Mixture Of Glycosaminoglycans Research Articles

Low Molecular Weight Heparin: An Evaluation of Current and Potential Clinical Utility in Surgery.

Heparin, a mixture of glycosaminoglycans of various sizes, is a potent natural anticoagulant. Low molecular weight heparins (LMWH) contain only the polymers of smaller size, which appear to possess most of the antithrombotic potential. Pharmacological differences between the two suggest a number of advantages with LMWH therapy. Our objective was to establish the utility of LMWHs in comparison to the current practice of anticoagulation in surgical patients. Articles were obtained through MEDLINE and CURRENT CONTENTS queries. The searches were limited to English and French-language articles and included published overviews containing relevant individual trials. We examined the current literature, consisting of 1,730 published reports from 1979-1998, regarding the biochemistry, pharmacology, physiology, and clinical applications of LMWH in comparison with current therapy. Studies were selected based on their relevance to LMWHs, the size and methods of trials, and their application to clinical care. Peer-reviewed published data were critically evaluated by independent extraction by several authors. Established rules for levels of evidence were used to objectively evaluate the strength of evidence supporting recommendations in each clinical area. LMWHs provide superior anticoagulation in the prophylaxis of DVT following orthopedic, general, and trauma surgery. Further studies should establish which other patients may benefit from such prophylaxis. Current evidence does not support the use of LMWHs in patients with mechanical heart valves or those on mechanical cardiac support devices; however, it may have a role in the maintenance of vascular graft patency. Further studies should examine the role of LMWHs in transplant atherosclerosis, and in patients requiring long-term anticoagulation at high risk for bleeding with warfarin therapy. The economic implications of LMWH administration remain unclear. On the basis of the information presented in this review, LMWHs are promising new agents in prophylaxis and treatment of both arterial and venous thrombosis. In the near future, LMWHs are likely to supplant UFH and perhaps warfarin in many applications.http://link.springer-ny.com/link/service/journals/00547/bibs/8n4p203.html</hea

Multivariate Analysis of Electron Detachment Dissociation and Infrared Multiphoton Dissociation Mass Spectra of Heparan Sulfate Tetrasaccharides Differing Only in Hexuronic acid Stereochemistry

The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.

Anticoagulant Therapy for Percutaneous Coronary Intervention

Received June 2, 2009; accepted November 16, 2009. Percutaneous coronary intervention (PCI) is the most commonly performed invasive therapeutic cardiac procedure and plays an important role in the treatment of ischemic heart disease. Since the first description of coronary angioplasty in a human by Gruntzig,1 the technique, equipment, and associated pharmacotherapy have undergone substantial evolution, leading to significant improvements in periprocedural complications.2 In particular, procedural anticoagulant therapy has been the focus of numerous clinical trials, and several options are now available and supported by practice guidelines; each agent has both advantages and disadvantages, and procedural pharmacotherapy continues to be a focus of drug development. The purpose of this review is to summarize the goals of anticoagulant therapy during PCI, the pharmacokinetics and pharmacodynamics of available agents, and the clinical data surrounding each agent and to identify new agents in development. The goals of pharmacotherapy during PCI are 2-fold: (1) to mitigate the sequelae of iatrogenic plaque rupture from balloon angioplasty or stenting and (2) to reduce the risk of thrombus formation on intravascular PCI equipment. Central to these thrombotic events is thrombin (factor IIa). Iatrogenic damage to the endothelium during PCI leads to increased expression of tissue factor, activation of the coagulation cascade, and formation of activated factor Xa. This ultimately leads to the generation of thrombin, conversion of fibrinogen to fibrin, and thrombus formation.3 In addition to its effects on fibrin, thrombin also directly activates platelets, enhances platelet aggregation, and is proinflammatory.4 Because of its multiple actions in promoting thrombosis, the focus of most anticoagulant agents is thrombin inhibition. Available agents for use include unfractionated heparin (UFH), low-molecular-weight heparins (LMWH, of which enoxaparin has the largest body of clinical data), the synthetic pentasaccharides (of which fondaparinux has the largest body of clinical data), and the …

Purified Glycosaminoglycans from Cooked Haddock May Enhance Fe Uptake Via Endocytosis in a Caco‐2 Cell Culture Model

