Van Der Poll Research Articles

Role of Lipopolysaccharide and Cecal Ligation and Puncture on Blood Coagulation and Inflammation in Sensitive and Resistant Mice Models

The hemostatic system is severely disturbed during endotoxemia, leading to a hypercoagulable state. However, it remains uncertain to what extent hypercoagulability is the critical factor in determining the clinical course rather than just the consequence of a severe systemic inflammatory response. To answer this question, we evaluated the evolution of hemostatic and inflammatory markers, as well as histological features, in mice sensitive and resistant to two models of endotoxemia: lipopolysaccharide-injection and cecal ligation puncture. Genetic (knockout mice) and pharmacological (PJ34) blockade of the nuclear enzyme PARP-1 was used to achieve resistance to the endotoxemia. In both models, endotoxemia resulted in antithrombin deficiency, decreased platelets, and fibrin deposition in organs, which were similar in all groups of mice. By contrast, proinflammatory mediators, inflammatory cell infiltration (especially that mediated by mononuclear cells), and organ degeneration were more intense in sensitive animals. Further studies supported a negative role for the triggering of the coagulation cascade in the mortality associated with the endotoxic shock. Hirudin had a minor effect on cell infiltration and organ damage, despite causing a potent inhibition of fibrin deposition. On the other hand, a sublethal dose of lipopolysaccharide yielded significant fibrin deposition but weak activation of the inflammatory response. Our results suggest that activation of coagulation by endotoxemia is severe and independent of the inflammatory response. However, such activation may act with fibrin deposition to have a minor influence on survival in sepsis.

A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Platelets at a crossroad of pathogenic pathways in sepsis

Platelets at a crossroad of pathogenic pathways in sepsis

Macrolides in the treatment of asthma and cystic fibrosis

Asthma and cystic fibrosis are two respiratory diseases characterized by chronic inflammation, leading to remodelling of the airways. Macrolides are widely used antibiotics, with a peculiar anti-inflammatory effect. On the basis of the methodologies used by the Cochrane collaboration, this review discusses the evidence for their long-term use as anti-inflammatory agents in these two diseases. Three randomized-controlled trials (RCTs) were identified for both asthma and cystic fibrosis. A positive effect of macrolides on reducing eosinophil numbers and markers of eosinophilic inflammation was demonstrated in patients with asthma. Data on cystic fibrosis demonstrated an effect on lung function with an increase of 5.4% in forced vital capacity (FVC) in patients treated with macrolide vs. placebo, but without a significant effect on FEV1. Side-effects were rare, mild and reversible on withdrawal of treatment. Although preliminary data from small studies are promising, the role of macrolides in the treatment of these chronic disorders needs to be more firmly established with larger, well-designed trials, targeted to investigate major clinical outcomes.

Measuring activated protein C in plasma to assess its role as a critical modulator of coagulation and inflammation

Measuring activated protein C in plasma to assess its role as a critical modulator of coagulation and inflammation

Essential Role of p38 Mitogen-activated Protein Kinase in Contact Hypersensitivity

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.

