Factor XII plays a pathogenic role in organ failure and death in baboons challenged with Staphylococcus aureus.

Activation of coagulation factor (F) XI promotes multiorgan failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus aureus. Here we used the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared with untreated control animals, repeated 5C12 administration before and at 8 and 24 hours after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa, and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes was also prevented. D-dimer levels exhibited a profound increase in the untreated animals but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1ß, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress, and multiorgan failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, whereas untreated control animals suffered irreversible multiorgan failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least 2 enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S aureus.

The Inflammatory and Hemostatic Response in Sepsis and Meningococcemia.

Meningococcemia is notorious for evasion of the host immune system and its rapid progression to fulminant disease, and serves as a unique model for pediatric sepsis. Illness severity is determined by complex interplays among host, pathogen, and environment. The inflammatory host response, including proinflammatory and anti-inflammatory responses in innate and adaptive immunity, skews toward a proinflammatory state. This leads to endothelial dysfunction and activation of the hemostatic response, which may lead to disseminated intravascular coagulation. This article reviews the pathogenesis of sepsis, in particular the inflammatory and hemostatic response in meningococcal sepsis.

Blood coagulation abnormalities in multibacillary leprosy patients.

BACKGROUND: Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities. PRINCIPAL FINDINGS: In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro. CONCLUSIONS: We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.

Blood coagulation abnormalities in multibacilary leprosy patients

Hemostatic illnesses are frequently associated with acute and chronic infections. In the present work we demonstrated that leprosy patients developed hemostatic abnormalities, like the formation of an atypical lipid clot mass during sera harvesting, a phenomenon previously observed and never unraveled. We characterize the nature of the "leprosum clot", formed during a protrombotic state developed by some patients. During the proteomic analysis of the leprosum clot we discovered a set of potential serum biomarkers to leprosy reactional episodes diagnosis, which at this moment is based only in clinical features. Taking together, our data suggest that leprosy patients are suffering from a procoagulant status, being beneficiated by the introduction of routine coagulation tests during their treatment, which will aloud physicians to prevent some of the acute clinical symptoms related with superficial vein thrombosis such as cyanosis and tissue necrosis observed during severe cases of leprosy reactional episodes. (AU)

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis.

Pathogenic bacteria of the genus Leptospira are the causative agent of leptospirosis, an emergent infectious disease that affects humans and animals worldwide. Severe forms of the disease in humans include jaundice, multiple organ failure and intense haemorrhage. Up to now, mechanisms associated with the haemorrhage foci are poorly understood. We report in this work that, despite the low levels of antithrombin III in convalescent human serum samples, virulent, culture-attenuated and saprophyte strains of Leptospira are unable to bind and/or degrade this thrombin inhibitor, suggesting an indirect mechanism of pathogenesis. Lower levels of prothrombin were found in serum samples at the onset and convalescent phase of the disease when compared to normal human sera. The concomitant decreased levels of antithrombin III and prothrombin suggest a process of stimulated coagulation, which is corroborated by the increase of prothrombin fragment F1+2 in the serum samples. Data obtained with hamsters experimentally infected with virulent Leptospira interrogans serovars Kennewicki and Canicola strongly point out that haemorrhage is correlated with decreased levels of thrombin inhibitors and prothrombin. Activated coagulation might lead to an overconsumption of coagulation factors ultimately leading to bleeding and organ failure.

Early Trauma-Induced Coagulopathy is Associated with Increased Ventilator-Associated Pneumonia in Spinal Cord Injury Patients.

INTRODUCTION: Early trauma-induced coagulopathy may increase susceptibility to nosocomial infections such as ventilator-associated pneumonia. However, the relationship between trauma- induced coagulopathy and the development of ventilator-associated pneumonia in spinal cord injury patients has not been evaluated. METHODS: We conducted a 5-year retrospective study of 300 spinal cord injury patients admitted to Level 1 trauma center. Standard coagulation studies were evaluated upon arrival, prior to fluid resuscitative efforts, and at 24  h after admission. Based on these studies, three groups of patients were identified: no coagulopathy, latent coagulopathy, and admission coagulopathy. Ventilator- associated pneumonia was identified utilizing Centers for Disease Control and Prevention criteria. Since we used the data in the trauma registry and did not have the information on FiO2 and PEEP, we elected to use the VAP terminology and not the VAE sequence. Demographic, injury, and clinical characteristics were compared among no coagulopathy, latent coagulopathy, and admission coagulopathy groups using chi-square test and ANOVA for categorical and continuous variables, respectively. A logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between coagulopathy and both ventilator-associated pneumonia and mortality. RESULTS: The incidence of ventilator-associated pneumonia was 54.5% (OR 4.01, 95% CI 1.76-9.15) in spinal cord injury patients with admission coagulopathy, compared with the 17.5% in spinal cord injury patients with no coagulopathy. Mortality was significantly higher in spinal cord injury patients with admission coagulopathy than in spinal cord injury patients with no coagulopathy (OR 6.14, 95% CI 1.73-21.73).After adjusting for age, race, injury mechanism, Injury Severity Score, base deficit at admission, the number of pRBC units transfused in the first 24  h, and hospital stay, only the association of ventilator-associated pneumonia among those with admission coagulopathy remained significant (OR 3.51, 95% CI 1.48-8.32). Compared with those with no coagulopathy, patients with admission coagulopathy had a higher odds of death (4.10, 95% CI 1.53-11.02), though this association lost significance after adjustment (OR 3.56, 95% CI 0.90-14.12). There was no statistical difference in mortality for latent coagulopathy compared with no coagulopathy patients. CONCLUSION: Coagulopathy on admission in patients with spinal cord injury is associated with a statistically significant increase in ventilator-associated pneumonia incidence. Additional research is warranted to further characterize this association.