This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High-sulfated GAGs were selectively purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe uptake (cell ferritin formation) from a Fe(+3)-containing solution, and Fe(+3)/ascorbic acid (AA) and Fe(+3)/GAGs mixtures. Mitochondria (MTT test) and endosomal/lysosomal activities (neutral red uptake, NR), intracellular accumulation of reactive oxygen species, and GSH concentration were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, divalent metal transporter-1 (DMT1), and duodenal cytochrome-b (DcytB) were also evaluated. The Fe uptake from Fe(+3)/GAGs mixture was up to 1.8-fold higher than from Fe(+3) alone. Both Fe(+3) alone and Fe(+3)/AA mixture produced highest increase in MTT conversion. In contrast, cell cultures exposed to the Fe(+3)/GAGs mixture exhibited highest NR uptake values. All Fe-containing solutions tested caused a sharp intramitochondrial accumulation of reactive oxygen species. Cell cultures exposed to the Fe(+3)/GAGs mixture exhibited a more preserved (by 8%) intracellular GSH concentration compared to cultures exposed to Fe(+3) or Fe(+3)/AA mixture. In addition to cell responses, the mRNA expression of Fe transporters may suggest that Fe could also be internalized into cells by endocytosis in addition to via DMT1 in Fe(+3)/GAGs mixtures. These aspects need to be confirmed in in vivo experiments to better establish nutritional interventional strategies.

Alzheimer's Disease: Another Target for Heparin Therapy

Alzheimer's disease (AD) is the leading cause of dementia and cognitive decline in the elderly. Brain tissue changes indicate that the two main proteins involved in AD are amyloid-β(A-β), which is associated with the formation of senile amyloid plaques, and tau, which is associated with the formation of neurofibrillary tangles. Although a central role for A-β in the pathogenesis of AD is indisputable, considerable evidence indicates that A-β production is not the sole culprit in AD pathology. AD is also accompanied by an inflammatory response that contributes to irreversible changes in neuronal viability and brain function, and accumulating evidence supports the pivotal role of complement and contact systems in its pathogenesis and progression. The complexity of AD pathology provides numerous potential targets for therapeutic interventions. Compounds that interact directly with A-β protein or interfere with its production and/or aggregation can reduce the inflammatory and neurotoxic effects of A-β, and heparin, a glycosaminoglycan mixture currently used in the prophylaxis and treatment of thrombosis, might be a candidate, as recent research has been extended to consider its nonanticoagulant properties, including its modulation of various proteases and anti-inflammatory activity.

Heparin-induced thrombocytopenia--therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4-heparin complexes.

Treatment of heparin-induced thrombocytopenia (HIT), a disorder in which anti-platelet factor 4 (PF4)-heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non-heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin-mimicking pentasaccharide). PF4-heparin complexes form at optimal stoichiometric ratios. To compare the effects of these various non-heparin anticoagulants in disrupting the formation of PF4-heparin complexes, and PF4-containing immune complexes. Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4-heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4-heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4-transfected HEK-293 EBNA cell line expressing the PF4 receptor CXCR3-B (P = 0.0408), reduced PF4-heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4-heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4-heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4-containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.

Parenteral Anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)

This chapter describes the pharmacology of approved parenteral anticoagulants, including the indirect anticoagulants, unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and danaparoid as well as the direct thrombin inhibitors hirudin, bivalirudin, and argatroban. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a unique pentasaccharide sequence and catalyze the inactivation of thrombin factor Xa and other clotting factors. Heparin also binds to cells and other plasma proteins, endowing it with unpredictable pharmacokinetic and pharmacodynamic properties, and can lead to nonhemorrhagic side effects, such as heparin-induced thrombocytopenia (HIT) and osteoporosis. LMWHs have greater inhibitory activity against factor Xa than thrombin and exhibit less binding to cells and proteins than heparin. Consequently, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties, have a longer half-life than heparin, and have a lower risk of nonhemorrhagic side effects. LMWHs can be administered once or twice daily by subcutaneous injection, without anticoagulant monitoring. Based on their greater convenience, LMWHs have replaced UFH for many clinical indications. Fondaparinux, a synthetic pentasaccharide, catalyzes the inhibition of factor Xa, but not thrombin, in an antithrombin-dependent fashion. Fondaparinux binds only to antithrombin; therefore, HIT and osteoporosis are unlikely to occur. Fondaparinux has excellent bioavailability when administered subcutaneously, has a longer half-life than LMWHs, and is given once daily by subcutaneous injection in fixed doses, without anticoagulant monitoring. Three parenteral direct thrombin inhibitors and danaparoid are approved as alternatives to heparin in HIT patients.

