α1-Antitrypsin activates protein phosphatase 2A to counter lung inflammatory responses.

RATIONALE: α1-Antitrypsin (A1AT) was identified as a plasma protease inhibitor; however, it is now recognized as a multifunctional protein that modulates immunity, inflammation, proteostasis, apoptosis, and cellular senescence. Like A1AT, protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, regulates similar biologic processes and plays a key role in chronic obstructive pulmonary disease. OBJECTIVES: Given their common effects, this study investigated whether A1AT acts via PP2A to alter tumor necrosis factor (TNF) signaling, inflammation, and proteolytic responses in this disease. METHODS: PP2A activity was measured in peripheral blood neutrophils from A1AT-deficient (PiZZ) and healthy (PiMM) individuals and in alveolar macrophages from normal (60 mg/kg) and high-dose (120 mg/kg) A1AT-treated PiZZ subjects. PP2A activation was assessed in human neutrophils, airway epithelial cells, and peripheral blood monocytes treated with plasma purified A1AT protein. Similarly, lung PP2A activity was measured in mice administered intranasal A1AT. PP2A was silenced in lung epithelial cells treated with A1AT and matrix metalloproteinase and cytokine production was then measured following TNF-α stimulation. MEASUREMENTS AND MAIN RESULTS: PP2A was significantly lower in neutrophils isolated from PiZZ compared with PiMM subjects. A1AT protein activated PP2A in human alveolar macrophages, monocytes, neutrophils, airway epithelial cells, and in mouse lungs. This activation required functionally active A1AT protein and protein tyrosine phosphatase 1B expression. A1AT treatment acted via PP2A to prevent p38 and IκBα phosphorylation and matrix metalloproteinase and cytokine induction in TNF-α-stimulated epithelial cells. CONCLUSIONS: Together, these data indicate that A1AT modulates PP2A to counter inflammatory and proteolytic responses induced by TNF signaling in the lung.

Leishmania inhibitor of serine peptidase 2 prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages.

Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.

Hereditary angioedema: the plasma contact system out of control.

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease.

Complete antithrombin deficiency in mice results in embryonic lethality.

Antithrombin is a plasma protease inhibitor that inhibits thrombin and contributes to the maintenance of blood fluidity. Using targeted gene disruption, we investigated the role of antithrombin in embryogenesis. Mating mice heterozygous for antithrombin gene (ATIII) disruption, ATIII(+/-), yielded the expected Mendelian distribution of genotypes until 14.5 gestational days (gd). However, approximately 70% of the ATIII(-/-) embryos at 15.5 gd and 100% at 16.5 gd had died and showed extensive subcutaneous hemorrhage. Histological examination of those embryos revealed extensive fibrin(ogen) deposition in the myocardium and liver, but not in the brain or lung. Furthermore, no apparent fibrin(ogen) deposition was detected in the extensive hemorrhagic region, suggesting that fibrinogen might be decreased due to consumptive coagulopathy and/or liver dysfunction. These findings suggest that antithrombin is essential for embryonic survival and that it plays an important role in regulation of blood coagulation in the myocardium and liver.

Practical genetics: alpha-1-antitrypsin deficiency and the serpinopathies.

Alpha-1-antitrypsin (alpha(1)-antitrypsin) is the archetypal member of the serine proteinase inhibitor or serpin superfamily. The most common severe deficiency variant is the Z allele, which results in the accumulation of mutant protein within hepatocytes. This 'protein overload' causes neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating plasma alpha(1)-antitrypsin results in early-onset panlobular emphysema. The mechanism underlying the deficiency of Z alpha(1)-antitrypsin is due to an aberrant conformational transition within the protein and the formation of chains of polymers that tangle within the secretory pathway of hepatocytes. This mechanism also underlies the plasma deficiency of other members of the serpin superfamily to cause a class of diseases called the serpinopathies. Specifically mutant alleles of antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin have been reported that favour the spontaneous formation of polymers and the retention of protein within hepatocytes. The consequent lack of plasma antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin results in thrombosis, angio-oedema and emphysema, respectively. Moreover, the polymerisation of mutants of neuroserpin results in the retention of polymers within neurones to cause the inclusion body dementia, familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the genetic and molecular basis and clinical features of alpha(1)-antitrypsin deficiency, and show how this provides a platform to understand the other serpinopathies.