Carboxypeptidase B2 deficiency reveals opposite effects of complement C3a and C5a in a murine polymicrobial sepsis model.

BACKGROUND AND OBJECTIVES: Carboxypeptidase B2 (CPB2) is a basic carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation. METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). RESULTS: Contrary to our hypothesis, Cpb2(-/-) mice had significantly improved survival, with reduced lung edema, less liver and kidney damage, and less disseminated intravascular coagulation. Hepatic pro-CPB2 was induced by CLP, leading to increased pro-CPB2 levels. Thrombomodulin present on mesothelium supported thrombin activation of pro-CPB2. Both wild-type and Cpb2(-/-) animals treated with a C5a receptor antagonist had improved survival, demonstrating that C5a was detrimental in this model. Treatment with a fibrinolysis inhibitor, tranexamic acid, caused a decrease in survival in both genotypes; however, the Cpb2(-/-) animals retained their survival advantage. Administration of a C3a receptor antagonist exacerbated the disease in both wild-type and Cpb2(-/-) mice and eliminated the survival advantage of Cpb2(-/-) mice. C5a receptor is expressed in both peritoneal macrophages and neutrophils; in contrast, C3a receptor expression is restricted to peritoneal macrophages, and C3a induced signaling in macrophages but not neutrophils. CONCLUSIONS: While C5a exacerbates the peritonitis, resulting in a deleterious generalized inflammatory state, C3a activation of peritoneal macrophages may limit the initial infection following CLP, thereby playing a diametrically opposing protective role in this polymicrobial sepsis model.

Pathogen safety of long-term treatments for bleeding disorders: (un)predictable risks and evolving threats.

Substantial improvements in the safety of blood and plasma products for the management of bleeding disorders have been achieved in recent decades. This has led some clinicians to believe that the infectious threat is over and that inhibitor formation is the foremost complication of hemophilia therapy. On the contrary, elimination of all microbes from blood is difficult, potentially impossible, and there are always threats from emerging pathogens. The risk of infection transmission is also increasing due to greater exposure to products, increasing prophylaxis and high-dose regimens for immune tolerance, and longevity of hemophilia patients. Current products can be considered "reasonably safe," but pathogen testing is not all-inclusive, and manufacturing and purification techniques are often not standardized. Although safer nonplasma-derived products are widely used, they are not available for all bleeding disorders, and so there is an ongoing need for plasma-derived products. This review will discuss the evolving risk from emerging pathogens in the context of the issues described. Reducing the risk from emerging infections requires global collaboration to devise ways to monitor and continue to improve blood safety.

Overexpression of activated protein C is detrimental during severe experimental gram-negative sepsis (melioidosis).

OBJECTIVE: The interplay between inflammation and blood coagulation is an essential part of host defense during severe pneumosepsis. Melioidosis, instigated by the Gram-negative bacterium Burkholderia pseudomallei, is a frequent cause of pneumosepsis in Southeast Asia. Patients with severe pneumosepsis, including melioidosis, have decreased circulating levels of protein C. Activated protein C has anticoagulant and anti-inflammatory properties. In this study, we aimed to investigate the effect of sustained elevated activated protein C levels on the host response during melioidosis. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Wild type and activated protein C overexpressing C57BL/6 mice. INTERVENTIONS: Mice were intranasally infected with viable B. pseudomallei and killed after 24, 48, or 72 hours for harvesting of lungs, liver, spleen, and blood. Additionally, survival studies were performed. MEASUREMENTS AND MAIN RESULTS: Plasma activated protein C concentrations in activated protein C overexpressing mice (median 18.1 ng/mL) were in the same range as previously measured in patients treated with recombinant human activated protein C. Activated protein C overexpressing mice demonstrated enhanced susceptibility to B. pseudomallei infection compared with wild type mice as evidenced by a strongly increased mortality accompanied by enhanced bacterial loads in the lungs, blood, and distant organs 48 hours after infection. Additionally, at this time point, activated protein C overexpressing mice showed elevated levels of proinflammatory cytokines in lungs and plasma, together with increased pulmonary histopathology scores and neutrophil influx. At 72 hours postinfection, decreased levels of thrombin-antithrombin complexes, reflecting inhibition of coagulation, were measured in lungs of activated protein C overexpressing mice. CONCLUSION: Constitutively enhanced expression of activated protein C impairs host defense during severe Gram-negative sepsis caused by B. pseudomallei.