Sulfated glycosaminoglycans restore glycocalyx barrier properties of cultured endothelial cells in hyperglycemia

Background: Microalbuminuria is a clinical parameter of vascular dysfunction in patients with diabetes. The endothelial glycocalyx plays a role in regulation of vascular permeability. Therapeutic interventions using a mixture of glycosaminoglycans (GAGs) containing 80% heparin and 20% dermatan sulfate have been shown to improve vascular barrier function by reducing microalbuminuria Hypothesis: Exogenous GAGs attenuate hyperglycemia-induced increases in endothelial permeability for albumin by restoring barrier properties of the endothelial glycocalyx. Materials and methods: Human umbilical vein endothelial cells (HUVECs) were cultured on semi-permeable inserts and exposed to normo- (5mM) or hyperglycemia (25mM) for 4 days, last 24h in presence of the GAG mixture. Endothelial permeability was assessed by determining FITC-labeled albumin transfer over the monolayer (3 hours). Additionally, the glycocalyx glucosamine sugar residues on the endothelial cells were visualized with LEA-lectin staining Results: Albumin permeability of endothelial cells under hyperglycemia was increased to 122 ±8% (p<0.01) compared to normoglycemia. GAG incubation restored permeability in hyperglycemia (122 ±8% vs 85 ±6%; p<0.05). Additionally, LEA-lectin revealed a 21% decrease in glucosamine staining in hyperglycemic cells which was restored following GAG mix incubation Conclusion: GAG supplementation reverses the increased trans-endothelial albumin leakage under hyperglycemic conditions by restoring the barrier properties of the endothelial glycocalyx layer in vitro

Effects of sulodexide in patients with type 2 diabetes and persistent albuminuria

Urinary albumin excretion frequently persists in diabetic patients who are treated with angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB). Sulodexide, a glycosaminoglycan mixture of 80% heparan sulfate and 20% dermatan sulfate, has been hypothesized to reduce persistent albuminuria. We have conducted a multi-center randomized double-blind pilot study in order to determine the effect of 6 months' therapy with sulodexide on urinary albumin excretion and to address logistical issues for a full-scale trial. A total of 149 patients with type 2 diabetes and an albumin:creatinine ratio (ACR) between 20 and 300 mg/g were randomized with equal allocation to either placebo, 200 mg of sulodexide or 400 mg of sulodexide. The primary endpoint was the achievement, at 6 months, of either 3(1) return to normoalbuminuria (ACR < 20 mg/g with a decrease of at least 25%) or (2) a decrease in ACR of at least 50% from the baseline value. All patients used a maximum tolerated recommended FDA approved dose of an ACEI or ARB for at least 60 days and had stable blood pressure prior to randomization. The primary efficacy endpoint was achieved in 25.3% of the patients in the two sulodexide groups combined versus 15.4% of the placebo-treated patients (P = 0.26). The primary endpoint was achieved in 33.3% (P = 0.075 for the comparison to placebo) in the sulodexide 200 mg group and 18.4% (P = 0.781) in the sulodexide 400 mg group. (No consistent patterns of side effects were observed. Based on the experience gained in this pilot study, one full-scale trial is currently being conducted to evaluate the effects of sulodexide on change in ACR in patients with persistent microalbuminuria, and a longer-term trial is underway to evaluate the effects of sulodexide on long-term renal disease progression in patients with overt proteinuria.

Structural Analysis of Bikunin Glycosaminoglycan

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

The Immunogenic Potential of Generic Version of Low-Molecular-Weight Heparins May Not be the Same as the Branded Products

