Inflammatory and Metabolic Syndrome Biomarker Analysis of Vascular Outcomes in End-stage Renal Disease.

End-stage renal disease (ESRD) presents a complex syndrome in which inflammatory and metabolic processes contribute to disease progression and development of comorbid conditions. Over $1 trillion is spent globally on ESRD care. Plasma samples collected from 83 ESRD patients prior to hemodialysis were profiled for metabolic and inflammatory biomarker concentrations. Concentrations were compared between groups with and without history of stroke, acute coronary syndrome (ACS), congestive heart failure (CHF), and coronary artery disease (CAD). The 25 patients (30.1%) with history of stroke demonstrated decreased plasma interferon-γ levels (p = 0.042) and elevated plasma resistin, interleukin (IL)-1α, and leptin levels (p = 0.008, 0.021, 0.026, respectively) when compared with ESRD patients without history of stroke. The 14 patients (16.9%) with history of ACS demonstrated elevated plasma IL-6 levels (p = 0.040) when compared with ESRD patients without history of ACS. The 30 patients (36.1%) with history of CHF demonstrated decreased plasma leptin levels (p = 0.031) and elevated plasma IL-1ß levels (p = 0.042) when compared with ESRD patients without history of CHF. Finally, the 39 patients (47.0%) with history of CAD demonstrated elevated plasma IL-1α levels (p = 0.049) when compared with ESRD patients without history of CAD. Plasma biomarker concentration disturbances were observed in ESRD patients with history of stroke, ACS, CHF, and CAD when compared with ESRD patients without such history. Proinflammatory biomarker elevations were seen in stroke, ACS, CHF and CAD, while adipocytokine aberrations were observed in both stroke and CHF. These studies demonstrate that biomarker profiling of vascular comorbidities in ESRD may provide useful diagnostic and prognostic information in the management of ESRD patients.

Effect of erythropoietin stimulating agents on vascular endothelial growth factor levels in patients with end stage renal disease.

BACKGROUND: End stage renal disease (ESRD) is a debilitating disease that not only impacts quality of life, but also increases the risk of cardiovascular disease, stroke, and overall mortality. By improving our understanding of the molecular patterns seen in progression of chronic renal disease (CRD), we may be able to slow down and possibly even prevent progression renal failure. The vascular endothelial growth factor (VEGF) has been implicated as a major contributor to CRD disease progression, thus our aim was to profile the VEGF levels in patients with ESRD and to determine the effects of the erythropoietin stimulating agents (ESAs). METHODS: Under institution review board (IRB) approval, plasma samples were collected from 77 patients prior to hemodialysis and heparin administration. Normal human plasma samples (female & male, 18-35 years old) were purchased from George King Biomedical Inc (Overland Park, KS). All samples were stored at -80 °C. Inflammatory biochips were purchased from RANDOX (Co. Antrim, Northern Ireland) to test VEGF on 77 ESRD and 48 normal samples. RESULTS: The VEGF was significantly elevated in ESRD vs. normal (P<0.0001), ESRD+ESA vs. normal (P<0.0001), and ESRD-ESA vs. normal (P<0.0001). No difference was seen between ESRD+ESA and ESRD-ESA. Hemoglobin and free iron were significantly elevated in ESRD-ESA compared to ESRD+ESA (PHemoglobin=0.0007, PIron<0.0001). CONCLUSIONS: Our finding that VEGF was significantly elevated in ESRD vs. normal, aligns with the literature and suggests that VEGF may in fact play a key role in CRD progression. However, further studies to evaluate VEGF isomer levels are needed. While we saw lack of difference in VEGF levels between ESRD+ESA and ESRD-ESA, this may be due to the treatment algorithm used. Further investigation is warranted to determine whether the ESAs and hemoglobin levels show any influence on or crosstalk with the VEGF levels.

Anticoagulant and Antithrombotic Actions of AVE5026, an Enriched Anti-Xa Hemisynthetic Ultra-Low-Molecular-Weight Heparin.

