Thrombin contributes to protective immunity in pneumonia-derived sepsis via fibrin polymerization and platelet-neutrophil interactions.

Essentials Immunity and coagulation are linked during sepsis but the role of thrombin is not fully elucidated. We investigated the effect of thrombin inhibition on murine Klebsiella pneumosepsis outcome. Thrombin is crucial for survival and limiting bacterial growth in pneumonia derived sepsis. Thrombin improves host defense via fibrin and enhancement of platelet-neutrophil interactions. SUMMARY: Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis-induced thrombocytopenia or organ damage. Conclusions These results suggest that thrombin plays an important role in protective immunity during pneumonia-derived sepsis by fibrin polymerization and enhancement of platelet-neutrophil interactions.

The role of platelet MyD88 in host response during gram-negative sepsis.

BACKGROUND: Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists. OBJECTIVE: Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae. METHODS: Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets. RESULTS: In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis. CONCLUSION: Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis.

Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis.

BACKGROUND: Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. METHODS: Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. RESULTS: Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. CONCLUSION: Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis.

Protease activated receptor 4 limits bacterial growth and lung pathology during late stage Streptococcus pneumoniae induced pneumonia in mice.

Streptococcus pneumoniae is a common causative pathogen of pneumonia and sepsis. Pneumonia and sepsis are associated with enhanced activation of coagulation, resulting in the production of several host-derived proteases at the primary site of infection and in the circulation. Serine proteases cleave protease activated receptors (PARs), which form a molecular link between coagulation and inflammation. PAR4 is one of four subtypes of PARs and is widely expressed by multiple cell types in the respiratory tract implicated in pulmonary inflammation, by immune cells and by platelets. In mice, mouse (m)PAR4 is the only thrombin receptor expressed by platelets. We here sought to determine the contribution of mPAR4 to the host response during pneumococcal pneumonia. Pneumonia was induced by intranasal inoculation with S. pneumoniae in mPAR4-deficient (par4-/-) and wild-type mice. Mice were sacrificed after 6, 24 or 48 hours (h). Blood, lungs, liver and spleen were collected for analyses. Ex vivo stimulation assays were performed with S. pneumoniae and mPAR4 activating peptides. At 48 h after infection, higher bacterial loads were found in the lungs and blood of par4-/- mice (p < 0.05), accompanied by higher histopathology scores and increased cytokine levels (p < 0.05) in the lungs. Ex vivo, co-stimulation with mPAR4 activating peptide enhanced the whole blood cytokine response to S. pneumoniae. Thrombin inhibition resulted in decreased cytokine release after S. pneumoniae stimulation in human whole blood. Our findings suggest that mPAR4 contributes to antibacterial defence during murine pneumococcal pneumonia.

Endogenous protein C has a protective role during Gram-negative pneumosepsis (melioidosis).

BACKGROUND: Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality. OBJECTIVES: We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia-derived sepsis (melioidosis). METHODS: We infected wild-type (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in southeast Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice. RESULTS: MPC1609, but not MAPC1591, significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871, suggesting that S1P1 does not play a major role in this model. CONCLUSIONS: These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC .

Effect of the factor V Leiden mutation on the incidence and outcome of severe infection and sepsis.

Activation of coagulation frequently occurs in severe infection and sepsis and may contribute to the development of multiple organ dysfunction. Factor V Leiden is a relatively common mutation resulting in a mild prohaemostatic state and consequently with an increased tendency to develop thrombosis. Hypothetically, patients with factor V Leiden may suffer from more severe coagulopathy in case of severe infection or sepsis. Aggravation of the procoagulant state in sepsis may subsequently result in more severe organ dysfunction and an increased risk of death. Here we discuss the experimental and clinical evidence regarding the relationship between the presence of a factor V Leiden mutation and the incidence and outcome of sepsis.

Plasminogen activator inhibitor type I contributes to protective immunity during experimental Gram-negative sepsis (melioidosis).

BACKGROUND: Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram-negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI-1), an important regulator of inflammation and fibrinolysis. OBJECTIVES: In this study, we aimed to investigate the role of PAI-1 during melioidosis. METHODS: Wild-type (WT) and PAI-1-deficient (PAI-1-/1(-/-) ) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72 h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed. RESULTS: PAI-1(-/-) mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, P < 0.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI-1(-/-) mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin-antithrombin complexes and D-dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea). CONCLUSIONS: PAI-1 has a protective role during severe Gram-negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.

Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia.

BACKGROUND: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-induced coagulation and has been evaluated in clinical trials involving patients with sepsis and community-acquired pneumonia. OBJECTIVE: To examine the effect of rh-TFPI on coagulation, inflammation and bacterial outgrowth in S. pneumoniae pneumonia in mice, with or without concurrent antibiotic treatment. METHODS: Pneumonia was induced by intranasal inoculation with S. pneumoniae. Mice were treated with placebo, rh-TFPI, ceftriaxone or rh-TFPI combined with ceftriaxone. Early (8 h) and late (24 h) initiated treatments were evaluated. Samples were obtained 24 or 48 h after infection, for early and late initiated treatment, respectively. In vitro, placebo or rh-TFPI was added to a suspension of S. pneumoniae. RESULTS: Rh-TFPI reduced pneumonia-induced coagulation; rh-TFPI with ceftriaxone further attenuated coagulation relative to ceftriaxone alone. Rh-TFPI inhibited accumulation of neutrophils in lung tissue and reduced the levels of several cytokines and chemokines in lungs and plasma in mice not treated with antibiotics; in these animals, rh-TFPI initiated 24 h after infection decreased pulmonary bacterial loads. In vitro, rh-TFPI also inhibited growth of S. pneumoniae. CONCLUSIONS: Therapeutic rh-TFPI attenuates coagulation, inflammation and bacterial growth during pneumococcal pneumonia, whereby the latter two effects only become apparent in the absence of concurrent antibiotic treatment.

Factor V Leiden mutation does not affect coagulopathy or outcome in lethal H1N1 influenza.

Influenza A is a major cause of mortality. Knowledge on coagulation activation in influenza infection is limited. The factor V Leiden (FVL) mutation is possibly subject to positive selection pressure. It is unknown whether this mutation impacts on the outcome of severe influenza. In the present study, the effect of lethal influenza on pulmonary and systemic coagulation activation and whether or not FVL mutation alters coagulation activation in and the course of lethal influenza, was determined. Wild-type mice, and mice heterozygous or homozygous for FVL were infected intranasally with a lethal dose of H1N1 (haemagglutinin 1 and neuraminidase 1) influenza A. Mice were sacrificed after 48 or 96 h for determination of coagulation activation, histopathology, pulmonary inflammatory parameters and viral load, or were observed in a survival study. Extensive local and systemic coagulation activation during lethal influenza was demonstrated by increased lung and plasma levels of thrombin-antithrombin complexes and fibrin degradation products, and by pulmonary fibrin deposition. FVL mutation did not influence the procoagulant response, lung histopathology or survival. FVL mice demonstrated elevated viral loads 48 h after infection. In conclusion, coagulation is activated locally and systemically during lethal murine influenza A infection. The FVL mutation does not influence coagulation activation, lung inflammation or survival in lethal influenza A.

Preferential protection of Borrelia burgdorferi sensu stricto by a Salp15 homologue in Ixodes ricinus saliva.

BACKGROUND: Ixodes ticks are the main vectors for Borrelia burgdorferi sensu lato. In the United States, B. burgdorferi is the sole causative agent of Lyme borreliosis and is transmitted by Ixodes scapularis. In Europe, 3 Borrelia species-B. burgdorferi, B. garinii, and B. afzelii-are prevalent, which are transmitted by Ixodes ricinus. The I. scapularis salivary protein Salp15 has been shown to bind to B. burgdorferi outer surface protein (Osp) C, protecting the spirochete from antibody-mediated killing. METHODS AND RESULTS: We recently identified a Salp15 homologue in I. ricinus, Salp15 Iric-1. Here, we have demonstrated, by solid-phase overlays, enzyme-linked immunosorbent assay, and surface plasmon resonance, that Salp15 Iric-1 binds to B. burgdorferi OspC. Importantly, this binding protected the spirochete from antibody-mediated killing in vitro and in vivo; immune mice rechallenged with B. burgdorferi preincubated with Salp15 Iric-1 displayed significantly higher Borrelia numbers and more severe carditis, compared with control mice. Furthermore, Salp15 Iric-1 was capable of binding to OspC from B. garinii and B. afzelii, but these Borrelia species were not protected from antibody-mediated killing. CONCLUSIONS: Salp15 Iric-1 interacts with all European Borrelia species but differentially protects B. burgdorferi from antibody-mediated killing, putatively giving this Borrelia species a survival advantage in nature.