translate into their safety and efficacy profiles in a given clinical indication. Therefore, the United States Food and Drug Administration (FDA) and other regulatory bodies consider each of the commercially available LMWHs as a distinct drug and require clinical validation for the approval of each of these in specific indications. The introduction of generic versions of the branded LMWHs has challenged the regulatory bodies to develop specific guidelines for their approval. The current guidelines for generic drug approval are not adequate because of the complex nature of the LMWHs due to biologic origins and chemical modifications of a polycomponent mixture of glycosaminoglycans. Although the incidence of HIT is rare with the use of LMWHs and heparinomimetics such as fondaparinux and idraparinux when used for prophylactic and therapeutic indications, these agents are capable of generating antibodies to heparin-PF4 complex, which are measurable by using immunologic methods. The relative prevalence of these antibodies is method-dependent owing to different capturing probes. Moreover, the relative proportion of antibody subtypes (immunoglobulin G, A, and M) differ from product to product. Up to 20% of the patients treated with LMWHs generate these antibodies. The pathophysiologic role of these antibodies remains unknown at this time. However, this immunogenic response is comparable to autoimmune responses observed in the case of the antiphospholipid syndrome. Additional data are needed to identify the role of these antibodies in various pathologic states and their impact on clinical outcomes. The FDA has been visionary in identifying the immunogenicity of LMWHs as an important criterion to differentiate and demonstrate the bioequivalence Heparin-induced thrombocytopenia (HIT) is a catastrophic adverse reaction associated with the use of heparins and related products. Although the incidence of HIT with low-molecularweight heparins (LMWHs) is much lower than with unfractionated heparin (UFH), these agents are capable of triggering immunogenic responses in 10% to 20% of the patients treated for different indications. The generation of these antibodies depends on the dosage, duration of treatment, patient population, and underlying comorbidities. Differences in the immunogenic responses among different branded LMWHs have been noted. These differences are due to the structural composition of the LMWH and the interactions with endogenous platelet factor 4 (PF4) and other proteins. Low-molecular-weight heparins have been differentiated on the basis of several biophysical, biochemical, and pharmacologic properties. These differences

Tags for the Stable Isotopic Labeling of Carbohydrates and Quantitative Analysis by Mass Spectrometry

Although stable isotopic labeling has found widespread use in the proteomics field, its application to carbohydrate quantification has been limited. Herein we report the design, synthesis, and application of a novel series of compounds that allow for the incorporation of isotopic variation within glycan structures. The novel feature of the compounds is the ability to incorporate the isotopes in a controlled manner, allowing for the generation of four tags that vary only in their isotopic content. This allows for the direct comparisons of three samples or triplicate measurements with an internal standard within one mass spectral analysis. Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy.

Heparin Oligosaccharides as Potential Therapeutic Agents in Senile Dementia

Heparin is a glycosaminoglycan mixture currently used in prophylaxis and treatment of thrombosis. Heparin possesses non-anticoagulant properties, including modulation of various proteases, interactions with fibroblast growth factors, and anti-inflammatory actions. Senile dementia of Alzheimer's type is accompanied by inflammatory responses contributing to irreversible changes in neuronal viability and brain function. Vascular factors are also involved in the pathogenesis of senile dementia. Inflammation, endogenous proteoglycans, and assembly of senile plagues and neurofibrillary tangles contribute directly and indirectly to further neuronal damage. Neuron salvage can be achieved by anti-inflammation and the competitive inhibition of proteoglycans accumulation. The complexity of the pathology of senile dementia provides numerous potential targets for therapeutic interventions designed to modulate inflammation and proteoglycan assembly. Heparin and related oligosaccharides are known to exhibit anti-inflammatory effects as well as inhibitory effects on proteoglycan assembly and may prove useful as neuroprotective agents.

In vivo antithrombotic synergy of oral heparin and arginine: Endothelial thromboresistance without changes in coagulation parameters

On the basis of suggested clinical efficacy in an uncontrolled study in ninety-seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular-weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28-day study with oral sulodexide (2.9 mg/kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine.

Sulodexide attenuates myocardial ischemia/reperfusion injury and the deposition of C-reactive protein in areas of infarction without affecting hemostasis.

Several glycosaminoglycans (GAGs) have been demonstrated to protect the ischemic heart against reperfusion injury, in part, by modulating activation of the complement cascade. The present study assessed the cardioprotective effects of sulodexide (KRX-101), a mixture of GAGs composed of 80% low-molecular mass heparin and 20% dermatan sulfate. KRX-101 differs from other GAGs (e.g., heparin) in that it has limited anticoagulant efficacy and can be administered orally. The experimental protocol was designed to determine whether KRX-101 could protect the ischemic myocardium. Anesthetized New Zealand white rabbits underwent 30 min of coronary artery occlusion. Intravenous doses of KRX-101 (0.5 mg/kg, n = 10) or drug diluent (n = 10) were administered at the end of regional ischemia and at each hour of reperfusion. Infarct size, as a percentage of the area at risk, was calculated for both groups. Myocardial infarct size was 31.3 +/- 4.1% in the vehicle- and 17.3 +/- 3.2% in the KRX-101-treated animals (p < 0.05 versus vehicle). Activated partial thromboplastin times determined at baseline (preischemia) and at each hour of reperfusion (n = 4) were not significantly different between vehicle- and KRX-101-treated groups (p = N.S.). Myocardial injury was further assessed by measuring serum levels of cardiac-specific troponin I. KRX-101 administration significantly reduced (p < 0.05) the serum concentration of troponin I during reperfusion. The results suggest that KRX-101 may be an effective adjunctive agent in myocardial revascularization procedures, without the risk of increased bleeding.

