Plasma kallikrein contributes to ambient particulate matter-induced lung injury.

Particulate matter (PM) is a key component of air pollutants and is associated with mortality of cardiovascular and respiratory diseases. PM-induced tissue injury involves inflammation and coagulation. Plasma prekallikrein (pKal), along with coagulation factor XII (FXII) and high-molecular-weight kininogen (HK), form the plasma kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory products in response to injury. When the KKS proteins contact with activation surface such as negatively charged molecules, this system becomes activated. Activated kallikrein (Kal) activates FXII to initiate the intrinsic coagulation pathway, and cleaves HK to release bradykinin to enhance vascular permeability and systemic inflammation. In his study we determined the role of plasma pKal in the PM2.5-induced lung injury. Using TALEN technology, we generated a new mouse strain lacking the gene for pKal. In PM2.5-induced lung injury model, Klkb1-/- mice exhibited a decrease in total protein, cells numbers in bronchoalveolar lavage fluid (BALF) and histologic lung injury score. The TNF-α and IL-6 levels in BALF were significantly decreased in PM2.5-treated Klkb1-/- mice. Plasma thrombin-antithrombin (TAT) complex levels were significantly decreased in PM2.5-treated Klkb1-/- mice. PM2.5 induces pKal activation, HK cleavage and bradykinin production. PM2.5-induced HK cleavage in plasma was completely blocked by a Kal inhibitor, as well as in pKal-deficient plasma. PM2.5 markedly induced thrombin generation in human plasma and wild-type mouse plasma, which was inhibited by both blockade and deficiency of pKal. Taken together, plasma pKal is activated by PM2.5 and the activated Kal plays an important role in PM2.5-induced lung injury.

Application of Isobaric Tags for Relative and Absolute Quantification (iTRAQ) Coupled with Two-Dimensional Liquid Chromatography/Tandem Mass Spectrometry in Quantitative Proteomic Analysis for Discovery of Serum Biomarkers for Idiopathic Pulmonary Fibrosis.

BACKGROUND The present study was performed to explore the presence of informative protein biomarkers of human serum proteome in idiopathic pulmonary fibrosis (IPF). MATERIAL AND METHODS Serum samples were profiled using iTRAQ coupled with two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) technique, and ELISA was used to validate candidate biomarkers. RESULTS A total of 394 proteins were identified and 97 proteins were associated with IPF. Four biomarker candidates generated from iTRAQ experiments - CRP, fibrinogen-α chain, haptoglobin, and kininogen-1 - were successfully verified using ELISA. CONCLUSIONS The present study demonstrates that levels of CRP and fibrinogen-a are higher and levels of haptoglobin and kininogen-1 are lower in patients with IPF compared to levels in healthy controls. We found they are useful candidate biomarkers for IPF.

The Plasma Kallikrein-Kininogen Pathway Is Critical in the Pathogenesis of Colitis in Mice.

The kallikrein-kinin system (KKS) consists of two serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-molecular-weight kininogen (HK). Upon activation of the KKS, HK is cleaved to release bradykinin. Although the KKS is activated in humans and animals with inflammatory bowel disease (IBD), its role in the pathogenesis of IBD has not been characterized. In the present study, we determined the role of the KKS in the pathogenesis of IBD using mice that lack proteins involved in the KKS. In two colitis models, induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), mice deficient in HK, pKal, or bradykinin receptors displayed attenuated phenotypes, including body weight loss, disease activity index, colon length shortening, histological scoring, and colonic production of cytokines. Infiltration of neutrophils and inflammatory monocytes in the colonic lamina propria was reduced in HK-deficient mice. Reconstitution of HK-deficient mice through intravenous injection of HK recovered their susceptibility to DSS-induced colitis, increased IL-1ß levels in the colon tissue and bradykinin concentrations in plasma. In contrast to the phenotypes of other mice lacking other proteins involved in the KKS, mice lacking FXII had comparable colonic inflammation to that observed in wild-type mice. The concentration of bradykinin was significantly increased in the plasma of wild-type mice after DSS-induced colitis. In vitro analysis revealed that DSS-induced pKal activation, HK cleavage, and bradykinin plasma release were prevented by the absence of pKal or the inhibition of Kal. Unlike DSS, TNBS-induced colitis did not trigger HK cleavage. Collectively, our data strongly suggest that Kal, acting independently of FXII, contributes to experimental colitis by promoting bradykinin release from HK.

