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.

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.

SummaryHeparin 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.

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.

4. Dahlbiick B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognised mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Nat! Acad Sci USA 1993; 90: 1004-8. 5. Dahlbiick B, Hildebrand B. Inherited resistance to activated protein Cis corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Nat! Acad Sci USA 1994; 91: 1396-400. ©F. K. Schattauer Verlagsgesellschaft mbH (Stuttgart) 72 (3) 481~90 (1994)

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).

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.

To investigate in vivo platelet function in acute falciparum malaria plasma concentrations of β-thromboglobulin (β-TG), platelet factor 4 (PF4) and thrombospondin (TSP) were determined in 10 severely-ill Thai patients and 11 healthy volunteers. 8 patients recovered. At presentation, the platelet counts of the 10 patients were significantly lower (p < 0.025) than those of the controls, and a slight but significant increase (p < 0.05) in β-TG/PF4 ratios in the patients suggested low-grade platelet activation. Presentation plasma β-TG and PF4 concentrations did not differ from control values, probably due to the opposing effects of decreased circulating platelet mass and increased activation. By contrast, admission concentrations of TSP in the surviving patients were markedly lower (p < 0.001) than those of the controls; β-TG/PF4 ratios, but not TSP levels, returned to normal during treatment. Hepatic dysfunction and oliguric renal failure probably contributed to a sustained increase in plasma β - TG and TSP in the 2 fatally ill patients, but associated elevated PF4 levels indicated concomitant platelet activation. Our results support the suggestion that in vivo platelet activation, which appears to be rapidly controlled by treatment, occurs in patients with severe, non-fatal falciparum malaria. TSP production, apparently from non-platelet sources, was decreased and/or its consumption was increased in these patients, perhaps by factors such as cytoadherence of infected erythrocytes and consequent endothelial damage.

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.

The aim of this study was to determine whether neutrophil activation occurs in the fetal circulation in pregnancy-induced hypertension and to correlate this with evidence of neutrophil activation in the maternal circulation. Twenty-one normal pregnancies and 23 complicated by pregnancy-induced hypertension were studied in the third trimester. The mean length of gestation at delivery was significantly shorter (P less than .01) and the mean birth weight percentile was significantly lower (P less than .05) in the hypertensive group; otherwise the groups were comparable. Blood was obtained before cesarean delivery or established labor in the mothers and immediately after delivery from the umbilical vein. Plasma neutrophil elastase, which is released after neutrophil activation, was measured by radioimmunoassay as a marker for neutrophil activation. The mean (+/- standard error) concentration of neutrophil elastase in maternal plasma in the hypertensive group (35.9 +/- 4.7 ng/mL) was significantly higher than in the normal group (20.8 +/- 0.87 ng/mL) (P less than .005). The concentration of neutrophil elastase in umbilical venous plasma was not significantly different between the normal and hypertensive groups. However, significantly higher concentrations of neutrophil elastase were found in the umbilical venous plasma of pregnancies delivered vaginally compared with those delivered by cesarean (P less than .05) regardless of diagnosis. There was no correlation between maternal venous and umbilical venous plasma neutrophil elastase concentrations, birth weight percentile, plasma urate, or platelet count. These data suggest that neutrophil activation is confined to the maternal circulation in pregnancy-induced hypertension where it may contribute to vascular damage and dysfunction in areas such as the placental bed.(ABSTRACT TRUNCATED AT 250 WORDS)

The observed increase in plasma von Willebrand factor antigen (vWF:Ag) in patients with the haemolytic-uraemic syndrome is presumed to be secondary to endothelial damage, which is a central event in these diseases. As the prognostic value of such changes has not been previously evaluated, vWF:Ag has been measured in plasma from children reported to a national survey of haemolytic-uraemic syndrome. Human neutrophil elastase was also measured, as an initial neutrophilia has been shown to have prognostic value in diarrhoea-associated haemolytic-uraemic syndrome. Despite a significant elevation of plasma vWF:Ag concentration in these patients at presentation, the values did not correlate with the period of thrombocytopenia, the need for dialysis, or outcome. However, children with a poor outcome had significantly greater plasma elastase concentrations compared to those who had a good outcome.