Heparin and Low-Molecular-Weight Heparin: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy

This article about unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) is part of the Seventh American College of Chest Physicians Conference on Antithrombotic and Thrombolytic Therapy: Evidence-Based Guidelines. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a pentasaccharide, catalyzing the inactivation of thrombin and other clotting factors. UFH also binds endothelial cells, platelet factor 4, and platelets, leading to rather unpredictable pharmacokinetic and pharmacodynamic properties. Variability in activated partial thromboplastin time (aPTT) reagents necessitates site-specific validation of the aPTT therapeutic range in order to properly monitor UFH therapy. Lack of validation has been an oversight in many clinical trials comparing UFH to LMWH. In patients with apparent heparin resistance, anti-factor Xa monitoring may be superior to measurement of aPTT. LMWHs lack the nonspecific binding affinities of UFH, and, as a result, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties. LMWHs have replaced UFH for most clinical indications for the following reasons: (1) these properties allow LMWHs to be administered subcutaneously, once daily without laboratory monitoring; and (2) the evidence from clinical trials that LMWH is as least as effective as and is safer than UFH. Several clinical issues regarding the use of LMWHs remain unanswered. These relate to the need for monitoring with an anti-factor Xa assay in patients with severe obesity or renal insufficiency. The therapeutic range for anti-factor Xa activity depends on the dosing interval. Anti-factor Xa monitoring is prudent when administering weight-based doses of LMWH to patients who weigh > 150 kg. It has been determined that UFH infusion is preferable to LMWH injection in patients with creatinine clearance of < 25 mL/min, until further data on therapeutic dosing of LMWHs in renal failure have been published. However, when administered in low doses prophylactically, LMWH is safe for therapy in patients with renal failure. Protamine may help to reverse bleeding related to LWMH, although anti-factor Xa activity is not fully normalized by protamine. The synthetic pentasaccharide fondaparinux is a promising new antithrombotic agent for the prevention and treatment of venous thromboembolism.

Promotion of fluorapatite crystallization by soluble-matrix proteins from Lingula anatina shells.

One of the most persistent enigmas in the field of biomineralization is the evolutionary selection of calcium carbonate and calcium phosphate biominerals in invertebrate and vertebrate skeletal tissues, respectively. Whereas bones and teeth are constructed with carbonate-substituted hydroxyapatite (Ca10(PO4)6(OH)2), most mollusc shells consist of polymorphs of calcium carbonate such as calcite and aragonite. One of the few modern exceptions to this carbonate–phosphate dichotomy in skeletal biomineralization is the bivalved shell of extant Linguliform brachiopods such as Lingula and Discinisca, which consists of carbonate-substituted fluorapatite, (Ca10(PO4)6F2, francolite, FAP). [2] Lingula shells appeared along with phosphatic biominerals in the Problematica at the base of the Cambrian, about 545 million years ago (Mya), possibly because of high concentrations of phosphorus in the seawater during the Precambrian and Cambrian eras (543 to 490 Mya). The subsequent decrease in phosphate levels by the end of the Cambrian era may account for the extinction of the phosphatic Problematica due to arrested shell mineralization, although a switch to a carbonate regime may also have occurred in these invertebrates during this era. The geological longevity of Lingula, in contrast, suggests that the lightly mineralized FAP shell has remained well adapted to evolutionary pressures even though by the middle of the Ordovician (ca. 470 Mya) phosphatic biomineralization was rare in invertebrates. Previous studies have shown that the Lingula shell is produced by a rhythmic alternation of organic and mineralized layers. 5] The latter are constructed from FAP granules, 4–8 nm in diameter, which are smaller at the anterior and periphery of the shell compared with those deposited in the central and posterior regions, and become progressively aligned with the organic matrix during mineralization. The matrix consists of a complex mixture of glycosaminoglycans, b-chitin, and proteins with molecular masses between 6 kDa to 46 kDa, many of which are enriched in acidic amino-acid residues. 11] Because acidic macromolecules are considered in general to play a pivotal role in the nucleation and growth of calcium-containing biominerals, we have investigated the potential role of Lingula shell proteins on the formation of carbonate-substituted FAP crystals in vitro. Herein we show for the first time that soluble macromolecules isolated from Lingula shells specifically promote FAP crystallization by the destabilization of an amorphous calcium phosphate precursor. Proteins were sequentially extracted from powdered L. anatina whole valves or from four equally sized sections from the posterior to the anterior of the shell. Fractionation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS PAGE) gave discrete bands that correspond to proteins with estimated molecular masses of 6, 8.5, 15.5, 21.5, 24, and 44 kDa for guanadine hydrochloride (GnHCl) and EDTA extracts (Figure 1A). The EDTA extract contained significantly higher concentrations of the lower-molecular-weight proteins as well as additional proteins of molecular mass 4.5, 28, 35, 40, 50, 57.5, 60, and 69 kDa. The 21.5 and 24 kDa proteins in both extracts were glycosylated as determined by using concanavalin A binding (Figure 1B,C). The influence of Lingula shell proteins on the in vitro deposition of carbonate-substituted FAP was investigated by adding mixtures of the extracted macromolecules at various total concentrations to buffered calcium phosphate/fluoride metastable solutions at constant temperature. In each case, metastability was induced by raising the solution pH from 6.2 to 7.5, which resulted in relatively slow growth kinetics that were monitored by a progressive reduction in solution pH with time due to deprotonation of [H2PO4] and [HPO4] 2