Proteomic biomarkers for psoriasis and psoriasis arthritis.

UNLABELLED: Although several new biomarkers have been recently proposed for psoriasis (Ps) and psoriasis arthritis (PsA), nothing is known about their diagnostic sensitivity and specificity, and their routine use. We therefore searched in-depth for new biomarker candidates using a biobank with EDTA-plasma from 158 individuals, patients and healthy controls. Samples from 6 selected pairs (patients against healthy controls) were searched proteomically using a workflow of extensive and precise design that is highly comprehensive. Subsequent verification was performed using ELISA and the entire biobank. By proteomic methods, 208 altered proteins were identified. Of these, 15 biomarker candidates were selected for verification. Of these 15, 4 individual parameters and 11 combinations significantly discriminated between patient and control groups. These individual parameters were Zn-α2-glycoprotein, complement C3, polymeric immunoglobulin receptor, and plasma kallikrein. Significant discrimination was obtained by combinations of 2 or 3 parameters. One combination seemed suitable for diagnosing PsA. Moreover, several candidates desmoplakin, complement C3, polymeric immunoglobulin receptor, and cytokeratin 17, correlated with PASI in all patients. This first comprehensive proteomic study on non-depleted plasma identified several biomarker candidates that have not been described before as well as some known from previous studies. BIOLOGICAL SIGNIFICANCE: Our non-gel proteomic analysis is based on the highly comprehensive and significantly optimized chromatographic protein pre-fractionation. The method allows a biomarker search in non-depleted plasma. The subsequent verification by ELISA identifies several biomarker-candidates for the unbiased diagnosis of psoriasis and psoriasis arthritis. Four of the identified candidate markers might be used individually. Combinations of several parameters improve the diagnostic sensitivity and specificity. The still not validated candidates form a reserve for further evaluation. Moreover, mass spectrometric data uncover several biomarker-candidates which show diverse protein species of the same protein with opposing changes in the same sample.

Determination of kininogens levels and kallikrein/kininase II activities in patients with thromboangiitis obliterans.

Some components of the kinin system such as plasma kallikrein levels, the activities of tissue kallikrein (including saliva) and kininase II and the concentrations of kininogen fractions (low-molecular weight/LKg and high-molecular weight/HKg) were evaluated in the plasma of patients with thromboangiitis obliterans (TAO) presenting clinical symptoms of the condition. Twenty TAO were diagnosed by means of the traditional Shionoya and Olin criteria and later classified into non-smokers (n = 11) and active smokers (n = 9). Fifty-three normal, non-smoking/smoking individuals (control) were also studied. Kininogen levels were determined by ELISA; the activities of kallikreins and kininase II were determined using selective substrates. The levels of enzymes (kallikreins and kininase II) and protein (kininogens) were significantly higher in patients with TAO who were active smokers compared to the control groups (no matter whether control individuals were active smokers or non-smokers, P < 0.001 for all comparisons). Interestingly, regardless of the time of disease onset, a significant increase in the levels of these components of the kinin system was also observed in patients when TAO active smokers were compared with TAO ex-smokers (P < 0.01 for all analysed parameters). Activation of the kinin system in patients with TAO may indicate the involvement of vasodilatation in an attempt to control vascular changes, thereby favouring the deposition of immune complexes at the vascular level because of nicotine stimulation. Moreover, our results corroborate the idea that TAO can be an autoimmune disorder with specific mechanisms.

The kinin system in patients with systemic lupus erythematosus exhibiting mucocutaneous lesions: a clinical study.

In the present study, we evaluated the kinin system components in the plasma of patients with systemic lupus erythematosus exhibiting mucocutaneous lesions. Fifteen women with active cutaneous lupus (P) and 15 normal healthy women (C) were studied. Low molecular (LKg) and high molecular (HKg) weight kininogen were determined by ELISA (expressed microg Bk/ml). The activities of tissue kallikrein (TKal), plasma kallikrein (PKal) and kininase II were assayed by their action on selective substrates. Statistical analysis was performed using the Mann-Whitney test. The patients presented increased plasma levels of LKg (P = 2.98, C = 0.79) and HKg (P = 1.78, C = 0.5) associated with the increased activity of PKal (P = 2.50, C = 1.63 U/ml), TKal (P = 1.87, C = 1.30 microM pNa/ml) and kininase II (P = 1.50, C = 0.51 microM Hys-Leu/ml), when compared to the values observed in the control group (P < 0.0001 for each comparison). Thus, the increased concentration of all parameters of the kinin system in these patients indicate an overactivity of the kinin system in the acute phase of lupus, corroborating with the participation of these mediators in lupus pathogenesis.

