Search

Objective: To investigate the role of bacterial DNA in development of an excessive inflammatory response and loss of gut barrier loss following systemic hypotension., Summary Background Data: Bacterial infection may contribute to development of inflammatory complications following major surgery; however, the pathogenesis is not clear. A common denominator of bacterial infection is bacterial DNA characterized by unmethylated CpG motifs. Recently, it has been shown that bacterial DNA or synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) are immunostimulatory leading to release of inflammatory mediators., Methods: Rats were exposed to CpG-ODN prior to a nonlethal hemorrhagic shock. The role of interferon-gamma (IFN-gamma) was investigated by administration of anti IFN-gamma antibodies., Results: Exposure to CpG-ODN prior to hemorrhagic shock significantly augmented shock-induced release of IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) (P < 0.05), interleukin (IL)-6 (P < 0.05), and nitrite levels (P < 0.05), while there was a defective IL-10 response (P < 0.05). Simultaneously, expression of Toll-like receptor (TLR) 4 in the liver was markedly enhanced. Furthermore, intestinal permeability for HRP significantly increased and bacterial translocation was enhanced in hemorrhagic shock rats pretreated with CpG-ODN. Interestingly, inhibition of IFN-gamma in CpG-treated animals reduced TNF-alpha (P < 0.05), IL-6 (P < 0.05), nitrite (P < 0.05), and intestinal permeability following hemorrhagic shock (P < 0.05) and down-regulated expression of TLR4., Conclusion: Exposure to bacterial DNA strongly aggravates the inflammatory response, disrupts the intestinal barrier, and up-regulates TLR4 expression in the liver following hemorrhagic shock. These effects are mediated via an IFN-gamma-dependent route. In the clinical setting, bacterial DNA may be important in development of inflammatory complications in surgical patients with bacterial infection.

Plasminogen activator inhibitor type-1 (PAI-1) levels are consistently elevated in patients with severe pneumonia and sepsis and highly predictive for an unfavorable outcome. In addition, pneumonia is associated with strongly elevated PAI-1 levels in the pulmonary compartment. However, whether PAI-1 causally affects antibacterial host defense in vivo remains unknown. We report here that pneumonia caused by the common respiratory pathogen Klebsiella pneumoniae is associated with local production of PAI-1 in the lungs of wild-type mice. PAI-1 deficiency impaired host defense as reflected by enhanced lethality and increased bacterial growth and dissemination in mice with a targeted deletion of the PAI-1 gene. Conversely, transgenic overexpression of PAI-1 in the lung using a replication-defective adenoviral vector markedly improved host defense against Klebsiella pneumonia and sepsis. PAI-1 deficiency reduced accumulation of neutrophils in the lungs during pneumonia, whereas PAI-1 overexpression in healthy lungs resulted in neutrophil influx, suggesting that PAI-1 protects the host against Klebsiella pneumonia by promoting neutrophil recruitment to the pulmonary compartment. These data demonstrate for the first time that PAI-1 is essential for host defense against severe Gram-negative pneumonia.

Sepsis is characterized by a concurrent activation of inflammation and coagulation. Recently, recombinant human activated protein C was shown to decrease mortality in patients with severe sepsis presumably due to a combined anti-inflammatory and anticoagulant effect. These promising findings led to a search for other products that influence both the inflammatory and the procoagulant response to severe infection. Ethyl pyruvate (EP) was recently identified as an experimental anti-inflammatory agent during endotoxemia and sepsis. The aim of the present study was to investigate whether EP influences coagulation besides its anti-inflammatory effects. For this we investigated the effects of EP on the expression and function of tissue factor (TF), the principal initiator of coagulation activation in sepsis, in human monocytic (THP-1) cell cultures. EP dose-dependently inhibited the production of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by lipopolysaccharide (LPS)-stimulated THP-1 cells at mRNA and protein level, thereby confirming its anti-inflammatory properties in this in-vitro system. In addition, EP dose-dependently attenuated the increases in TF mRNA levels, TF-protein-surface expression and cell-surface-associated TF activity in LPS-stimulated THP-1 cells. These results demonstrate for the first time that EP is a compound with combined anti-inflammatory and anticoagulant effects.