BJOG: An International Journal of Obstetrics & GynaecologyVolume 98, Issue 1 p. 105-106 Neutrophil activation in small for gestational age pregnancies T. A. Johnston, Corresponding Author T. A. Johnston University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghT. A. Johnston.Search for more papers by this authorI. A. Greer, I. A. Greer University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghSearch for more papers by this authorJ. Dawes, J. Dawes MRC/SNBTS Blood Components Assay Group, Forrest Road, EdinburghSearch for more papers by this authorA. A. Calder, A. A. Calder University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghSearch for more papers by this author T. A. Johnston, Corresponding Author T. A. Johnston University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghT. A. Johnston.Search for more papers by this authorI. A. Greer, I. A. Greer University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghSearch for more papers by this authorJ. Dawes, J. Dawes MRC/SNBTS Blood Components Assay Group, Forrest Road, EdinburghSearch for more papers by this authorA. A. Calder, A. A. Calder University Department of Obstetrics, Centre for Reproductive Biology, 37 Chalmers Street, EdinburghSearch for more papers by this author First published: January 1991 https://doi.org/10.1111/j.1471-0528.1991.tb10321.xCitations: 17Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume98, Issue1January 1991Pages 105-106 RelatedInformation

1 The pharmacokinetics of dermatan sulphate MF701 were studied in 12 healthy males after administration of single intravenous bolus (200 mg), intramuscular (100 and 300 mg) and oral (1 g) doses. The study was conducted according to a within-subject crossover design in two paired blocks.2 Plasma drug concentrations were measured using a competitive binding assay and a range of biological activity assays, including a sensitive catalysed thrombin inhibition test.3 Following intravenous administration, plasma concentrations of dermatan sulphate determined by competitive binding assay were described by a two-compartment open model with an initial t1/2, in of 0.6 h and a t1/2,z of 7.5 h. Biological activity assays were insufficiently sensitive to detect the second phase, and therefore yielded apparent monoexponential kinetics.4 After intramuscular injection the apparent bioavailability of dermatan sulphate was 16-20%. Plasma drug concentrations increased in proportion to dose when measured by competitive binding assay. Low concentrations persisted for more than 24 h at the higher dose, and these may prove therapeutically relevant on chronic administration.5 We confirm that dermatan sulphate is the only glycosaminoglycan known to generate significant plasma concentrations following oral administration. Oral bioavailability was estimated to be 7%.

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

Dose-ranging studies with a batch of factor IX concentrate have been performed in a canine non-stasis model of thrombogenicity. Doses between 50 and 200 IU/kg were infused over a 30 min period, and beagles were found to be more sensitive than greyhounds with regard to subsequent alterations in haemostatic parameters over a 150 min period. In beagles we detected significant increases in plasma fibrin(ogen) degradation products and reduction in fibrinogen concentrations in a dose-related manner after infusion of factor IX concentrate over the range 50-150 IU/kg. Plasma fibrinopeptide A was the most sensitive marker of activation of coagulation with significantly increased levels after factor IX at 50 IU/kg compared with control infusions of albumin. Recovery of infused factor IX was similar to values reported in man. In these experiments, measurement of urinary fibrinopeptide A did not prove to be a useful indicator of thrombogenicity. In conclusion, the beagle non-stasis model will provide a sensitive method to quantify the unwanted thrombogenic activities associated with the use of high doses of certain factor IX concentrates.

Summary. Human neutrophil elastase may be a major mediator of vascular damage and could contribute to the vascular damage seen in women with pregnancy‐induced hypertension (PTH). Elevated plasma levels of this substance will reflect neutrophil activation in vivo. To determine neutrophil activation in PIH, we studied 30 normal nonpregnant women, 32 women with normal pregnancies, 19 with mild/moderate PIH and 16 with severe PIH between 28 and 39 weeks gestation. Plasma neutrophil elastase was measured by radioimmuno‐assay. There was a significantly higher concentration of plasma neutrophil elastase in both mild/moderate and severe PIH than in normotensive pregnancies and this may contribute to the vascular lesion associated with PIH. Concentrations were also significantly higher in normal pregnancy than in non‐pregnant women which suggests that neutrophil activation and degranulation are increased in normal pregnancy.