Transgenic tissue-type plasminogen activator expression improves host defense during Klebsiella pneumonia.

BACKGROUND: Severe pneumonia is associated with a local inhibition of fibrinolysis in the lung as reflected by strongly reduced pulmonary plasminogen activator activity. OBJECTIVES: To study the effect of elevation of local plasminogen activator activity during pneumonia caused by the common respiratory pathogen Klebsiella pneumoniae. METHODS: Female C57Bl/6 mice were inoculated intranasally with a replication-defective adenoviral vector expressing human tissue-type plasminogen activator or a control vector 24 h before intranasal infection with K. pneumoniae. RESULTS: Mice infected with Klebsiella via the airways developed overt pneumonia, which was accompanied by a downregulation of pulmonary tissue-type plasminogen activator levels at protein and mRNA levels. Pulmonary overexpression of human tissue-type plasminogen activator resulted in increased fibrinolytic activity in the lungs during pneumonia, as indicated by higher D-dimer levels and reduced fibrin deposition. Interestingly, overexpression of tissue-type plasminogen activator markedly improved host defense against pneumonia: mice treated with the tissue-type plasminogen activator vector displayed less bacterial growth and dissemination, attenuated distant organ injury and a reduced mortality. CONCLUSIONS: These data demonstrate that local elevation of plasminogen activator activity in the lungs improves host defense against severe gram-negative pneumonia and sepsis.

Activation of coagulation with concurrent impairment of anticoagulant mechanisms correlates with a poor outcome in severe melioidosis.

BACKGROUND: Melioidosis, which is caused by infection with the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of sepsis in South-East Asia with a mortality of up to 40%. Knowledge of the involvement of coagulation and fibrinolysis in the pathogenesis of melioidosis is highly limited. OBJECTIVE: To define the involvement of the coagulation and fibrinolytic systems in patients with severe melioidosis. METHODS: Parameters of coagulation and fibrinolysis were measured in 34 patients with culture proven septic melioidosis and 32 healthy controls. RESULTS: Patients demonstrated strong activation of the coagulation system, as reflected by high plasma levels of soluble tissue factor, the prothrombin fragment F(1+2) and thrombin-antithrombin complexes (TATc), and consumption of coagulation factors resulting in a prolonged prothrombin time and activated partial thromboplastin time. Concurrently, anticoagulant pathways were downregulated in patients: protein C, protein S, and antithrombin levels were all decreased when compared to controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis, as reflected by elevated concentrations of tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1, plasmin-alpha2-antiplasmin complexes (PAPc) and D-dimer. High TATc/PAPc ratios in patients pointed to a predominance of the prothrombotic pathway in melioidosis. Furthermore, soluble thrombomodulin levels were increased. The extent of coagulation activation correlated with mortality; patients who went on to die had higher TATc, F(1+2), tPA and PAPc and lower protein C and antithrombin levels on admission than patients who survived. CONCLUSIONS: The coagulation system is strongly activated during melioidosis. A high degree of activation of the coagulation system is an indicator of poor outcome in patients with melioidosis.

Identification of Salp15 homologues in Ixodes ricinus ticks.

The 15-kDa Ixodes scapularis salivary gland protein Salp15 protects Borrelia burgdorferi sensu stricto from antibody-mediated killing and facilitates infection of the mammalian host. In addition, Salp 15 has been shown to inhibit T-cell activation. We determined whether Ixodes ricinus, the major vector for Lyme borreliosis in Western Europe, also express salp15-related genes. We show that engorged I. ricinus express salp15 and we have identified three Salp15 homologues within these ticks by reverse transcriptase-polymerase chain reaction (RT-PCR). One of the predicted proteins showed 80% similarity to I. scapularis Salp15, evenly distributed over the entire amino acid sequence, whereas the two other predicted proteins showed approximately 60% similarity, mainly confined to the signal sequence and C-terminus. Comparison of the DNA and protein sequences with those deposited in several databases indicates that these proteins are part of a Salp15 family of which members are conserved among different Ixodes species, all capable of transmitting B. burgdorferi sensu lato. This suggests that these Salp15 homologues could also play a role in the transmission of diverse Borrelia species and in inhibition of T-cell activation.

Natural anticoagulants limit lipopolysaccharide-induced pulmonary coagulation but not inflammation.