Sepsis: from basics to clinical practice - Editorial

Severe sepsis and septic shock are common clinical problems from the newborn to elderly people. The patients often need to be treated in intensive care units. The mortality in septic shock is high. It has been estimated that about 150,000 and 200,000 yearly deaths are due to sepsis in the European Union and the United States, respectively. New developments in the treatment possibilities of sepsis have, however, been achieved during the last few years, and it may be easier to tackle this major clinical problem more effectively than before. This issue of the Journal includes a special section on sepsis. In the seven articles, both molecular and clinical aspects are discussed in the light of recent discoveries and modern clinical practice. Animal models for sepsis have revealed promising immunomodulating agents for the treatment of sepsis. Most models for sepsis lack an infectious focus. Many studies have addressed a variety of immunomodulating strategies, but the results have often been discouraging. The differences in the sepsis models as well as their advantages and disadvantages are discussed by Schultz and van der Poll (p. 573±81). They conclude that the use of a combination of models to test new therapeutic agents would be the most likely successful route for preclinical studies. Of the new molecules studied, activated protein C has been shown to be particularly effective in preventing microvascular thrombosis. Dr. Esmon provides a comprehensive review of the biology of the protein C pathway and of the features making it unique in controlling both microvascular coagulation and acute in ammatory response (p. 598±605). The review describes the complex molecular pathways, in which protein C is involved in these two contexts. Recent data suggest that activated protein C reduces the death rate from sepsis. Some concerns have also been raised, and the clinical studies published need further support. Accordingly, several such studies are underway. Another potential concern is the high cost, which may restrict the world-wide use of activated protein C. We may expect that the next few years will clarify the role of activated protein C in clinical practice. Both the in ammatory reaction and concurrent activation of the anti-in ammatory mechanisms in the host are central to the pathophysiology of sepsis. The balance between the two opposing groups of reactions may be critical for the outcome of the sepsis patient. Activated phagocytes and high circulating levels of pro-in ammatory cytokines occur in patients at risk for multi-organ failure. Impaired natural (innate) and adaptive immune reactions also predispose the patients to the development of secondary infections. As well as therapies aiming at modifying the natural course of in ammatory responses, the in ammatory markers may also offer tools for the diagnosis of sepsis and the degree of organ failure associated with it. Takala et al. (p. 614± 23) summarize their own results and provide evidence that markers like CD11b on phagocytes and serum interleukin-8 can discriminate patients at high risk for organ dysfunction. Such discrimination would be useful in targeting treatment for severe infections and in focusing anti-in ammatory therapies on the right type of patients. Severe sepsis is today a common disease accounting for up to 10% of hospital admissions. Dr. Vincent and co-workers give a practical view on sepsis and septic shock covering the supportive measures such as uid resuscitation and vasopressors (p. 606±13). They also review the use of activated protein C in the world-wide PROWESS trial. Three other articles focus on more speci®c issues in the treatment of sepsis, namely the treatment of impaired perfusion of the affected organs (Ruokonen et al. (p. 590±7)), the use of corticosteroids (Chadda and Annane (p. 582± 9)) and the management of meningoccocal disease (Wall (p. 624±34)). The main causes of septic death are multiple organ failure and concurrent hypotension. The treatment of haemodynamics consists of appropriate uid therapy and vasoactive drugs aiming to correct hypotension and inappropriately low cardiac output (Ruokonen et al. (p. 590±7)). While norepinephrine is the drug of choice for low vascular resistance, insuf®cient myocardial function is commonly treated with dobutamine. The use of norepi-

Bronchoalveolar activation of coagulation and depression of fibrinolysis as pathogenetic pathway of ARDS in experimental endotoxemia in chimpanzees: M. Levi, T. van der Poll, B.J. Biemond, H. ten Cate, B. Kuipers, J.W. ten Cate. Centre for Haemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Centre, Amsterdam and Department of Pulmonology, Sophia Hospital, Zwolle, Netherlands

Bronchoalveolar activation of coagulation and depression of fibrinolysis as pathogenetic pathway of ARDS in experimental endotoxemia in chimpanzees: M. Levi, T. van der Poll, B.J. Biemond, H. ten Cate, B. Kuipers, J.W. ten Cate. Centre for Haemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Centre, Amsterdam and Department of Pulmonology, Sophia Hospital, Zwolle, Netherlands

Effect of Tumor Necrosis Factor in Normal Subjects

To the Editor: Considering that there is now a consensus on the central role of tumor necrosis factor in the pathogenesis of septic shock, the study by van der Poll et al. (June 7 issue),1 in which normal subjects were administered tumor necrosis factor, is both timely and important. I agree with their deduction — based on the increased levels of the activation peptide of factor X and prothrombin fragment F1+2 — that the common coagulation pathway is probably activated initially by tissue factor. However, their conclusion that the intrinsic pathway, particularly the contact system, was not activated is.