INTRODUCTION: AVE5026 represents a new generation of ultra-low-molecular-weight heparin (LMWH) with high anti-Xa and low anti-IIa activities (anti Xa-IIa ratio >30). In addition, AVE5026 exhibits a relatively higher proportion of AT components. MATERIALS AND METHODS: The anticoagulant, antiplatelet, antithrombotic, and bleeding effects of AVE5026 in comparison to other heparins were investigated in this study. RESULTS: AVE5026 demonstrated weak effects in the global clotting assays; however, in the amidolytic anti-Xa assay, AVE5026 produced strong inhibitory effects. AVE5026 showed no cross-reactivity with the heparin-induced thrombocytopenia antibodies in the platelet aggregation system. AVE5026 produced a dose-dependent antithrombotic response after intravenous (IV) and subcutaneous (SC) administration in thrombosis models. The relative bleeding effects of AVE5026 in a rat tail bleeding and rabbit blood loss model were negligible after both IV and SC administration. CONCLUSIONS: This superior safety efficacy index in animal models in comparison with other LMWH may translate into improved antithrombotic efficacy with decreased bleeding risk.

The association of pain with protein inflammatory biomarkers: a review of the literature.

BACKGROUND: Pain is a key diagnostic criterion in many medical conditions. In the absence of self-reported pain, measurement of a proxy for pain, such as an inflammatory biomarker, could aid in diagnosis and disease management. OBJECTIVES: The aim was to determine if there is an association between inflammatory biomarkers and self-reported pain in individuals with medical conditions associated with the symptom of pain and to clarify whether inflammatory biomarkers might aid in the diagnostic process. METHODS: An integrative literature review was conducted. PubMed, CINAHL, and Cochrane databases were searched for articles published between January 2000 and September 2012. Inclusion criteria were original research testing a relationship between inflammatory biomarkers and pain, pain measurement, laboratory measure of inflammatory biomarkers, and a prospective single-group experimental design or comparative nonrandomized or randomized design. Excluded were studies describing an association between inflammatory biomarkers and treatment, risk, and generation; pathophysiology; or genetic polymorphisms/transcripts. Ten studies meeting inclusion criteria were reviewed. RESULTS: In most of the studies, baseline elevations in both proinflammatory and anti-inflammatory cytokines were reported in painful conditions compared with healthy controls. In half of the studies, higher levels of proinflammatory markers (C-reactive protein, tumor necrosis factor-alpha, interleukin-2 [IL-2], IL-6, IL-8, IL-10, and CD40 ligand) were associated with greater pain. Proinflammatory cytokines decreased after treatment for pain in only two studies. DISCUSSION: The association between inflammatory markers varied in the direction and magnitude of expression, which may be explained by differences in designs and assays, disease condition and duration, variations in symptom severity, and timing of measurement. Elevation in anti-inflammatory cytokines in the presence of pain represents a homeostatic immune response. Further study is required to determine the value of cytokines as biomarkers of pain.

A heparin-like compound isolated from a marine crab rich in glucuronic acid 2-O-sulfate presents low anticoagulant activity.

A natural heparin-like compound isolated from the crab Goniopsis cruentata was structurally characterized and its anticoagulant and hemorrhagic activities were determined. Enzymatic and nuclear magnetic resonance analysis revealed that its structure is rich in disulfated disaccharides, possessing significant amounts of 2-O-sulfated-ß-D-glucuronic acid units. Furthermore, low amounts of trisulfated disaccharide units containing 2-O-sulfated-α-L-iduronic acid were detected, when compared to mammalian heparin. In addition, this heparin-like structure showed negligible in vitro anticoagulant activity and low bleeding potency, facts that make it a suitable candidate for the development of structure-driven, heparin based therapeutic agents with fewer undesirable effects.

Proinflammatory cytokines in the prefrontal cortex of teenage suicide victims.

Teenage suicide is a major public health concern, but its neurobiology is not well understood. Proinflammatory cytokines play an important role in stress and in the pathophysiology of depression-two major risk factors for suicide. Cytokines are increased in the serum of patients with depression and suicidal behavior; however, it is not clear if similar abnormality in cytokines occurs in brains of suicide victims. We therefore measured the gene and protein expression levels of proinflammatory cytokines interleukin (IL)-1ß, IL-6, and tissue necrosis factor (TNF)-α in the prefrontal cortex (PFC) of 24 teenage suicide victims and 24 matched normal control subjects. Our results show that the mRNA and protein expression levels of IL-1ß, IL-6, and TNF-α were significantly increased in Brodmann area 10 (BA-10) of suicide victims compared with normal control subjects. These results suggest an important role for IL-1ß, IL-6, and TNF-α in the pathophysiology of suicidal behavior and that proinflammatory cytokines may be an appropriate target for developing therapeutic agents.

Pharmacology of argatroban.

Argatroban is a synthetic, small-molecule direct thrombin inhibitor that is approved in the USA, the EU and Japan for prophylaxis or treatment of thrombosis in patients with heparin-induced thrombocytopenia (HIT), and for anticoagulation of HIT patients undergoing PCI. Argatroban binds reversibly to, and inhibits both soluble and clot-bound thrombin. Argatroban does not generate antibodies, is not susceptible to degradation by proteases and is cleared hepatically. It has a predictable anticoagulant effect and there is a good correlation between dose, plasma concentration and pharmacodynamic effect. Initial clinical studies suggest that further investigations to establish the use of argatroban in ischemic stroke, acute coronary syndrome, hemodialysis, blood oxygenation, off-pump cardiac surgery and other clinical indications are warranted.

In vivo neutralization of unfractionated heparin and low-molecular-weight heparin by a novel salicylamide derivative.

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are widely used anticoagulants for surgical and interventional use. Currently, the anticoagulant and bleeding effects of heparin are neutralized by protamine sulfate. There are several problems associated with the use of protamine sulfate, including allergic reactions, cardiovascular effects, heparin rebound, and incomplete neutralization of LMWHs. The objective of this investigation is to characterize the effectiveness of a novel salicylamide-derived heparin antagonist, PMX 60056, in neutralizing the antithrombotic and bleeding effects of UFH and LMWHs. Animals were first anticoagulated using an intravenous injection of UFH or LMWH followed by protamine sulfate or PMX 60056. Established animal models of hemorrhage (rat-tail transection) and thrombosis (jugular vein clamping) were then performed. Blood samples were collected for ex vivo analysis using activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa assays. We demonstrate that PMX 60056 neutralized the antithrombotic, anticoagulant, and bleeding effects of heparins as effectively as protamine sulfate and may be slightly more efficacious against LMWHs. These results suggest that PMX 60056 may provide an improved approach for the neutralization of UFH and LMWHs.

Comparative studies on the topical administration of mucopolysaccharide and heparin ointments in nonhuman primates.

Mucopolysaccharide polysulfate (MPS) represents a mammalian-derived sulfated polysaccharide. Because the origin and structure of heparins is similar to MPS, this study was conducted to compare 2 ointment formulations containing MPS or heparin with a placebo ointment on tissue factor pathway inhibitor (TFPI) released in nonhuman primates (Macaca mulatta). A primate colony composed of 18 animals, housed at Loyola University Medical Center, was used in compliance with an Institutional Animal Care and Use Committee (IACUC)-approved protocol. Mucopolysaccharide polysulfate (4.5%), heparin (4.5%), and a placebo ointment were topically applied to individual groups of primates in a crossover study for periods of up to 2 weeks. Blood samples were drawn on days 1, 2, 5, 7, and 10. The anticoagulant effects (activated partial thromboplastin time [APTT], Heptest, thrombin time [TT]), TFPI antigen and functional levels, thrombin activatable fibrinolytic inhibitor (TAFI), and antiheparin platelet factor 4 antibodies (AHPF4 abs) were measured in citrated plasma. All data were compiled as mean +/- 1 standard deviation and compared in groups. Topical administration of both the MPS and heparin ointments resulted in no measurable anticoagulant effects in the primate model; however, MPS produced a concentration-dependent release of TFPI antigen and a functional activity that was stronger than the effects observed with heparin. A decrease in TAFI activation was also observed in the MPS-treated primates. In addition, in the heparin-treated group, a slight increase in AHPF4 abs was observed. In conclusion, MPS showed a stronger release of TFPI than heparin that was not associated with a strong anticoagulant effect. Moreover, MPS downregulated TAFI, resulting in an enhanced fibrinolytic effect.