Pulmonary coagulopathy and hyperinflammation may contribute to an adverse outcome in sepsis. The present study determines the effects of natural inhibitors of coagulation on bronchoalveolar haemostasis and inflammation in a rat model of endotoxaemia. Male Sprague-Dawley rats were randomised to treatment with normal saline, recombinant human activated protein C (APC), plasma-derived antithrombin (AT), recombinant human tissue factor pathway inhibitor (TFPI), heparin or recombinant tissue plasminogen activator (tPA). Rats were intravenously injected with lipopolysaccharide (LPS), which induced a systemic inflammatory response and pulmonary inflammation. Blood and bronchoalveolar lavage were obtained at 4 and 16 h after LPS injection, and markers of coagulation and inflammation were measured. LPS injection caused an increase in the levels of thrombin-AT complexes, whereas plasminogen activator activity was attenuated, both systemically and within the bronchoalveolar compartment. Administration of APC, AT and TFPI significantly limited LPS-induced generation of thrombin-AT complexes in the lungs, and tPA stimulated pulmonary fibrinolytic activity. However, none of the agents had significant effects on the production of pulmonary cytokines, chemokines, neutrophil influx and myeloperoxidase activity. Natural inhibitors of coagulation prevent bronchoalveolar activation of coagulation, but do not induce major alterations of the pulmonary inflammatory response in rat endotoxaemia.

Platelets and phospholipids in tissue factor-initiated thrombin generation.

The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 microM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 microM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 x 10(8)/ml to 0.5 x 10(8)/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient's blood was accelerated from approximately 10 min to 5 min when the platelet concentration increased from 0.05 x 10(8)/ml to 0.11 x 10(8)/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 x 10(8)/ml (range 0.5-3.1 x 10(8)/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 x 10(8)/ml to 3.1 x 10(8)/ml (4.0+/-0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 x 10(8)/ml.

Systemic anti-inflammatory mediators in COPD: increase in soluble interleukin 1 receptor II during treatment of exacerbations.

BACKGROUND: The aim of this study was to test the hypothesis that the chronic inflammatory process present in chronic obstructive pulmonary disease (COPD) is due to a defective endogenous anti-inflammatory mechanism. METHODS: Systemic levels of the anti-inflammatory mediators soluble interleukin 1 receptor II (sIL-1RII), soluble tumour necrosis factor receptor p55 (sTNF-R55) and sTNF-R75, and of C reactive protein (CRP) and lipopolysaccharide binding protein (LBP) were analysed in 55 patients with stable COPD (median forced expiratory volume in one second (FEV(1)) 34% predicted (range 15-78)) and compared with levels in 23 control subjects. In addition, changes in these mediators were studied in 13 patients with COPD (median FEV(1) 34% predicted (range 19-51)) during the first 7 days in hospital with an exacerbation of the disease. RESULTS: Patients with stable COPD were characterised by a systemic inflammatory process indicated by an increased leucocyte count (7.2 (4.7-16.4) v 4.8 (3.5-8.3) x 10(9)/l), raised levels of CRP (11.8 (1.1-75.0) v 4.1 (0.6-75.0) microg/ml) and LBP (45.6 (8.1-200.0) v 27.9 (14.1-71.5) microg/ml), and moderate increases in both sTNF-Rs. In contrast, the sIL-1RII level did not differ between patients and controls (4.53 (2.09-7.60) v 4.63 (3.80-5.93) ng/ml). During treatment of disease exacerbations, systemic levels of both CRP (at day 3) and LBP (at day 7) were significantly reduced compared with day 1, whereas sIL-1RII levels increased. CONCLUSIONS: These data suggest an imbalance in systemic levels of pro- and anti-inflammatory mediators in patients with stable COPD. The increase in the anti-inflammatory mediator sIL-1RII during treatment of exacerbations may contribute to the clinical improvement.

Activated caspase-1 is not a central mediator of inflammation in the course of ischemia-reperfusion.

BACKGROUND: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury. METHODS: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared. RESULTS: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis. CONCLUSIONS: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury.

Apoptosis and chemokine induction after renal ischemia-reperfusion.

BACKGROUND: One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R. METHODS: The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction. RESULTS: We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion. CONCLUSIONS: These data for the first time show that chemokine induction following I/R is dependent on caspase activation.

Contribution of platelet-derived factor Va to thrombin generation on immobilized collagen- and fibrinogen-adherent platelets.

Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e.g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 +/- 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.