Gene targeting in hemostasis. tissue factor pathway inhibitor.

Tissue Factor Pathway Inhibitor (TFPI) is a serine protease inhibitor of the Factor VIIa/Tissue Factor (FVIIa/TF)-initiated clotting cascade. Mice expressing a mutant form of TFPI, in which its Kunitz-1 domain has been deleted (TFPIKu1delta/delta), die prematurely in embryogenesis between E9.5dpc and birth. These results provide a rationale for the absence of TFPI-deficient patients. This early mortality can be ameliorated by an accompanying heterozygous or homozygous deficiency in FVII. Thus, diminishment of FVII activity precludes the requirement for TFPI-mediated inhibition of the FVIIa/TF pathway during embryogenesis.

Antithrombin: molecular basis of deficiency.

Antithrombin is a plasma protein regulator of coagulation proteinase activity, particularly that of thrombin. Its deficiency is a risk factor for venous thromboembolism. Considerable progress has been made in understanding the organisation and function of the antithrombin gene and protein, and the molecular basis of deficiency, all of which are reviewed, but briefly, here.

Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HC II Tokushima.

We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bhv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC I1 in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC I1 mRNA was transcribed at a similar level as that of wild-type. Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.

Pharmacokinetic study of alpha1-antitrypsin infusion in alpha1-antitrypsin deficiency.

OBJECTIVES: To ascertain how long 120 mg/kg alpha1-antitrypsin concentrate (alpha1-AT-C), administered I.V. every 2 weeks, can maintain alpha1-antitrypsin (alpha1-AT) serum levels above 70 to 80 mg/dL. Secondary objectives were to summarize the nature, severity, and relationship of a plasma-derived alpha1-AT-C infusion to any side effects. METHODS: This was an open-label uncontrolled pharmacokinetic study. Alpha1-AT-C was administered I.V. every 2 weeks for 10 infusions in 23 patients with PIZ alpha1-AT deficiency. Serum alpha1-AT levels and neutralizing elastase activity were measured preinfusion, postinfusion, and at nadir. During two infusion periods, daily serum alpha1-AT and neutralizing elastase activities were measured on the seventh to 14th days. Five patients received BAL assays for alpha1-AT and neutralizing elastase activity. Adverse events were recorded in a patient diary and by a nurse at each infusion visit. RESULTS: The 120-mg/kg dose of alpha1-AT-C could not maintain nadir serum protective levels above 70 or 80 mg/dL for the entire 14-day dosing interval in most patients. None of the patients had alpha1-AT levels above 80 mg/dL for all 14 days. The serum alpha1-AT and neutralizing elastase levels correlated suggesting functional activity. The BAL alpha1-AT and neutralizing elastase activities were low and did not correlate with serum levels. CONCLUSION: Alpha1-AT-C at 120 mg/kg administered every 2 weeks did not maintain nadir serum alpha1-AT levels above 70 to 80 mg/dL for a 14-day dosing interval. Higher doses every 2 weeks or decreased interval between infusions may be required.

Decompensation in proteinase-inhibitor system and application of proteinase inhibitors in pemphigus and pemphigoid.

The goal of this study was to determine levels of serine proteinases and their inhibitors in serum and blister fluid of patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP) in the course of treatment with prednisolone and proteinase inhibitors such as Contrycal and epsilon-aminocaproic acid. Assessment of the total proteolytic activity of serine proteinases (TPASP) and total antitryptic activity (TATA) is sera and blister fluids from 53 PV and 38 BP patients showed abnormalities in the proteinase and proteinase inhibitor levels in the acute stage of these bullous dermatoses. In PV, the level of TRASP was raised to 495 +/- 73 mU/ml (control 246 +/- 48 mU/ml; P < 0.001) in serum and to 849 +/- 96 mU/ml (control 189 +/- 34 mU/ml; P < 0.001) in blister fluid. In BP, the level of TRASP was raised to 472 +/- 101 mU/ml (P < 0.001) and 796 +/- 103 mU/ml (P < 0.001) in serum and blister fluid, respectively. In both dermatoses, the increase in serine proteinase activity developed against the background of decreased or unchanged antiproteolytic activity. TATA levels dropped to 16 +/- 5 inhibitory units (iU)/ml (control 26 +/- 5 iU/ml; P < 0.001) in PV serum and to 6 +/- 1 iU/ml (control 20 +/- 2 iU/ml: P < 0.001) in PV blister fluid. In BP, serum TATA levels were within normal values, whereas blister fluid TATA levels were significantly decreased (14 +/- 3 iU/ml; P < 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: a controlled double-blind, randomized, multicenter study.