Coinfection with Staphylococcus aureus increases risk of severe coagulopathy in critically ill children with influenza A (H1N1) virus infection.

OBJECTIVES: H1N1 influenza with coinfections has been implicated to have high morbidity and mortality. We hypothesized that critically ill children with 2009 H1N1 and coinfections are at a higher risk of developing disseminated intravascular coagulation. DESIGN: The chart review included demographics, length-of-stay, severity of illness score (Pediatric Risk of Mortality III acute physiology score), clinical laboratories, and outcomes at hospital day 90 data. Patients were classified as having methicillin-sensitive or -resistant Staphylococcus aureus, other, or no coinfections. SETTING: Single-center pediatric intensive care unit. PATIENTS: Sixty-six consecutive patients with 2009 H1N1 and influenza A infection. INTERVENTIONS: None. MAIN RESULTS: : There were 12, 22, and 32 patients with methicillin-sensitive or -resistant Staphylococcus aureus, other, and no coinfections, respectively. Pediatric critical care unit length-of-stay was 11, 10, and 5.5 days (median), and survival at day 90 was 83%, 96%, and 91% in patients with methicillin-sensitive or -resistant Staphylococcus aureus, other, and no coinfections. Patients with methicillin-sensitive or -resistant Staphylococcus aureus coinfections compared to patients with other, and no coinfections had higher Pediatric Risk of Mortality III acute physiology scores (14 [6-25] vs. 7 [2-10], p = .052 and 6 [2.5-10], p = .008; median [interquartile range]), higher D-dimer (16.1 [7.9-19.3] vs. 1.6 [1.1-4], p = .02 and 2.3 [0.8-8.7] µg/mL, p = .05), longer prothrombin time (19.3 [15.4-25.9] vs. 15.3 [14.8-17.1], p = .04 and 16.6 [14.7-20.4] secs, p < .39) at admission, and lower day-7 platelet counts (90K [26-161K] vs. 277K [98-314], p = .03 and 256K [152-339]/mm, p < .07). Patients with methicillin-sensitive or -resistant Staphylococcus aureus coinfections compared to patients without coinfections were more likely to be sicker with Pediatric Risk of Mortality III acute physiology score >10 vs. <10 (relative risk 2.4; 95% confidence interval 1.2-4.7; p = .035) and have overt disseminated intravascular coagulation (relative risk 4.4; 95% confidence interval 1.3-15.8, p = .025). CONCLUSIONS: During the 2009-2010 H1N1 pandemic, pediatric patients with influenza A and methicillin-sensitive or -resistant Staphylococcus aureus coinfections were sicker and more likely to develop disseminated intravascular coagulation than patients with other or no coinfections.

Plasma-derived human antithrombin attenuates ventilator-induced coagulopathy but not inflammation in a Streptococcus pneumoniae pneumonia model in rats.

BACKGROUND: Mechanical ventilation exaggerates pneumonia-associated pulmonary coagulopathy and inflammation. We hypothesized that the administration of plasma-derived human antithrombin (AT), one of the natural inhibitors of coagulation, prevents ventilator-induced pulmonary coagulopathy, inflammation and bacterial outgrowth in a Streptococcus pneumoniae pneumonia model in rats. METHODS: Forty-eight hours after induction of S. pneumoniae pneumonia rats were subjected to mechanical ventilation (tidal volume 12 mL kg(-1), positive end-expiratory pressure 0 cmH(2)O and inspired oxygen fraction 40%). Rats were randomized to systemic treatment with AT (250 IU administered intravenously (i.v.) before the start of mechanical ventilation) or placebo (saline). Non-ventilated, non-infected rats and non-ventilated rats with pneumonia served as controls. The primary endpoints were pulmonary coagulation and inflammation in bronchoalveolar lavage fluid (BALF). RESULTS: Pneumonia was characterized by local activation of coagulation and inhibition of fibrinolysis, resulting in increased levels of fibrin degradation products and fibrin deposition in the lung. Mechanical ventilation exaggerated pulmonary coagulopathy and inflammation. Systemic administration of AT led to supra-normal BALF levels of AT and decreased ventilator-associated activation of coagulation. AT neither affected pulmonary inflammation nor bacterial outgrowth from the lungs or blood. CONCLUSIONS: Plasma-derived human AT attenuates ventilator-induced coagulopathy, but not inflammation and bacterial outgrowth in a S. pneumoniae pneumonia model in rats.

Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B.

Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ~20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.

Basilar artery occlusion after multifactor coagulopathy including Rhizopus oryzae infection in burns.

The authors report a case of a 29-year-old male patient with severe burn injuries (54% TBSA) and inhalation injury. He developed a candidemia and a cutaneous zygomycotic superinfection with Rhizopus oryzae while he received burn intensive care. Despite aggressive surgery, sepsis persisted, and therapy was limited by uncontrollable coagulopathy and catecholamine refractory shock after 15 days. Autopsy revealed a thromboembolic occlusion of the basilar artery that resulted in liquefactive necrosis of the basal brain tissue and the brain stem. Because cerebral vessel occlusions after burn injuries are reported rarely, the current literature was reviewed, and possible pathophysiological aspects are discussed.

Naturally occurring lipid A mutants in neisseria meningitidis from patients with invasive meningococcal disease are associated with reduced coagulopathy.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

A case of reversible posterior leukoencephalopathy syndrome caused by transient hypercoagulable state induced by infection.

We report a normotensive case of reversible posterior leukoencephalopathy syndrome caused by transient hypercoagulable state. Hypertension is the main risk factor for reversible posterior leukoencephalopathy syndrome, which is believed to occur as a result of high blood pressure-related dysfunction of cerebrovascular endothelial cells, because it commonly appears in hypertensive emergency. However, in this completely normotensive case, the typical clinical findings of reversible posterior leukoencephalopathy syndrome were triggered by transient hypercoagulable state without any blood pressure variation. The case was successfully treated with anticoagulation therapy using heparin. Thus, this case indicates that reversible posterior leukoencephalopathy syndrome is induced by cerebrovascular endothelial dysfunction, which is induced not only by high blood pressure but also hemostatic dysfunction.

Effect of interleukin-6 blockade on tissue factor-induced coagulation in human endotoxemia.

OBJECTIVE: Clinical trials show that interleukin (IL)-6 represents a predictive marker in human sepsis. Furthermore, IL-6 has been proposed as a candidate mediator for endotoxin (lipopolysaccharide)-induced coagulation activation: In a primate model, an (alphaIL-6 antibody (alphaIL-6 Ab) almost abolished lipopolysaccharide-induced coagulation activation. Therefore, we wished to determine if an alphaIL-6 Ab (B-E8) may also attenuate lipopolysaccharide-induced activation of coagulation in humans. DESIGN: The study was a randomized, double blind, placebo-controlled parallel group trial (n = 12 per group). SETTING: University medical center. PATIENTS: Healthy volunteers. INTERVENTIONS: Healthy volunteers were randomized to receive either 80 mg of a monoclonal anti-IL-6 Ab (B-E8) or placebo intravenously before bolus infusion of 2 ng/kg lipopolysaccharide. MEASUREMENTS AND MAIN RESULTS: B-E8 effectively decreased IL-6 bioactivity as measured by a Bg-bioassay in vitro and concentrations of C reactive protein. However, B-E8 did not decrease lipopolysaccharide-induced tissue factor-messenger RNA transcription or plasma concentrations of downstream coagulation variables (prothrombin fragment 1 + 2, thrombin-antithrombin III complexes, and D-dimer concentrations). Similarly, tumor necrosis factor-alpha concentrations, fibrinolytic activity (plasmin-antiplasmin complexes), endothelial activation (soluble E-selectin), and IL-10 were unaffected. CONCLUSION: IL-6 does not appear to mediate early-phase lipopolysaccharide-induced coagulation activation in humans.

A probable cause of paradoxical thrombosis in zygomycosis.

Invasive zygomycoses (syn. mucormycoses) are rather rare but life-threatening diseases which often take a peracute course. Particularly endangered are diabetics and patients suffering from siderophilia. Zygomycosis is regularly complicated by thrombosis and subsequent necrosis. Usually it evolves from sinusitis in a rhinocerebral form. With the use of a clinical isolate (Rhizopus microsporus) and sera of the same female survivor, we investigated possible sources of the typical blood clotting. The results suggest that coagulation is probably initiated in a bimodal manner by an extracellular serine proteinase of the fungus and by elastase from the patients' leukocytes. The former causes a partial hydrolysis of fibrinogen, while the latter activates coagulation factor XIII (fibrin stabilizing factor). Both proteinases were present in the patient at the site of infection, and in vitro they jointly bring about regular clotting of fibrinogen.