Background: Nontypable Haemophilus influenzae (NTHi) is a common bacterial pathogen causing human respiratory tract infections under permissive conditions such as chronic obstructive pulmonary disease. Inhalation of beta2-receptor agonists is a widely used treatment in patients with chronic obstructive pulmonary disease. The aim of this study was to determine the effect of inhalation of beta2 agonists on the host immune response to respiratory tract infection with NTHi., Methods: Mouse alveolar macrophages were stimulated in vitro with NTHi in the presence or absence of the beta2 receptor agonists salmeterol or salbutamol. In addition, mice received salmeterol or salbutamol by inhalation and were intranasally infected with NTHi. End points were pulmonary inflammation and bacterial loads., Results: Both salmeterol and salbutamol inhibited NTHi induced tumor necrosis factor-alpha (TNFalpha) release by mouse alveolar macrophages in vitro by a beta receptor dependent mechanism. In line, inhalation of either salmeterol or salbutamol was associated with a reduced early TNFalpha production in lungs of mice infected intranasally with NTHi, an effect that was reversed by concurrent treatment with the beta blocker propranolol. The clearance of NTHi from the lungs was impaired in mice treated with salmeterol or salbutamol, an adverse effect that was prevented by propranolol and independent of the reduction in TNFalpha., Conclusion: These data suggest that inhalation of salmeterol or salbutamol may negatively influence an effective clearance of NTHi from the airways.

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9(-/-)) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9(-/-) neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9(-/-) mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9(-/-) mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9(-/-) mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9(-/-) mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.

Objective: Nosocomial pneumonia is a feared complication in the critically ill patient. Aspiration pneumonitis is frequently complicated by infections. The objective of this study was to determine the influence of aspiration pneumonitis on the host response to a common nosocomial respiratory pathogen., Design: Controlled, in vivo laboratory study., Setting: Research laboratory of a health sciences university., Subjects: Female C57Bl/6 mice., Interventions: Mice received hydrochloric acid or saline intratracheally followed 16 hrs later by Klebsiella pneumoniae., Measurements and Main Results: Hydrochloric acid induced a mild aspiration pneumonitis. Nonetheless, hydrochloric acid aspiration resulted in a markedly increased inflammatory response in the lung on infection with K. pneumoniae. This enhanced inflammatory reaction was accompanied by a greatly increased outgrowth of K. pneumoniae in lungs of mice previously exposed to hydrochloric acid. Preexisting aspiration pneumonitis also triggered mouse lungs in vivo and alveolar macrophages ex vivo for enhanced release of proinflammatory mediators on stimulation with Klebsiella lipopolysaccharide. Inhibition of tumor necrosis factor-alpha resulted in an increased inflammatory reaction and enhanced bacterial outgrowth in mice with primary K. pneumoniae pneumonia, whereas it had no effect in mice with preexisting aspiration pneumonitis., Conclusions: These data indicate a) that aspiration pneumonitis renders the host more susceptible to respiratory tract infection with K. pneumoniae, concurrently priming the lung for an exaggerated inflammatory response; and b) that although tumor necrosis factor-alpha plays a major role in the host response to primary infection, it does not affect lung inflammation or defense after aspiration pneumonitis.

Objective: Acute pancreatitis is frequently complicated by Gram-negative sepsis. Mammalian cells recognize lipopolysaccharide from Gram-negative bacteria via Toll-like receptor (TLR) 4. The objective of this study was to determine the role of TLR4 in the defense against Gram-negative sepsis in previously healthy mice and in animals with preexisting pancreatitis., Design: A controlled, in vivo laboratory study., Setting: Research laboratory of a health sciences university., Subjects: Female C3H/HeJ (nonfunctional TLR4 mutant) and C3H/HeN (wild-type) mice., Interventions: Abdominal sepsis was induced by the intraperitoneal injection of Escherichia coli. Pancreatitis was induced by 12 hourly intraperitoneal injections of cerulein., Measurements and Main Results: The following experiments were performed. First, healthy TLR4 mutant mice demonstrated an enhanced bacterial load and dissemination of the infection relative to wild-type mice after intraperitoneal injection with E. coli, associated with a reduced early release of proinflammatory cytokines and an attenuated influx of neutrophils into the peritoneal fluid. Second, wild-type mice in which acute pancreatitis was induced by repeated cerulein injections showed an increased bacterial load and dissemination of E. coli relative to wild-type mice without pancreatitis, which was accompanied by a blunted proinflammatory cytokine response by peritoneal macrophages ex vivo and a diminished early cytokine and neutrophil response in vivo. Third, whereas the severity of cerulein-induced pancreatitis was similar in TLR4 mutant and wild-type mice, the important contribution of TLR4 to an effective host defense against E. coli sepsis observed in previously healthy mice was no longer present in mice with preexisting pancreatitis., Conclusions: These data suggest that TLR4 deficiency and acute pancreatitis act similarly in reducing host defense against E. coli peritonitis and that the role of TLR4 in severe Gram-negative infection depends, at least in part, on the presence of preexisting critical illness.