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS.

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.

Increased activity of plasma and tissue kallikreins, plasma kininase II and salivary kallikrein in pemphigus foliaceus (fogo selvagem).

BACKGROUND: Pemphigus foliaceus (PF) is an autoimmune blistering disease of unknown aetiology, which is endemic in Brazil. Although the pathogenesis of PF is still unknown, proteins of the contact system have been implicated. OBJECTIVES: As the components of the kinin system may interact with those of the contact system, in this study we evaluated the plasma levels of high-molecular-weight kininogen (HK) and low-molecular-weight kininogen (LK), and the activity of plasma kallikrein, tissue kallikrein and kininase II in plasma of patients with PF presenting with Nikolsky's sign. As kidneys and salivary glands are relevant sources of tissue kallikrein for plasma, we also evaluated urinary/salivary kallikrein and urinary kininase II activities. METHODS: Fifteen patients and 15 age- and sex-matched controls were studied. Kininogen levels were determined by enzyme-linked immunosorbent assay, and the activities of kallikreins and kininase II were determined using selective chromogenic substrates. RESULTS: Compared with controls, plasma HK levels were decreased (P = 0.031), whereas the activities of plasma kallikrein, tissue kallikrein and kininase II in plasma, and the activity of salivary kallikrein, were increased in patients (P < 0.001 for each comparison). Plasma levels of LK and the activities of urinary kallikrein and urinary kininase II were not significantly different from controls. CONCLUSIONS: Diminished levels of HK associated with increased activities of plasma kallikrein and kininase II indicate that the kinin system is activated at the systemic level in PF. As active plasma kallikreins may act on some proteins of the contact system, it is possible that the enzyme may contribute to blister formation. The further observation of an increased tissue kallikrein activity at the systemic and saliva levels may be interpreted as a systemic reflex of skin inflammation. Whether the activation of the kinin system is a cause or a consequence of blister formation needs further clarification.

Kallikrein modification with affinity to IgG present in higher amounts than normal in plasma from patients with Crohn's disease.

This study presents immunological and functional evidence to suggest that the activation of prekallikrein (PK) in human plasma yields two modifications of kallikrein. One of these modifications showed amidolytic properties strongly deviating from those registered for the main part of the enzyme. The substrates were S-2302, Bz-Pro-Phe-Arg-pNA, S-2366 and S-2222. In PAGE immunoblots the PK heavy chain mAb 13G11 was found to detect the kallikrein 85 kD double band and bands with mol. weights of about 152 and 135 kD. Such a 152 kD band could be removed together with an IgG fraction on a Protein G column. In this study the kallikrein identity was confirmed by an estimation of the levels obtained in amidolytic assays of mixtures of normal plasma and plasma deficient in FXI, which in immunological assays showed a PK level of 140-150%, of normal. A comparison in amidolytic assays of normal plasma and plasma from patients with Crohn's disease showed that patients' plasma contained a significantly higher level than normal of modified kallikrein. An IgG removal procedure removed all modified kallikrein, and did not affect ordinary kallikrein levels.

Effect of short-term losartan treatment in patients with primary aldosteronism and essential hypertension.

