Soluble syndecan-1 and glycosaminoglycans in preeclamptic and normotensive pregnancies.

Preeclampsia, an important cause of maternal and fetal morbidity and mortality, is associated with increased sFLT1 levels and with structural and functional damage to the glycocalyx contributing to endothelial dysfunction. We investigated glycocalyx components in relation to preeclampsia in human samples. While soluble syndecan-1 and heparan sulphate were similar in plasma of preeclamptic and normotensive pregnant women, dermatan sulphate was increased and keratan sulphate decreased in preeclamptic women. Dermatan sulphate was correlated with soluble syndecan-1, and inversely correlated with blood pressure and activated partial thromboplastin time. To determine if syndecan-1 was a prerequisite for the sFlt1 induced increase in blood pressure in mice we studied the effect of sFlt1 on blood pressure and vascular contractile responses in syndecan-1 deficient and wild type male mice. The classical sFlt1 induced rise in blood pressure was absent in syndecan-1 deficient mice indicating that syndecan-1 is a prerequisite for sFlt1 induced increase in blood pressure central to preeclampsia. The results show that an interplay between syndecan-1 and dermatan sulphate contributes to sFlt1 induced blood pressure elevation in pre-eclampsia.

Heparin Anti-Xa Activity, a Readily Available Unique Test to Quantify Apixaban, Rivaroxaban, Fondaparinux, and Danaparoid Levels.

BACKGROUND: Despite their usefulness in perioperative and acute care settings, factor-Xa inhibitor-specific assays are scarcely available, contrary to heparin anti-Xa assay. We assessed whether the heparin anti-Xa assay can (1) be used as a screening test to rule out apixaban, rivaroxaban, fondaparinux, and danaparoid levels that contraindicate invasive procedures according to current guidelines (>30 ng·mL-1, >30 ng·mL-1, >0.1 µg·mL-1, and >0.1 IU·mL-1, respectively), (2) quantify the anticoagulant level if found significant, that is, if it exceeded the abovementioned threshold. METHODS: In the derivation cohort then in the validation cohort, via receiver operating characteristics (ROC) curve analysis, we evaluated the ability of heparin anti-Xa assay to detect levels of factor-Xa inhibitors above or below the abovementioned safety thresholds recommended for an invasive procedure (screening test). Among samples with relevant levels of factor-Xa inhibitor, we determined the conversion factor linking the measured level and heparin anti-Xa activity in a derivation cohort. In a validation cohort, the estimated level of each factor-Xa inhibitor was thus inferred from heparin anti-Xa activity. The agreement between measured and estimated levels of factor-Xa inhibitors was assessed. RESULTS: Among 989 (355 patients) and 756 blood samples (420 patients) in the derivation and validation cohort, there was a strong linear relationship between heparin anti-Xa activities and factor-Xa inhibitors measured level (r = 0.99 [95% confidence interval {CI}, 0.99-0.99]). In the derivation cohort, heparin anti-Xa activity ≤0.2, ≤0.3, <0.1, <0.1 IU·mL-1 reliably ruled out a relevant level of apixaban, rivaroxaban, fondaparinux, and danaparoid, respectively (area under the ROC curve ≥0.99). In the validation cohort, these cutoffs yielded excellent classification accuracy (≥96%). If this screening test indicated relevant level of factor-Xa inhibitor, estimated and measured levels closely agreed (Lin's correlation coefficient close to its maximal value: 95% CI, 0.99-0.99). More than 96% of the estimated levels fell into the predefined range of acceptability (ie, 80%-120% of the measured level). CONCLUSIONS: A unique simple test already widely used to assay heparin was also useful for quantifying these 4 other anticoagulants. Both clinical and economic impacts of these findings should be assessed in a specific study.

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Quality performance for indirect Xa inhibitor monitoring in patients using international external quality data.

Objectives Chromogenic anti-activated factor X (FXa) assays are currently the "gold standard" for monitoring indirect anticoagulants. However, anti-FXa has been shown to vary according to the choice of reagents. In the present study, the performance of anti-FXa measurement was evaluated in order to gain more insight into the clinical applications. Furthermore, the longitudinal coefficient of variation (CV) was studied to investigate whether there is improvement over time. Methods Laboratory tests results were evaluated for samples spiked with unfractionated heparin (UFH), low-molecular-weight-heparin (LMWH), fondaparinux and danaparoid sodium. External quality assessment (EQA) data from multiple years were used from more than 100 laboratories. Results Comparison of the results for all methods showed significant differences in measured values between the frequently used methods (ANOVA: p < 0.001). The largest differences were observed for LMWH and UFH measurements. These differences may be caused by differences in method composition, such as the addition of dextran sulphate. Substantial interlaboratory variation in anti-FXa monitoring was observed for all parameters, particularly at low concentrations. Our results showed that below 0.35 IU/mL, the CVs for UFH and LMWH increase dramatically and results below this limit should be used with caution. Conclusions Our study demonstrates that the choice of the anti-FXa method is particularly important for UFH and LMWH measurement. The variation in measurements may have an effect on clinical implications, such as therapeutic ranges. Furthermore, the longitudinal EQA data demonstrated a constant performance and, in at least 50% of the cases, improvement in the CV% of the anti-Xa results over time.

Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.

Glycosaminoglycan Neutralization in Coagulation Control.

The glycosaminoglycans (GAGs) heparan sulfate, dermatan sulfate, and heparin are important anticoagulants that inhibit clot formation through interactions with antithrombin and heparin cofactor II. Unfractionated heparin, low-molecular-weight heparin, and heparin-derived drugs are often the main treatments used clinically to handle coagulatory disorders. A wide range of proteins have been reported to bind and neutralize these GAGs to promote clot formation. Such neutralizing proteins are involved in a variety of other physiological processes, including inflammation, transport, and signaling. It is clear that these interactions are important for the control of normal coagulation and influence the efficacy of heparin and heparin-based therapeutics. In addition to neutralization, the anticoagulant activities of GAGs may also be regulated through reduced synthesis or by degradation. In this review, we describe GAG neutralization, the proteins involved, and the molecular processes that contribute to the regulation of anticoagulant GAG activity.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients. METHODS: In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS). RESULTS: Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine. CONCLUSIONS: Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

A Fluorescent Probe for Glycosaminoglycans Applied to the Detection of Dermatan Sulfate by a Mix-and-Read Assay.

Glycosaminoglycans are complex biomolecules of great biological and medical importance. The quantification of glycosaminoglycans, in particular in complex matrices, is challenging due to their inherent structural heterogeneity. Heparin Red, a polycationic, fluorescent perylene diimide derivative, has recently emerged as a commercial probe for the convenient detection of heparins by a mix-and-read fluorescence assay. The probe also detects glycosaminoglycans with a lower negative charge density than heparin, although with lower sensitivity. We describe here the synthesis and characterization of a structurally related molecular probe with a higher positive charge of +10 (vs. +8 of Heparin Red). The superior performance of this probe is exemplified by the quantification of low dermatan sulfate concentrations in an aqueous matrix (quantification limit 1 ng/mL) and the detection of dermatan sulfate in blood plasma in a clinically relevant concentration range. The potential applications of this probe include monitoring the blood levels of dermatan sulfate after administration as an antithrombotic drug in the absence of heparin and other glycosaminoglycans.

Glycosaminoglycan levels in dried blood spots of patients with mucopolysaccharidoses and mucolipidoses.

Mucopolysaccharidoses (MPSs) and mucolipidoses (ML) are groups of lysosomal storage disorders in which lysosomal hydrolases are deficient leading to accumulation of undegraded glycosaminoglycans (GAGs), throughout the body, subsequently resulting in progressive damage to multiple tissues and organs. Assays using tandem mass spectrometry (MS/MS) have been established to measure GAGs in serum or plasma from MPS and ML patients, but few studies were performed to determine whether these assays are sufficiently robust to measure GAG levels in dried blood spots (DBS) of patients with MPS and ML. MATERIAL AND METHODS: In this study, we evaluated GAG levels in DBS samples from 124 MPS and ML patients (MPS I=16; MPS II=21; MPS III=40; MPS IV=32; MPS VI=10; MPS VII=1; ML=4), and compared them with 115 age-matched controls. Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Subsequently, dermatan sulfate (DS), heparan sulfate (HS-0S, HS-NS), and keratan sulfate (mono-sulfated KS, di-sulfated KS, and ratio of di-sulfated KS in total KS) were measured by MS/MS. RESULTS: Untreated patients with MPS I, II, VI, and ML had higher levels of DS compared to control samples. Untreated patients with MPS I, II, III, VI, and ML had higher levels of HS-0S; and untreated patients with MPS II, III and VI and ML had higher levels of HS-NS. Levels of KS were age dependent, so although levels of both mono-sulfated KS and di-sulfated KS were generally higher in patients, particularly for MPS II and MPS IV, age group numbers were not sufficient to determine significance of such changes. However, the ratio of di-sulfated KS in total KS was significantly higher in all MPS patients younger than 5years old, compared to age-matched controls. MPS I and VI patients treated with HSCT had normal levels of DS, and MPS I, VI, and VII treated with ERT or HSCT had normal levels of HS-0S and HS-NS, indicating that both treatments are effective in decreasing blood GAG levels. CONCLUSION: Measurement of GAG levels in DBS is useful for diagnosis and potentially for monitoring the therapeutic efficacy in MPS.

Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry.

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis. METHODS: This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns. RESULTS: The cutoffs were as follows: HS-0S > 90 ng/mL; HS-NS > 23 ng/mL, DS > 88 ng/mL; mono-sulfated KS > 445 ng/mL; di-sulfated KS > 89 ng/mL and ratio di-KS in total KS > 32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS. CONCLUSIONS: Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

Diet-induced hypercholesterolemia imparts structure-function changes to erythrocyte chondroitin sulphate/dermatan sulphate.

Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4-O-sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding.

Plasma and urinary glycosaminoglycans in the course of juvenile idiopathic arthritis.