Objective: To investigate i) whether the Dialyzable Leukocyte Extract (DLE) modulates the production of proinflammatory cytokines in leukocytes activated by the bacterial cell wall components lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN); ii) the effect of DLE on LPS-stimulated endothelial cells; and iii) whether the regulatory effect of DLE on inflammatory mediators is related to the modulation of Toll-like receptors (TLRs), NF-kappaB and cAMP signaling pathways., Methods: Leukocytes were stimulated with LPS, LTA, and PGN in the presence of DLE. Endothelial cells were stimulated with LPS and treated with DLE. The levels of Tumor Necrosis Factor-alpha(TNFalpha), Interleukin-6 (IL-6), and IL-8 in culture supernatants were evaluated by ELISA. The expression of Toll-like receptor 2 (TLR2) and 4 (TLR4), NF-kappaB activity and cAMP levels were evaluated by flow cytometry, EMSA, and EIA, respectively., Results: The addition of DLE to leukocytes stimulated with cell wall constituents suppressed the production of TNFalpha. However, DLE induced IL-8 release in monocytes and enhanced IL-6 and IL-8 production by activated monocytes and endothelial cells. Also, DLE induced TLR2 and TLR4 expression, and increased cAMP levels, whereas NF-kappaB activity was inhibited., Conclusions: The present data indicate the differential regulation by DLE of the production of TNFalpha, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-kappaB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.

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.

Several platelet inhibitors were examined in a tissue factor (TF)-initiated model of whole blood coagulation. In vitro coagulation of human blood from normal donors was initiated by 25 pM TF while contact pathway coagulation was suppressed using corn trypsin inhibitor. Products of the reaction were analyzed by immunoassay. Preactivation of platelets with the thrombin receptor activation peptide did not influence significantly the clotting time or thrombin-antithrombin III complex (TAT) formation. Addition of prostaglandin E(1) (5 microM) caused a significant delay in clotting (10.0 minutes) versus control (4.3 minutes). The prolonged clotting time is correlated with delays in platelet activation, formation of TAT, and fibrinopeptide A (FPA) release. In blood from subjects receiving acetylsalicylic acid (ASA or aspirin), none of the measured products of coagulation were significantly affected. Similarly, no significant effect was observed when 5 microM dipyridamole (Persantine) was added to the blood. Antagonists of the platelet integrin receptor glycoprotein (gp) IIb/IIIa had intermediate effects on the reaction. A 1- to 2-minute delay in clot time and FPA formation was observed with addition of the antibodies 7E3 and Reopro (abciximab) (10 microg/mL), accompanied by a 40% to 70% reduction in the maximal rate of TAT formation and delay in platelet activation. The cyclic heptapetide, Integrilin (eptifibatide), at 5 microM concentration slightly prolonged clot time and significantly attenuated the maximum rate of TAT formation. The disruption of the gpIIb/IIIa-ligand interaction not only affects platelet aggregation, but also decreases the rate of TF-initiated thrombin generation in whole blood, demonstrating a potent antithrombotic effect superimposed on the antiaggregation characteristics.

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.

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.

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.

