Combined inhibition of CD14 and complement, two main inducers of the inflammatory response, have proved particularly effective in attenuating Gram-negative bacteria-induced inflammation. Approaching possible clinical relevance, we investigated the effect of such inhibition in a post-challenge setting. Human whole blood was anti-coagulated with lepirudin. Anti-CD14, compstatin (C3 inhibitor) and the combination thereof were added 5 min prior to or 5, 15 or 30 min after adding Escherichia coli. Total incubation time with Escherichia coli was 120 min. Cytokines, myeloperoxidase (MPO) and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. Delayed combined inhibition significantly attenuated the inflammatory response. IL-1β, IL-8 and TNF-α were significantly inhibited in the range of 20-40%, even when adding the inhibitors with up to 30 min delay. IL-6 was significantly inhibited with 15 min delay, and MIP-1α and MPO with 5 min delay. Complement activation (TCC) was blocked completely at each time point compstatin was added, whereas the cytokines and MPO increased steadily between the time points. The combined regimen was significantly more effective than single inhibition in the pre-challenge setting. The attenuation of Escherichia coli-induced inflammation in a post-challenge setting suggests a potential therapeutic window for this treatment in sepsis.

To review published literature regarding use of strategies to prevent thrombotic events in patients with nephrotic syndrome (NS).The MEDLINE/PubMed, EMBASE, and Cochrane databases were queried from 1980 to December 2012 for articles in English using the search terms nephrotic syndrome, thrombosis, thromboembolism, anticoagulation, warfarin, heparin, low-molecular-weight heparin, enoxaparin, dalteparin, tinzaparin, statin, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, aspirin, direct thrombin inhibitor, rivaroxaban, argatroban, lepirudin, bivalirudin, dabigatran, factor Xa inhibitor, fondaparinux, rivaroxaban, clopidogrel, ticlopidine, and prasugrel.All relevant original studies, meta-analyses, systematic reviews, guidelines, and review articles were assessed for inclusion. References from pertinent articles were examined for additional content not found during the initial search.NS leads to multiple complications, including hypercoagulability. A small prospective cohort study used enoxaparin for primary prophylaxis and demonstrated successful prevention of thrombotic events with minimal adverse events. Additional information has come in the form of decision analyses, which show potential decreased morbidity and mortality when primary prophylaxis for thrombotic events is used; however, all data have numerous limitations. Other strategies for thrombus prevention, including statins and antiplatelet agents, also have been investigated.When patients with NS are admitted to the hospital, develop an acute medical illness, or acquire an additional thrombotic events risk factor such as surgery, active malignancy, or pregnancy, consideration for primary pharmacologic prophylaxis with appropriately dosed low-molecular-weight heparin or other indicated anticoagulant should include the potential for increased thrombotic events risk in this patient population. Consideration may also be given to the use of primary pharmacologic prophylaxis with low-molecular-weight heparin or oral vitamin K antagonist in patients with membranous nephropathy once the albumin level drops below 2.0-2.5 g/dL. Short-term use of pharmacologic prophylaxis during the first 6 months following diagnosis warrants further investigation.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and crosstalk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this crosstalk by investigating the effects of C1-INH on Escherichia coli (E. coli)-induced hemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultra-purified E. coli LPS in the absence or presence of C1-INH or protease inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using RT-qPCR, and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1.2 (PTF1.2) and long-pentraxin 3 (PTX3) were measured by ELISA. Cytokines were analysed using a multiplex kit. C1-INH (1.25 to 5 mg/ml) efficiently and dose-dependently reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1.2 in plasma (P<0.05). Both LPS and E. coli-induced marked upregulation of TF mRNA levels and surface expression on whole blood monocytes. This upregulation was efficiently reduced by treatment with C1-INH (P<0.05). C1-INH reduced the release of PTX3 (P<0.05) and virtually all cytokines measured (P<0.05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH more efficiently inhibited most of the other readouts, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and hemostatic responses.

Heparin-induced thrombocytopenia (HIT) is a drug-mediated, prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After their binding to PF4/heparin complexes on the platelet surface, HIT antibodies are capable of intravascular platelet activation by cross-linking Fcγ receptor IIA leading to a platelet count decrease and/or thrombosis. Diagnosis of HIT is often difficult. This, and the low specificity of the commercially available immunoassays, leads currently to substantial overdiagnosis of HIT. Timing of onset, the moderate nature of thrombocytopenia, and the common concurrence of thrombosis are very important factors, which help to differentiate HIT from other potential causes of thrombocytopenia. A combination of a clinical pretest scoring system and laboratory investigation is usually necessary to diagnose HIT. Although HIT is considered to be a rare complication of heparin treatment, the very high number of hospital inpatients, and increasingly also hospital outpatients receiving heparin, still result in a considerable number of patients developing HIT. If HIT occurs, potentially devastating complications such as life-threatening thrombosis make it one of the most serious adverse drug reactions. If HIT is strongly suspected, all heparin must be stopped and an alternative nonheparin anticoagulant started at a therapeutic dose to prevent thromboembolic complications. However, the nonheparin alternative anticoagulants bear a considerable bleeding risk, especially if given to patients with thrombocytopenia due to other reasons than HIT. While established drugs for HIT are disappearing from the market (lepirudin, danaparoid), bivalirudin, fondaparinux and potentially the new anticoagulants such as dabigatran, rivaroxaban and apixaban provide new treatment options.