Additive thrombin inhibition by fast moving heparin and dermatan sulfate explains the anticoagulant effect of sulodexide, a natural mixture of glycosaminoglycans

Introduction: The aim of the study was to evaluate the mechanism of the anticoagulant action of sulodexide, a mixture of glycosaminoglycans (GAGs) composed of dermatan sulfate (DS) and fast moving heparin (FMH), in vitro. Materials and methods: Thrombin clotting time (TCT) was measured in human platelet poor plasma (PPP). A chromogenic substrate assay was used to determine the pseudo-first order constant kinetic of thrombin inhibition ( k′= k obs/min) either in defibrinated PPP or antithrombin (AT) or heparin cofactor II (HCII) depleted defibrinated PPP in the absence and presence of sulodexide or its components, alone and in combination. The interaction between DS and FMH was analysed by both the algebraic fractional and isobole graphical methods. Results: Sulodexide, DS and FMH produced a dose-dependent prolongation of TCT with unclottable TCT at sulodexide above 4 μg/ml and at DS or FMH above 5 μg/ml. Sulodexide and its components alone and in combination produced a dose-dependent linear increase in the rate of thrombin inhibition in defibrinated PPP. The algebraic fractional and the isobole graphical methods indicated an additive effect between DS and FMH. In AT depleted PPP, the dose-dependent increase in k′ produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k′ produced by DS was similar to the increase produced in PPP. In HCII depleted PPP, the dose-dependent increase in k′ produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k′ produced by FMH was similar to the increase produced in PPP. Conclusions: Thrombin inhibition produced by sulodexide is due to the additive effect of its components, namely, HCII catalysis by DS and AT catalysis by FMH.

Availability of Danaparoid, an Anticoagulant, for Routine Hematological Tests.

In the Multiphasic Health Check-up System, medical laboratory testing has been changing lately to utilize a small and compact type of testing systems. The system uses heparinized blood for chemistry tests to reduce laboratory's workload and sample volume. The dana-paroid, a mixture of glycosamino-glycans (Heparan sulfate), has been used clinically for treatment of patients with disseminated intravascular coagulopathy instead of heparin sulfate. Heparin has been used for routine laboratory tests, although it has a strong activity of platelet aggregation. This study aimed to examine availability of Danaparoid for routine hematology tests as a substitute of heparin. Although Danaparoid induced platelet aggregation directly, the reaction was weaker than that of heparin. The platelet count of Danaparoid blood was decreased after five minutes of mixing. Addition of MgSO4 or of kanamycin sulfate to Danaparoid blood was able to prevent this decrease. When Danaparoid blood was applied to chemistry tests, major chemistry test data showed acceptable results, although level differences were observed slightly. It was concluded that Danaparoid would be a good candidate for a universal anticoagulant for both chemical and hematological laboratory tests.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.