Members of the Toll-like receptor (TLR) family probably play a fundamental role in pathogen recognition and activation of innate immunity. The present study used a systematic approach to analyze how different human leukocyte populations express specific transcripts for the first five characterized TLR family members. TLR1 was expressed in all leukocytes examined, including monocytes, polymorphonuclear leukocytes, T and B cells, and NK cells. In contrast TLR2, TLR4, and TLR5 were expressed in myelomonocytic elements. Exposure to bacterial products, such as LPS or lipoarabinomannan, or to proinflammatory cytokines increased TLR4 expression in monocytes and polymorphonuclear leukocytes, whereas IL-10 blocked this effect. TLR3 was only expressed in human dendritic cells (DC) wherein maturation induced by bacterial products or cytokines was associated with reduced expression. TLR3 mRNA expression was detected by in situ hybridization in DC and lymph nodes. These results demonstrate that TLR1 through TLR5 mRNAs are differentially expressed and regulated in human leukocytes. In particular, expression of TLR3 transcripts is restricted to DC that are the only elements which express the full TLR repertoire. These data suggest that TLR can be classified based on expression pattern as ubiquitous (TLR1), restricted (TLR2, TLR4, and TLR5 in myelomonocytic cells), and specific (TLR3 in DC) molecules.

We have explored the molecular basis of the clinical therapeutic effect of factor VIIa in hemophilia A using empirical reconstituted in vitro thrombin generation models. Tissue factor acts as a receptor and activator of preexistent but virtually inactive two-chain plasma factor VIIa. However, most of the factor VII circulates as a single-chain inactive zymogen (10 nM) and a trace (approximately 10-100 pM) circulates in the active two-chain form. Empirical reconstitution (purified factors VIIa, X, IX, VIII, V, prothrombin, and relipidated tissue factor) showed that plasma concentrations of factor VII (10 nM) prolong the initiation phase of thrombin generation significantly at low concentrations of tissue factor and 100 pM factor VIIa. Thus, we show for the first time that the zymogen factor VII may have a very significant inhibitory action on thrombin generation at physiologic ratios of factor VII to factor VIIa. The inhibition kinetics of factor Xa generation by low concentrations of tissue factor indicate that factor VII inhibits the reaction by competition for the initial binding of factor VIIa to tissue factor. Physiological concentrations of factor VII also inhibit the maximal rate of thrombin generation by 100 pM factor VIIa in the absence of factor VIII. Increasing the concentration of factor VIIa to 2 nM in this hemophilia A model overcame the inhibition of thrombin generation by 10 nM factor VII. Increasing the concentration up to 10 nM factor VIIa in the absence of factor VIII completely normalized the thrombin generation profile to that observed in the presence of factor VIII and 10 nM factor VII/100 pM factor VIIa. The levels of factor VIIa that overcome the inhibitory effect of factor VII and that normalize thrombin generation in our model are consistent with the observed plasma levels of factor VIIa needed to manage hemophilia A. Our data strongly indicate that the therapeutic mechanism of factor VIIa in the medical treatment of hemophiliacs with inhibitors is in large part based on overcoming the inhibitory effect of factor VII on thrombin generation.