OBJECTIVES: The aim of the study was to perform analyses of plasma and urinary glycosaminoglycan isolated from juvenile idiopathic arthritis (JIA). METHODS, RESULTS: Chondroitin/dermatan sulfate (CS/DS), heparan sulfate/heparin (HS/H) and hyaluronic acid (HA) were evaluated in samples obtained from JIA patients before and after treatment. Electrophoretic analysis of GAGs identified the presence of CS, DS and HS/H in plasma of healthy subjects and JIA patients. CS were the predominant plasma GAGs constituent in all investigated subject. The plasma CS level in untreated patients was significantly decreased. Therapy resulted in an increase in this glycan level. However, plasma CS concentration still remained higher than in controls. Increased levels of DS and HA in untreated JIA patients were recorded. Anti-inflammatory treatment led to normalization of these parameters concentrations. Plasma and urinary concentrations of HS/H were similar in all groups of individuals. Urinary CS/DS and HA were decreased only in untreated patients. CONCLUSIONS: The data presented indicate that changes in plasma and urinary glycosaminoglycan occur in the course of JIA. There are probably the expression of both local articular cartilage matrix and systemic changes in connective tissue remodeling.

Newborn screening and diagnosis of mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5-99.4%; positive predictive value, 54.5-75%; false positive rate, 0.62-1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.

Plasmatic dermatan sulfate and chondroitin sulfate determination in mucopolysaccharidoses.

The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (~8 µg/mL). A highly significant decrease was observed for 1-5-year-old (∼-33%) and 5-10-year-old (∼-65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.

Plasmatic kinetics of dermatan sulfate during enzyme replacement therapy with iduronate-2-sulfatase in a mucopolysaccharidosis II patient.

Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10 months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3 h, followed by a continuous decrease in the 8-15 days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80-85 %, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long-term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans.

INTRODUCTION: Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels. METHODS: Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test. RESULTS: The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state. CONCLUSIONS: HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.

Large inter-individual variation of the pharmacodynamic effect of anticoagulant drugs on thrombin generation.

Anticoagulation by a standard dosage of an inhibitor of thrombin generation presupposes predictable pharmacokinetics and pharmacodynamics of the anticoagulant. We determined the inter-individual variation of the effect on thrombin generation of a fixed concentration of direct and antithrombin-mediated inhibitors of thrombin and factor Xa. Thrombin generation was determined by calibrated automated thrombinography in platelet-poor plasma from 44 apparently healthy subjects which was spiked with fixed concentrations of otamixaban, melagatran, unfractionated heparin, dermatan sulfate and pentasaccharide. The variability of the inhibitory effect of the different anticoagulants within the population was determined using the coefficient of variation, i.e. the standard deviation expressed as a percentage of the mean. The inter-individual coefficients of variation of the endogenous thrombin potential and peak height before inhibition were 18% and 16%, respectively and became 20%-24% and 24%-43% after inhibition. The average inhibition of endogenous thrombin potential and peak height (ETP, peak) brought about by the anticoagulants was respectively: otamixaban (27%, 83%), melagatran (56%, 63%), unfractionated heparin (43%, 58%), dermatan sulfate (68%, 57%) and pentasaccharide (25%, 67%). This study demonstrates that the addition of a fixed concentration of any type of anticoagulant tested causes an inhibition that is highly variable from one individual to another. In this respect there is no difference between direct inhibitors of thrombin and factor Xa and heparin(-like) inhibitors acting on the same factors.

Heparan sulfate and dermatan sulfate derived disaccharides are sensitive markers for newborn screening for mucopolysaccharidoses types I, II and III.

INTRODUCTION: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three of the MPSs and is being developed for the other types. Early initiation of treatment, before the onset of irreversible tissue damage, clearly provides a favorable disease outcome. However, early diagnosis is difficult due to the rarity of these disorders in combination with the wide variety of clinical symptoms. Newborn screening (NBS) is probably the optimal approach, and several screening techniques for different MPSs have been studied. Here we describe a relatively simple and sensitive method to measure levels of dermatan and heparan sulfate derived disaccharides in dried blood spots (DBS) with HPLC-MS/MS, and show that this reliably separates MPS I, II and MPS III newborns from controls and heterozygotes. METHODS: Newborn DBS of 11 MPS I, 1 MPS II, and 6 MPS III patients, with phenotypes ranging from severe to relatively attenuated, were collected and levels of dermatan and heparan sulfate derived disaccharides in these DBS were compared with levels in DBS of newborn MPS I and MPS III heterozygotes and controls. RESULTS: The levels of dermatan and heparan sulfate derived disaccharides were clearly elevated in all newborn DBS of MPS I, II and III patients when compared to controls. In contrast, DBS of MPS I and III heterozygotes showed similar disaccharide levels when compared to control DBS. CONCLUSIONS: Our study demonstrates that measurement of heparan and dermatan sulfate derived disaccharides in DBS may be suitable for NBS for MPS I, II and MPS III. We hypothesize that this same approach will also detect MPS VI, and VII patients, as heparan sulfate and/or dermatan sulfate is also the primary storage products in these disorders.

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components.

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.