BACKGROUND: ATIII is decreased in sepsis and/or shock and its baseline value correlates with mortality. The efficacy of ATIII therapy on mortality was assessed in a selected group of patients admitted to the intensive care unit (ICU) in a double-blind, randomized, multicenter study. METHODS: 120 patients admitted to the ICU with an ATIII concentration < 70% were randomized to receive ATIII (total dose 24000 units) or placebo treatment for 5 days; 56 patients had septic shock. RESULTS: ATIII concentrations in the treated group remained constant throughout the treatment period (range 97-102%). The Kaplan-Meier analysis showed no difference in overall survival between the two groups: 50 and 46% for ATIII and placebo, respectively. Septic shock and hemodynamic support were unbalanced in the two groups at admission. Therefore the Cox analysis was carried out after adjusting for these two variables. Treatment with ATIII decreases the risk of death with an odds ratio (OR) of 0.56. Of the covariates analyzed, septic shock and the baseline multiple organ failure score were negatively associated with survival and plasma activity level was positively associated with survival with an OR of 0.97 for each 1% increase in the ATIII plasma concentration at baseline. CONCLUSIONS: The results of ATIII treatment in this population of patients suggests that replacement therapy reduces mortality in the subgroup of septic shock patients only.

Sweet's syndrome leading to acquired cutis laxa (Marshall's syndrome) in an infant with alpha 1-antitrypsin deficiency.

BACKGROUND: Marshall's syndrome is a rare pediatric skin disease that is characterized by acquired, localized neutrophilic dermatitis (Sweet's disease), followed by loss of elastic tissue in the dermis and cutis laxa. The cause of this syndrome is unknown. alpha 1-Antitrypsin (alpha 1-AT) deficiency is a codominantly inherited disorder of alpha 1-AT, the major serum antiprotease active against a number of serine-type proteases. OBSERVATIONS: The first patient with classic Marshall's syndrome who had coexisting alpha 1-AT deficiency and a review of other cases of Marshall's syndrome are presented, and pathogenic mechanisms are discussed. CONCLUSIONS: A deficiency of alpha 1-AT may allow proteases such as neutrophil elastase to destroy dermal elastin and, thus, produce cutis laxa in Marshall's syndrome. Other cases of acquired cutis laxa should be screened for alpha 1-AT deficiency to further evaluate this association and to enable patients and their families to be counseled about possible systemic complications of alpha 1-AT deficiency.

Alpha 1-antitrypsin deficiency in a child with X-linked lymphoproliferative disease.

An 18-month-old white male infant with X-linked lymphoproliferative disease was evaluated for persistent hepatic dysfunction following primary Epstein-Barr virus infection. A liver biopsy revealed cirrhosis with a dense mononuclear cell infiltrate. These findings were confounding because cirrhosis is not a typical finding in either normal or immunodeficient individuals following infection with Epstein-Barr virus. An alpha 1-antitrypsin level obtained shortly after biopsy was spuriously within the lower limits of the physiologic range. Further investigation demonstrated a homozygous Z phenotype, the classic protease inhibitor variant described in alpha 1-antitrypsin deficiency. A repeat liver biopsy confirmed the presence of a second hereditary disease. This is a unique concurrence of two uncommon genetic disorders.

[Hemostatic disorders in liver disease]. / Anomaliile hemostazei in suferintele hepatice.