The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.

To report a case of heparin-induced thrombocytopenia (HIT) in a patient with concurrent liver dysfunction and a prolonged baseline activated partial thromboplastin time (aPTT) in whom argatroban therapy was monitored with aPTT and a novel plasma-diluted thrombin assay.An 80-year-old man with HIT and liver dysfunction was treated with argatroban, which was initiated at a dose of 0.5 μg/kg/min and gradually decreased to 0.09 μg/kg/min. The patient had a mildly prolonged aPTT at baseline (37.5 seconds). He was concurrently monitored with aPTT, per institution protocol, and plasma-diluted thrombin time. Plasma-diluted thrombin times were consistently lower than aPTTs, but mirrored the trend of the aPTTs. Eleven hours after argatroban was stopped, the aPTT remained elevated (53.9 seconds), while the plasma-diluted thrombin time returned to normal range (26.4 seconds). The patient's therapy was transitioned to warfarin and he had a hospital course with no thrombotic or bleeding complications.Plasma-diluted thrombin time is a novel laboratory test consisting of 1 part patient plasma diluted with 3 parts normal plasma. Plasma-diluted thrombin time has been shown to blunt the sensitivity of the thrombin time and may be more accurate for drug monitoring. A MEDLINE search revealed 2 studies using the plasma-diluted thrombin time assay. The first study compared aPTT and plasma-diluted thrombin times in blood samples mixed with argatroban, bivalirudin, or lepirudin at 3 different concentrations. Blood samples contained lupus inhibitors, vitamin k deficiency, or normal baseline aPTTs. The aPTT overestimated drug concentrations in all samples with lupus anticoagulant and vitamin k deficiency, while the plasma-diluted thrombin time correctly estimated drug concentrations in nearly all samples. The second study looked at monitoring dabigatran with plasma-diluted thrombin time and found a linear relationship between the plasma-diluted thrombin time and the dabigatran dose-response curve.Plasma-diluted thrombin time may be an alternative for direct thrombin inhibitor monitoring in patients with elevated aPTT values at baseline. Further randomized control trials are needed to determine its applicability in clinical practice.

Type II heparin-induced thrombocytopenia is currently managed by withdrawing heparin and replacing it with danaparoid sodium. Argatroban, a direct thrombin inhibitor anticoagulant (like lepirudin), is now authorised for this indication in France, following authorisation in several other countries since the early 2000s. Argatroban has not been compared with danaparoid in clinical trials. About 700 patients treated with argatroban in 2 trials were compared to historical controls managed by simple withdrawal of heparin and, in some cases, switching to an oral anticoagulant. Argatroban had no apparent advantages in terms of death or the need for amputation. Argatroban did not appear to increase the risk of bleeding in these trials, but evidence provided by historical comparisons is weak. The adverse effect profile includes hepatic disorders (notably fulminant hepatitis). The risk of pharmacokinetic interactions appears to below. In practice, given the absence of a proven therapeutic advantage, it is better to continue to use danaparoid for first-line treatment, reserving argatroban for the rare situations in which danaparoid is inappropriate.

The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood.Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody.The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb.Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

DNA aptamer RA36 with a molecular weight of 10000 is direct-acting anticoagulant whose efficacy is lower than that of recombinant hirudin and unfractionated heparin (UFH) in blood clotting time (BCT), activated blood recalcification time (ABRT), recalcification time (RT), prothrombin time (PT), and activated partial thromboplastin time (APTT) tests. The anticoagulant effect of RA36 is comparable with that of UFH in the thrombin time (TT) test, but is lower than the effect of recombinant hirudin. Analysis of the blood and plasma anticoagulant activity during intravenous bolus administration of aptamer RA36 in rabbits and rats is based on the use ABRT (in blood case) and APTT/RT (in plasma case) tests. The range of doses for evaluation of pharmacodynamic parameters of RA36 during intravenous bolus administration in rabbits and rats is 3 - 34 mg/kg and 1 - 27 mg/kg, respectively. Accordingly, designed dose range for humans is 1 -29 mg/kg.