAGs was inhibited by chlorate, barrier function was compromised and was not restored by exogenous heparin. Thus heparin enhances the barrier function of resting endothelium, but cannot completely overcome the increased permeability resulting from exposure to IL-1 or substitute for endogenous GAGs.

Albumins↗
PubMed1995

Role of the liver and kidney in the desulphation of heparin in vivo.

Heparin radiolabelled with 125I was given intravenously to intact, bilaterally nephrectomised or completely hepatectomised rats in the presence and absence of 1 mg unlabelled heparin/kg. Plasma samples were collected and analysed by gel filtration chromatography and affinity chromatography on Polybrene-Sepharose, which binds sulphated glycosaminoglycans. Radiolabel in the plasma was associated with both intact heparin and fully desulphated macromolecular carbohydrate chains. Levels of intact heparin in plasma from control rats decreased with time at both doses with a concomitant increase in desulphated material. Livers accumulated the greatest amounts of radiolabel on a per organ basis. Hepatectomy both increased levels of radiolabel in plasma and decreased amounts of desulphated heparin produced, indicating that the liver was the major site of desulphation. Even after removal of the liver, the desulphated metabolite comprised approximately 15% of radiolabelled material at all times and doses, suggesting an additional site of GAG desulphation which may be the vascular endothelium. The kidneys contributed significantly to in vivo desulphation of heparin only at the high dose.

Animals↗
PubMed1994

Conformational change in antithrombin induced by heparin probed with a monoclonal antibody against the 1C/4B region.

A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Desulphation of dextran sulphate during kidney ultrafiltration.

The renal clearance of [3H]dextran sulphate by the isolated perfused rat kidney was associated with desulphation of the molecule, as demonstrated by ion-exchange and affinity chromatography of material resident in both glomeruli and urine samples. This process also occurred in vivo. The molecular size distribution of glomerular dextran sulphate in the perfused kidney was indistinguishable from that in the perfusate, and although urinary material was smaller it remained macromolecular. Sulphatase activity was not detected in urine or in the perfusate of perfused kidneys, but was detected in glomerular and non-glomerular cortex fractions isolated by a sieving procedure. The identification of significant biochemical changes to dextran sulphate demonstrates that it does not function as an inert transport probe, and supports the concept of cellular involvement in the process of renal charge selectivity.

Animals↗
PubMed1994

Transendothelial transport of modified low-density lipoproteins.

Human umbilical vein endothelial cell monolayers were grown as monolayers on porous filters and their transcellular transport and degradation of 125I-labelled native and modified forms of LDL, supplied to either the intimal or the luminal face, were measured. Intact native, acetylated and oxidized LDL were all transported in both directions across the cell monolayers by receptor-independent mechanisms, and all forms of LDL were transported at similar rates. However, the mass of intact LDL transported from the intimal to the luminal face of the monolayer was always fourfold more than that transported in the opposite direction under similar conditions. In addition to LDL transport, endothelial cell monolayers also degraded native and modified forms of LDL by predominantly receptor-dependent routes, in that these could be inhibited (> 70%) by the addition of a 20-fold excess of the same form of (but unlabelled) LDL. The measured amounts of lipoprotein degraded were the same whether supplied to the intimal or the luminal face. Incubation of endothelial cells with oxidized LDL led to intracellular accumulation of a pool of macromolecular apo B which was apparently resistant to lysosomal proteolysis.

Biological Transport↗
PubMed1994

The effects of hyperthermia on human endothelial monolayers: modulation of thrombotic potential and permeability.

The effects of hyperthermia on potentially prothrombotic endothelial function were investigated by measuring levels of von Willebrand factor, thrombospondin, tissue plasminogen activator and plasminogen activator inhibitor-1 secreted by unstimulated human umbilical vein endothelial cells cultured at 37 degrees C, 39 degrees C, 41 degrees C and 43 degrees C for 24 h. Endothelial barrier function at 43 degrees C was compared with that at 37 degrees C by measuring permeability to radiolabelled human serum albumin and low density lipoprotein. Thrombospondin levels were unaffected by a temperature of 39 degrees C; they increased after 3 h at 41 degrees C and subsequently declined to values significantly below the 37 degrees C control. At 43 degrees C, secretion exhibited a time-dependent decrease. Secretion of von Willebrand factor was not discernibly affected by exposure to 39 degrees C or 41 degrees C. Its response to 43 degrees C resembled that of thrombospondin to 41 degrees C. In contrast, elevated temperatures markedly increased plasminogen activator inhibitor-1 while decreasing t-PA secretion, though after prolonged exposure to 43 degrees C the levels of both returned to control values. After 12-24 h at 43 degrees C, endothelial permeability to both albumin and low density lipoprotein increased markedly. Vascular endothelium may contribute to the thrombotic tendency associated with heat stroke by increasing access to the prothrombotic subendothelium and reducing fibrinolysis.