Hemorrhagic complications are common in patients with liver diseases and contribute to the morbidity and mortality associated to this condition. The liver plays a central role in the hemostatic process as here all clotting factors and their inhibitors are synthetized. Liver damage is commonly associated with variable impairment of hemostasis due to multiple causes: decreased synthesis of clotting and inhibitor factors, decreased clearance of activated factors, hyperfibrinolysis, accelerated intravascular coagulation, quantitative and qualitative platelet defects. Their clinical implications remain to be elucidated, so further studies addressing this issue are needed.

Hereditary deficiency of protein C, protein S and antithrombin III.

It is estimated that 5% of patients with deep vein thrombosis and 50% of those with recurrent thrombosis have an inherited abnormality of coagulation, most commonly deficiency of protein C, protein S or antithrombin III. These disorders should be suspected when venous thrombosis occurs in a young person, if there is a family history of thrombosis, if thrombosis occurs at an unusual site or if there is recurrent thrombosis with no predisposing factors. Affected patients are treated with lifelong anticoagulation therapy. Thromboembolism and its sequelae often produce abnormal findings on radiologic examinations, and therefore the radiologist who is familiar with these abnormalities is in a position to be the first to suggest the diagnosis.

Deficient antithrombin III activity and enhanced fibrinolysis in patients with liver disease: evidence against a cause-effect relationship.

To investigate the pathogenesis of fibrinolysis in liver disease, antithrombin III (AT III) activity, prothrombin fragment (F1 + 2) and d-dimer (D-DI) were measured in 50 patients with liver disease and in 17 healthy controls. Moreover, 4 patients with cirrhosis were randomly assigned to receive either an intravenous infusion of AT III (at two different dosages) or placebo, with a crossover design. Increased levels of D-DI were detected in patients with cirrhosis and hepatocellular carcinoma in comparison both with control subjects and with patients with acute hepatitis or mild chronic liver disease. An inverse correlation was observed between AT III and D-DI (r = -0.755, P < 0.001, simple linear regression), while no correlation was found between D-DI or AT III and F1 + 2. The correlation of the deficiency of AT III activity by infusion of human AT III did not result in any significant change (P0.10, analysis of variance for repeated measures) of the plasma concentration of either D-DI or F1 + 2, in comparison to placebo. Thus, advanced forms of chronic liver disease, but not acute hepatitis and mild forms of chronic liver disease, are associated with increased plasma concentrations of markers of fibrinolysis, which are inversely correlated with AT III activity. However, the correction of the deficient AT III activity does not affect the plasma concentration of either D-DI or F1 + 2, thence not supporting the hypothesis that enhanced fibrinolysis in advanced liver disease is the result of low-grade disseminated intravascular coagulation.

Protein C, protein S, and antithrombin III in acute ocular occlusive diseases.

Hereditary and acquired deficiencies of protein C, protein S, and antithrombin III are important risk factors of thrombosis, especially in peripheral veins. In a prospective study, concentrations of these factors were measured to determine the prevalence of deficiencies of these proteins in ocular vascular occlusive disease. A total of 167 patients with acute retinal arterial (28%) or venous occlusion (55%) or ischemic optic neuropathy (17%) were included. Exclusion criteria were anticoagulant therapy, renal insufficiency, and hepatic disease. The mean values obtained for all patients were in normal range (protein C, 102 +/- 25%; protein S, 109 +/- 22%; antithrombin III, 23.6 +/- 3.1 IU/ml). Antithrombin III was pathologically reduced in one patient with branch venous occlusion. Proteins C and S were severely altered in two patients with central venous occlusion, in one individual with ischemic optic neuropathy, and in one patient with branch arterial occlusion. Subnormal values were found in 21 patients for antithrombin III (16.1-19.9 IU/ml), in 7 patients for protein C (55-65%). Two of these five patients with pathologic findings showed severe vascular manifestations in the form of current deep-vein thrombosis and multiple retinal occlusions. Their age was 30 and 53 years, respectively, and differed considerably from the mean age of the entire group (65 +/- 12 years). This study suggests that these proteins were important factors for the development of ocular vascular occlusive diseases in single patients. Although the prevalence is low, measurement of these parameters in young patients may be useful in preventing other vascular complications.

Molecular and cellular basis for type I heparin cofactor II deficiency (heparin cofactor II Awaji).

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.