We have investigated the influence of alterations in plasma coagulation factor levels between 50% and 150% of their mean values for prothrombin, factor X, factor XI, factor IX, factor VII, factor VIII, factor V, protein C, protein S, antithrombin III (AT-III), and tissue factor pathway inhibitor (TFPI) as well as combinations of extremes, eg, 50% anticoagulants and 150% procoagulants or 50% procoagulants and 150% anticoagulants in a synthetic "plasma" system. The reaction systems were constructed in vitro using purified, natural, and recombinant proteins and synthetic phospholipid vesicles or platelets with the reactions initiated by recombinant tissue factor (TF)-factor VIIa complex (5 pmol/L). To investigate the influence of the protein C system, soluble thrombomodulin (Tm) was also added to the reaction mixture. For the most extreme situations in which the essential plasma procoagulants (prothrombin, and factors X, IX, V, and VIII) and the stoichiometric anticoagulants (AT-III and TFPI) were collectively and inversely altered by 50%, a 28-fold difference in the total available thrombin generated was observed. Variations of most of these proteins 50% above and below the "normal" range, with the remainder at 100%, had only modest influences on the peak and total levels of thrombin generated. The dominant factors influencing thrombin generation were prothrombin and AT-III. When these 2 components were held at 100% and all other plasma procoagulants were reduced to 50%, there was a 60% reduction in the available thrombin generated. No increase in the thrombin generated was observed when the 150% level of all plasma procoagulants other than prothrombin was evaluated. When only prothrombin was raised to 150%, and all other factors were maintained at 100%, the thrombin generated increased by 71% to 121%. When AT-III was at 50% and all other constituents were at 100%, thrombin production was increased by 104% to 196%. The additions of protein C and protein S over the 50% to 150% ranges with Tm at 0.1 nmol/L concentration had limited influence on thrombin generation. Individual variations in factors VII, XI, and X concentrations had little effect on the duration of the initiation phase, the peak thrombin level achieved, or the available thrombin generated. Paradoxically, increases in factor IX concentration to 150% led to lowered thrombin generation, while decreases to 50% led to enhanced thrombin generation, most likely a consequence of factor IX as a competitive substrate with factor X for factor VIIa-TF. Reductions in factor V or factor VIII concentration led to prolongations of the initiation phase, while the reduction of TFPI to 50% led to shortening of this phase. However, none of these alterations led to significant changes in the available thrombin generated. Based on these data, one might surmise that increases in prothrombin and reductions in AT-III, within the normal range, would be potential risk factors for thrombosis and that algorithms that combine normal factor levels may be required to develop predictive tests for thrombosis.

Tissue injury as a consequence of ischemia followed by reperfusion is characterized by early as well as late signs of inflammation. The latter, among others, involves IFN-gamma-dependent up-regulation of MHC class I and II Ag expression. Employing a murine model of renal ischemia, we show that renal IL-18 mRNA up-regulation coincides with caspase-1 activation at day 1 following ischemia. IFN-gamma and IL-12 mRNA are subsequently up-regulated at day 6 following ischemia. Combined, but not separate, in vivo neutralization of the IFN-gamma inducing cytokines IL-12 and IL-18 reduces IFN-gamma-dependent MHC class I and II up-regulation to a similar extent as IFN-gamma neutralization, suggesting the involvement of functional IL-12, IL-18, and IFN-gamma protein. These results reveal a novel relationship between tissue injury of nonmicrobial origin and the induction of IL-12 as well as IL-18. The collaboration observed between endogenous IL-12 and IL-18 in the induction of IFN-gamma after renal ischemia/reperfusion, resembles the immune response to bacterial infections.

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.

The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 micromol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 micromol/L phospholipid) yielded rates of 2 to 8 nmol Flla x L(-1) x s(-1) in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (< or =1.0 nmol FIIa x L(-1) x s[-1]). For comparison, VLDL at 2 mmol/L triglyceride yielded approximately 50% the activity of 2X10(8) thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylsenne vesicles (approximately 50 nmol FIIa x L(-1) x s[-1]), the prothrombin Km values were similar, 0.8 and 0.5 micromol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa x L(-1) x s[-1]). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.

Three model systems have been used to study the dynamics of the blood clotting process initiated by tissue factor (TF): synthetic plasma mixtures prepared with purified coagulation proteins and inhibitors; mathematical models based on the reaction constants, stoichiometries and thermodynamics of individual catalyst and inhibitor reactions; and contact suppressed whole blood induced to clot in vitro by the addition of exogenous TF. In the three models, the generation of thrombin can be described in terms of an initiation phase in which pmol/l concentrations of the coagulation serine proteases are generated and the cofactor proteins factor V (FV) and FVIII are activated. Subsequently, explosive thrombin generation occurs during a propagation phase. The complementary inhibitory pathways extinguish the generation of thrombin. Tissue factor pathway inhibitor (TFPI), present in low concentrations, primarily influences the duration of the initiation phase and has little influence on the propagation phase. Antithrombin III (ATIII), present in higher concentrations, has little influence during the initiation phase, but decreases the rate of thrombin generation during the propagation phase. The protein C pathway cannot act in the absence of thrombin and therefore only influences the duration of the propagation phase by inactivating activated FV. Thus combinations of TFPI plus ATIII and TFPI plus protein C pathway components contribute to the synergistic inhibitory processes. As a consequence of the roles of pro, and anti-coagulants, the generation of thrombin by the TF pathway becomes a threshold limited process.

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis.