hirudin and Sepsis

Incidence of deep vein thrombosis in critically ill patients depends on the underlying disease but may be as high as 60%. The Surviving Sepsis Campaign clearly recommends administering anticoagulation in the absence of specific contraindications in patients with severe sepsis or septic shock. The article discusses risk factor for thromboembolic events in critical illness as well as means of non-pharmacologic and pharmacologic thrombosis prophylaxis. Peripheral vasoconstriction, edema, shock, and administration of catecholamines may reduce the bioavailability and efficacy of subcutaneous administration of low molecular weight heparin. This article further elaborates on the problem and pathophysiology of heparin resistance. Continuous intravenous administration of new anticoagulants may be a promising alternative to indirect anticoagulants. Severity of illness and SAPS II-score determine dosing of the direct thrombin inhibitor argatroban which needs to be about 10-times lower than in patients without critical illness.

Topics: Anticoagulants; Arginine; Biological Availability; Chromosome Breakage; Chromosome Disorders; Critical Care; Dose-Response Relationship, Drug; Drug Resistance; Heparin; Heparin, Low-Molecular-Weight; Hirudins; Humans; Infusions, Intravenous; Injections, Subcutaneous; Pipecolic Acids; Recombinant Proteins; Risk Factors; Sepsis; Severity of Illness Index; Shock, Septic; Sulfonamides; Thrombin; Thrombocytopenia; Venous Thrombosis

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Topics: Antibodies, Blocking; Antibodies, Monoclonal, Humanized; Blood Cells; Blood Coagulation; Cells, Cultured; Complement C5; Disaccharides; Escherichia coli; Escherichia coli Infections; Female; Hemostasis; Hirudins; Humans; Lipopolysaccharide Receptors; Male; Platelet Function Tests; Receptor Cross-Talk; Recombinant Proteins; Sepsis; Sugar Phosphates; Thrombelastography; Thromboplastin; Toll-Like Receptor 4

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

Topics: Antithrombins; Blood Coagulation; C-Reactive Protein; Complement C3; Escherichia coli; Hirudins; Humans; Lipopolysaccharide Receptors; Lipopolysaccharides; Peptide Fragments; Peptides, Cyclic; Prothrombin; Recombinant Proteins; RNA, Messenger; Sepsis; Serum Amyloid P-Component; Thromboplastin; Up-Regulation

Neisseria meningitidis causes fulminant meningococcal sepsis with a massive activation of the coagulation and complement cascades. Bacterial cell envelope molecules from N. meningitidis, particularly lipopolysaccharide (LPS), induce tissue factor (TF) expression. In meningococcal sepsis, TF can be detected on circulating monocytes and microparticles (MPs) within the bloodstream. During infection, Nm activates C5 and C5a, which also is able to induce TF. We evaluated the effect of eculizumab, a C5-blocking monoclonal antibodies (mAb), on cell- and MP-associated TF. Using a lepirudin-anticoagulated whole blood model, we activated the coagulation and complement cascades by N. meningitidis, and investigated the interaction between the cascade systems with special focus on cell-associated TF-expression (mRNA and protein) and MP-associated TF-dependent thrombin and fibrin generation in platelet-free plasma. We also examined the ability of TF-positive MPs to support clot formation in whole blood. In addition, the effect of corn trypsin inhibitor and time-dependent changes on MP-associated functional TF activity was examined. Inhibition of C5 reduced cell-associated TF expression at both gene and protein level, and reduced MP-associated TF-dependent thrombin and fibrin generation in platelet-poor plasma, MP-induced TF-dependent clot formation in whole blood, implying that the complement and coagulation cascades are interplayers in N. meningitidis-mediated activation of these cascades.

Topics: Antibodies, Monoclonal, Humanized; Anticoagulants; Blood Coagulation; Blood Platelets; Cell-Derived Microparticles; Complement Activation; Complement C5; Hirudins; Humans; Meningococcal Infections; Neisseria meningitidis; Recombinant Proteins; Sepsis; Thromboplastin

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.

Topics: Adult; Anticoagulants; Complement Inactivating Agents; Complement System Proteins; Cytokines; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Flow Cytometry; Hirudins; Humans; Lipopolysaccharide Receptors; Monocytes; Real-Time Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Sepsis; Thromboplastin; Up-Regulation

Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo.. Human endothelial cells were stimulated with thrombin (0.1-10 U/mL), CD40L (0.25-1 μg/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, α-CD40L, and α-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun N-terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L.. Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antibodies; Blood Coagulation; Case-Control Studies; CD40 Antigens; CD40 Ligand; Cell-Derived Microparticles; Cells, Cultured; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelial Cells; Endotoxemia; Female; Gene Expression Regulation, Enzymologic; Hirudins; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Male; Matrix Metalloproteinase 10; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Mitogen-Activated Protein Kinase 8; Multivariate Analysis; p38 Mitogen-Activated Protein Kinases; Peptides; Protein Kinase Inhibitors; Receptor, PAR-1; Risk Assessment; Risk Factors; RNA, Messenger; Sepsis; Signal Transduction; Spain; Survival Analysis; Thrombin; Time Factors; Up-Regulation

To investigate the effects of antithrombin on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) production in three different in vitro cellular systems: whole blood, human umbilical vein endothelial cells, and mononuclear cells.. Laboratory in vitro study of the effects of antithrombin on procoagulant activity and cytokine release by LPS-stimulated endothelial and peripheral blood cells.. In vitro whole blood, isolated human umbilical vein endothelial cell, and mononuclear cell cultures.. Addition of antithrombin to LPS-treated whole blood, human umbilical vein endothelial cells, and mononuclear cells.. Citrated whole blood, isolated human umbilical vein endothelial cells, or mononuclear cells were stimulated with LPS for 4-6 hrs in the presence or absence of antithrombin. Tissue factor activity was estimated by a tissue factor-dependent clotting or chromogenic assay and IL-6 was measured by specific ELISA. Antithrombin was found to inhibit tissue factor and IL-6 production in all three systems in a dose-dependent manner (0-40 IU/mL). Flow-through fractions of immunoadsorbed antithrombin concentrate were found to be ineffective. Five different batches of the same antithrombin concentrate were tested and the inhibitory activity was found to be consistent throughout all batches. Up to 40 microM of recombinant hirudin, a specific thrombin inhibitor, did not inhibit the production of tissue factor or IL-6 in either of the three cell systems, suggesting that the observed inhibition by antithrombin was not due solely to its ability to inhibit thrombin.. Apart from the inhibition of thrombin and other activated clotting factors, antithrombin may also down-regulate the cellular expression of proinflammatory cytokines. Consequently, antithrombin concentrates may have value in the treatment of sepsis-induced disseminated intravascular coagulation.

Topics: Antithrombins; Blood; Cells, Cultured; Endothelium, Vascular; Hirudins; Humans; In Vitro Techniques; Interleukin-6; Leukocytes, Mononuclear; Lipopolysaccharides; Sepsis; Thromboplastin; Umbilical Veins

The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels. In a model of septic peritonitis-cecal ligation and puncture-TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented. To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase. Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees. Under these experimental conditions, antibiotics prevented death. In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization. These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.

Topics: Animals; Cecum; Colony Count, Microbial; Disease Models, Animal; Heparin; Hirudins; Immunity, Innate; Ligation; Male; Mice; Peritonitis; Sepsis; Tissue Adhesions; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Rats which were infected with the gramnegative pathogen Klebsiella pneumoniae develop disseminated intravascular coagulation (DIC), multi-organ failure (MOF) and finally die in a septic shock. We investigated the therapeutic effect of antibiotic (tobramycin) treatment combined with the infusion of the highly specific thrombin inhibitor rec. hirudin. Although administration of 2 mg/kg tobramycin alone leads to a decrease of the bacterial burden, DIC could not be prevented. Infusion of rec. hirudin (0.25 mg/kg x h) for 4 h (start of treatment 1 h post infection), in addition to a bolus administration of tobramycin, led to an amelioration of DIC parameters as fibrinogen, thrombin-antithrombin complex (TAT) and platelets. Serum transaminase levels (GOT, GPT) as a marker of MOF were significantly improved by rec. hirudin, the T50 value increased from 17 h in the tobramycin group to 42 h in the tobramycin+rec. hirudin group, mortality rates were 90% or 60%, respectively. Combination of heparin (100 U/kg x h) and tobramycin was not effective on survival.

Topics: Animals; Disseminated Intravascular Coagulation; Drug Therapy, Combination; Female; Hirudin Therapy; Hirudins; Klebsiella Infections; Klebsiella pneumoniae; Multiple Organ Failure; Rats; Recombinant Proteins; Sepsis; Tobramycin