Transition from argatroban to oral anticoagulation with phenprocoumon or acenocoumarol: effect on coagulation factor testing.

Treatment with the thrombin inhibitor argatroban is often followed by vitamin K-antagonist treatment. In this study, the behavior of coagulation factors measured under these treatment regimens is shown. Healthy subjects received infusions of 1.0, 2.0, or 3.0 microg/kg/hr argatroban before and during phenprocoumon or acenocoumarol dosing. Quantitation of factors II, VII, IX, and X by clot-based assays resulted in dose dependent, approximately 20%, lower than expected values in the presence of argatroban. On the contrary, values for the inhibitors, protein C and protein S, were higher. Cotherapy exaggerated the effect by vitamin K-antagonist alone. However, testing by immunologic and chromogenic assays did not show any effect by argatroban. Coupled with a lack of bleeding in the subjects, these data suggests that argatroban does not affect coagulation proteins and that the observations are only an assay artifact. Assay interferences must be considered when measuring coagulation proteins in patients receiving thrombin inhibitors.

Survival of heparins, oral anticoagulants, and aspirin after the year 2010.

The conventional management of thrombotic and cardiovascular disorders is based on the use of heparin, oral anticoagulants, and aspirin. Despite remarkable progress in life sciences, these drugs still remain a challenge and a mystery to us, and their use is far from optimized. The development of low-molecular-weight heparins and the synthesis of heparinomimetics, such as the chemically synthesized pentasaccharide, represent a refined use of heparin. Additional drugs from this knowledge will continue to develop; however, none of these drugs will ever match the polypharmacology of heparin. Aspirin still remains the leading drug in the management of thrombotic and cardiovascular disorders. The newer antiplatelet drugs such as adenosine diphosphate receptor inhibitors, glycoprotein IIb/IIIa inhibitors, and other specific inhibitors have limited effects and have been tested in patients who have already been treated with aspirin. Warfarin provides a convenient and affordable approach in the long-term outpatient management of thrombotic disorders. The optimized use of these drugs still remains as the approach of choice to manage thrombotic disorders. The new anticoagulant targets, including specific sites in the hemostatic network such as tissue factor, individual clotting factors (IIa, VIIa, IXa, Xa, XIIa, and XIIIa), recombinant forms of serpins (antithrombin, heparin cofactor II, and tissue factor pathway inhibitors), recombinant activated protein C, thrombomodulin, and site-specific serine protease inhibitor complexes have also been developed. There is a major thrust on the development of orally bioavailable anticoagulant drugs (anti-Xa and anti-IIa agents), which are slated to replace oral anticoagulants. Both the anti-factor Xa and antithrombin agents have been developed for oral use and have provided impressive clinical outcomes in sponsor trials for the postsurgical prophylaxis of venous thrombosis; however, safety concerns related to liver enzyme elevations and thrombosis rebound have been reported with their use. For these reasons, the U.S. Food and Drug Administration did not approve the orally active antithrombin agent ximelagatran for several indications. The synthetic pentasaccharide (fondaparinux) has undergone an aggressive clinical development. Unexpectedly, fondaparinux also produced major bleeding problems at minimal dosages. Fondaparinux represents only one of the multiple pharmacologic effects of heparins. Thus, its therapeutic index will be proportionately narrower. The newer antiplatelet drugs have added a new dimension in the management of thrombotic disorders. The favorable clinical outcomes with aspirin and clopidogrel have validated cyclooxygenase (COX)-1 and P2Y (12) receptors as targets for new drug development. Prasugrel, a novel thienopyridine, cangrelor, and AZD 6140 represent newer P2Y (12) antagonists. Cangrelor and AZD 6140 are direct inhibitors, whereas prasugrel requires metabolic activation. Though clinically effective, recent results have prompted a closure of a large clinical trial with prasugrel due to bleeding. The newer anticoagulant and antiplatelet drugs are attractive for several reasons; however, none of these are expected to replace the conventional drugs in polytherapeutic approaches. Heparins, warfarin, and aspirin will continue to play a major role in the management of thrombotic and cardiovascular disorders beyond 2010.