Body Temperature Regulation↗
PubMed1994

Low-affinity material does not contribute to the antithrombotic activity of Orgaran (Org 10172) in human plasma.

Orgaran is a LMW heparinoid composed of heparan sulphate (83% w/w) of which 4-5% has high affinity for antithrombin, dermatan sulphate (12% w/w) and chondroitin sulphate (5% w/w). To examine the contribution of the low-affinity fraction to Orgaran's antithrombotic activity we have quantitated the binding of plasma proteins to Orgaran and its component fractions in whole, hirudin-anticoagulated human plasma. Antithrombin, largely bound to the high-affinity fraction, and histidine-rich glycoprotein, interacting with low-affinity components, were the dominant proteins bound to Orgaran. Vitronectin, fibrinogen, fibronectin, heparin cofactor II, and apolipoprotein B were also detected in small amounts. The ratio of bound antithrombin, histidine-rich glycoprotein and vitronectin to GAG was negatively correlated with the Orgaran concentration in plasma, implying that the efficacy of Orgaran may not be linearly related to dose. Binding of antithrombin to the high-affinity fraction was not decreased by other plasma proteins or affected by addition of low-affinity material. Moreover, the antithrombin and anti-factor Xa activities of the high-affinity material were unaltered by low-affinity GAGs. On the basis of our results we conclude that the low-affinity material does not contribute to the antithrombotic activity of Orgaran by binding non-anticoagulant plasma proteins and releasing the high-affinity chains to interact with antithrombin and its target proteinases.

Anticoagulants↗
PubMed1994

Characterisation of persistent anti-Xa activity following administration of the low molecular weight heparin enoxaparin sodium (Clexane).

It is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected 125I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

Animals↗
PubMed1993

Interaction of immobilised unfractionated and LMW heparins with proteins in whole human plasma.

The profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (M(r) = 12,000), whereas heparin cofactor II constituted < 1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipoprotein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-alpha-trypsin inhibitor, alpha 2-macroglobulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (M(r) = 4,500) also bound antithrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (M(r) = 2,200).

Blood Proteins↗
PubMed1993

Cross-linked fibrin degradation products, progression of peripheral arterial disease, and risk of coronary heart disease.

Haemostatic and rheological factors may predict cardiovascular disease. We studied patients with intermittent claudication to see if the progression of peripheral arterial disease and the risks of coronary events could be predicted by baseline packed cell volume, plasma fibrinogen, blood and plasma viscosites, von Willebrand factor antigen, cross-linked fibrin degradation products (XLFDP), urinary fibrinopeptide A, and plasma leucocyte elastase. In 617 patients with claudication followed up for one year, baseline XLFDP was related most strongly to coronary events, relative risk 4.4 (95% CI 1.3-19.0) between top and bottom quintiles. Plasma fibrinogen was the strongest independent predictor of death from coronary disease. XLFDP was the only factor, in addition to age and cigarette smoking, that was independently associated (p = 0.008) with deterioration in peripheral arterial disease. We conclude that, in patients with peripheral arterial disease, plasma concentration of XLFDP, a measure of ongoing fibrin formation and degradation, is a strong predictor of both disease progression and future coronary risk. These results accord with the hypothesis that fibrin formation contributes to progression of coronary and peripheral atherosclerosis.

Adult↗
PubMed1993

The pharmacokinetics of LMW dermatan sulphate: long-term persistence of intact material.

An iodinated derivative of the low molecular weight dermatan sulphate Desmin 370 was administered to rats by intravenous, subcutaneous and intramuscular routes in conjunction with unlabelled Desmin 370. Following intravenous injection radiolabel was cleared from the plasma according to a tri-exponential function with elimination half-lives of 45 min and 13 h; there was a high concentration in tissues, particularly the kidney, compared with plasma. The apparent bioavailability following s.c. injection was 52-65%, but 84-100% after i.m. administration. Undegraded as well as desulphated molecules were identified in plasma, and intact 125I-Desmin 370 comprised > 85% of the radiolabel in urine 48 h post-injection. Thus intact Desmin 370 persists for a long period after a single injection. The pharmacological significance of these findings, however, remains to be determined.

Animals↗