Differentiating low-molecular-weight heparins based on chemical, biological, and pharmacologic properties: implications for the development of generic versions of low-molecular-weight heparins.

Low-molecular-weight heparins (LMWHs) are polypharmacologic drugs used to treat thrombotic and cardiovascular disorders. These drugs are manufactured using different chemical and enzymatic methods, resulting in products with distinct chemical and pharmacologic profiles. Generic LMWHs have been introduced in Asia and South America, and several generic suppliers are seeking regulatory approval in the United States and the European Union. For simple small-molecule drugs, generic drugs have the same chemical structure, potency, and bioavailability as the innovator drug. Applying this definition to complex biological products such as the LMWHs has proved difficult. One major issue is defining appropriate criteria to demonstrate bioequivalence; pharmacopoeial specifications alone appear to be inadequate. Whereas available generic versions of LMWHs exhibit similar molecular and pharmacopoeial profiles, marked differences in their biological and pharmacologic behavior have been noted. Preliminary studies have demonstrated differences in terms of anti-Xa activity and tissue factor pathway inhibitor release after subcutaneous administration, as well as antiplatelet and profibrinolytic effects. The current data emphasize the need to consider multiple functional parameters when defining bioequivalence of biologic drugs with complex structures and activities and also underscore the importance of further pharmacologic studies involving animal models and human clinical trials. The U.S. Food and Drug Administration and the European Medicine Evaluation Agency are currently developing guidelines for the acceptance of biosimilar agents including LMWHs. Until such guidelines are complete, generic interchange may not be feasible.

The future of anticoagulation.

The conventional management of thrombotic disorders is based on the use of heparin, oral anticoagulants, and aspirin. The development of low molecular weight heparins and the synthesis of heparinomimetics such as the chemically synthesized pentasaccharide represent a refined use of heparin. Aspirin still remains the lead drug in the management of thrombotic and cardiovascular disorders. The newer antiplatelet drugs such as the adenosine diphosphate receptor inhibitors, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors and other specific inhibitors have limited effects and have been tested in patients who have already been treated with aspirin. The oral anticoagulants such as warfarin provide a convenient and affordable approach in the long-term outpatient management of thrombotic disorders. The optimized use of these drugs still remains the approach of choice to manage thrombotic disorders. The new anticoagulant drugs target specific sites in the hemostatic network. There is a major thrust on the development of orally bioavailable anticoagulant drugs to replace oral anticoagulants. Heparin and low molecular weight heparins have been considered with various chemical enhancers for absorption. Both the factor Xa and antithrombin agents have been developed for oral use and some of these agents are in clinical development. Besides the limited bioavailability, the therapeutic indices of some of these drugs have been rather disappointing. Factor Xa inhibitors such as the pentasaccharides have undergone aggressive clinical development. The newer antiplatelet drugs have added a new dimension in the management of thrombotic disorders. The newer drugs are attractive for several reasons; however, none of these are expected to completely replace the conventional drugs in polytherapeutic approaches. It is conceivable that some of the newer drugs in combined modalities may mimic the broad therapeutic spectrum of heparins and warfarin. However, clinical validation is required for the therapeutic interchange for specific indications.

The relationship between the antiphospholipid syndrome and heparin-induced thrombocytopenia.

Antiphospholipid syndrome (APS) and heparin-induced thrombocytopenia (HIT) are immune-mediated thrombotic conditions caused by antibodies targeted to a protein-antigen complex. Although each disorder is attributed to two distinct antibodies, these autoimmune disorders are characterized by a similar pathogenesis that includes a hypercoagulable state, platelet activation, damage to the vascular endothelium, and inflammation. APS and HIT share similarities in the clinical presentation because each is associated with thrombocytopenia, a high risk of thrombosis in all venous and arterial sites, and catastrophic thrombotic outcomes occur if untreated. Understanding the disease process for one disorder could potentially aid in understanding the other disorder.

Laboratory evaluation of the antiphospholipid syndrome.

Antiphospholipid syndrome (APLS) is among the most common acquired blood protein defects that have been identified as leading to thrombosis. This article describes the laboratory diagnosis of APLS, including the detection of lupus anticoagulants, anticardiolipin antibodies, and subtypes of antiphospholipid antibodies.

Comparison of platelet function and morphology in patients undergoing percutaneous coronary intervention receiving bivalirudin versus unfractionated heparin versus clopidogrel pretreatment and bivalirudin.

We hypothesized that direct thrombin inhibition could attenuate platelet activation and release of soluble CD40 ligand (sCD40L), a marker of inflammation, during percutaneous coronary intervention (PCI). To assess platelet function under flow conditions with bivalirudin versus unfractionated heparin (UFH), we employed the cone and plate(let) analyzer (CPA) assay in drug-spiked blood samples from volunteers (n = 3) in vitro, and then in PCI patients who received bivalirudin alone (n = 20), UFH alone (n = 15), and clopidogrel pretreatment plus bivalirudin (n = 15). Scanning electron microscopy was employed to image bivalirudin or UFH-treated platelets to determine whether platelet function observations had a morphologic explanation. Enzyme immunoassay was used to measure sCD40L levels in PCI patients. In vitro, bivalirudin decreased platelet surface coverage; UFH increased platelet surface coverage. In PCI patients, bivalirudin alone decreased platelet surface coverage, UFH alone increased platelet surface coverage, and clopidogrel pretreatment plus bivalirudin additively reduced platelet surface coverage. Unlike UFH, bivalirudin did not activate platelets in SEM studies. Bivalirudin alone or coupled with clopidogrel significantly reduced plasma sCD40L in PCI patients. In conclusion, our findings suggest that under flow conditions, bivalirudin alone or coupled with clopidogrel may have an antiplatelet effect versus UFH alone during PCI. These data suggest that bivalirudin and UFH may confer an anti-inflammatory effect by reducing sCD40L during PCI.

Isolation and characterization of heparin from tuna skins.

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using (13)C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 microg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 microg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.

Relative neutralization of the biological actions of sulfaminoheparosans (K5 derivatives) and heparins by protamine sulfate.

Biosynthetic, semisynthetic, and synthetic analogues of heparins are currently developed as substitute antithrombotic agents for heparin. Sulfaminoheparosan (SAH) represents a bacterial polysaccharide (K5)-derived antithrombotic polymer from which pharmacologically active products with varying molecular weights (5-25 kDa) can be derived. These agents have been shown to exhibit pharmacologic effects comparable to heparins. The objective of this investigation is to determine the relative neutralization profile of various SAH derivatives, also called as bioheparins, by protamine sulfate. Four SAH fractions with varying molecular weights (20, 9, 7, and 6 kDa), a low molecular weight heparin (LMWH), tinzaparin, and unfractionated heparin (UFH) were supplemented to normal human pool plasma over a concentration range of 6.2 to 100 microg/mL. A fixed amount of protamine sulfate at 25 microg/mL (final concentration) was supplemented to determine the neutralization profile by performing tests such as prothrombin time (PT), activated partial thromboplastin time (APTT), Heptest, prothrombin-induced clotting time (PiCT), and amidolytic anti-Xa and anti-IIa assays. Protamine sulfate produced varying degrees of neutralization of all bioheparin fractions in the clotting assays. In the amidolytic anti-IIa assay relatively stronger inhibition was noted for all agents than inhibition of FXa. On a cumulative basis the neutralization profile of SAHs was comparable with heparins. These results suggest that the anticoagulant activities of SAH derivatives can be antagonized by protamine sulfate. These studies warrant further in vivo investigation to validate the relative neutralization profile of sulfaminoheparosans.