The aim of this study was to investigate the effect of short-term treatment with losartan, a selective and competitive angiotensin II (AngII) receptor blocker, on vascular endothelial growth factor (VEGF), active renin and kallikrein activity (KA) in patients with essential hypertension and primary aldosteronism. Nine patients with primary aldosteronism (5 with Conn adenoma and 4 with idiopathic hyperaldosteronism) and 9 patients with essential hypertension were included in the study. Systolic and diastolic blood pressure decreased significantly after losartan treatment in both patient groups. Plasma and urinary Kallikrein activity were significantly higher in primary aldosteronism in comparison with essential hypertension. There were no significant changes in the active renin and aldosterone in patients with primary aldosteronism after treatment. Plasma and urinary KA decreased significantly after losartan administration in both groups. Serum VEGF levels in primary aldosteronism were not significantly different from those in essential hypertension and did not change significantly after treatment in either group. In conclusion, losartan, in usual therapeutic doses, lowers blood pressure in patients with primary aldosteronism and essential hypertension. This marked antihypertensive effect in primary aldosteronism could be explained predominantly by blockade of tissue renin-angiotensin system (RAS). The variations in KA could be due to hemodynamic changes. VEGF is not likely to be involved in the action of losartan.

Immunolocalization of plasma kallikrein in human brain.

Plasma kallikrein (PK) is a cofactor in blood coagulation and modulates inflammation through the release of bradykinin. Previously it was believed that plasma prekallikrein (PPK), the precursor of PK and a member of the serine protease superfamily, was synthesized exclusively by hepatocytes and secreted into circulation. However, recent studies show that the human brain contains a high level of PPK mRNA. In this study we sought to determine which areas of the brain express PK. Tissue from the spinal cord and 13 different regions of the human brain were collected at autopsy within 24h from death. Sections were probed using polyclonal antibodies (characterized by Western blotting) specific for PK. PK concentrations in extracts of these tissues were measured by ELISA. Immunolabeling of PK was observed in the cell bodies of the neurons of the hypothalamus, thalamus, spinal cord, cerebral cortex and brainstem. Positive PK immuno-reactivity was also demonstrated in the cytoplasm of the ependymal cells in sections of the hypothalamus and spinal cord. In addition, some fibre tracts of the pons, medulla and hippocampus as well as secretory cells of the pituitary gland also labeled. No immunoreactive PK was visualized in the choroid plexus or cerebellum. Our data demonstrate the cellular localization of PK in human brain. This work is supported by other studies that demonstrate PK mRNA in human heart, lung, trachea and brain. The cellular distribution of PK and kinin receptors in specific brain areas suggests a role for PK in the nervous system.

Plasma and tissue kallikrein in arthritis and inflammatory bowel disease.

To ascertain the participation of the plasma kallikrein-kinin system (KKS) in arthritis and inflammatory bowel disease, we used two rat models resembling rheumatoid arthritis and Crohn's disease. Proteoglycan-polysaccharide from group A streptococcus (PG-APS) produced chronic destructive inflammation and systemic response in the genetically susceptible Lewis rat, in the joints when injected intraperitoneally and in the bowel when injected into the gut wall. In both models, the KKS is activated, as evidenced by decreased prekallikrein, factor XI and high molecular weight kininogen. A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroarginine, reverses the plasma changes as well as the clinical gross and microscopic pathology of both the experimental arthritis and the inflammatory bowel disease in the genetically susceptible rats. We have also shown that the tissue kallikrein system is involved in the intestinal inflammatory changes. Intestinal tissue kalikrein (ITK) is localized in goblet cells in both normal and inflamed tissue. In chronic granulomatous inflammation, ITK is localized in macrophages. ITK decreases in chronic inflammation, probably due to secretion, since the mRNA is unchanged. Kallikrein binding protein, the ITK inhibitor, decreases due to enzyme-inhibitor complexes. Both plasma and tissue kallikrein are appealing targets for drug therapy of rheumatoid arthritis and Crohn's disease.

The possible role of plasma kallikrein-kinin system and leukocyte elastase in pathogenesis of schizophrenia.

The main purpose of this paper is to study the possible causes of blood-brain barrier (BBB) damage during the acute condition of schizophrenia (Sch), which makes brain antigens accessible to the immunocompetent cells. The development of autoimmune reactions in this disease has to be preceded by the damage of BBB. We have studied the level of activity of plasma kallikrein-kinin (KKS) and complement systems, C-reactive protein (CRP) concentration, proteinase inhibitory potential as well as the oxidized and degranulating activity of neutrophils as the main factors affected the permeability of tissue-blood barrier. Our results suggested that the acute stage of Sch was accompanied by the activation of KKS on the background of enhance in the functional activity of the alpha-1-proteinase inhibitor. The increased level of CRP, the high haemolytic activity of complement and significant degranulating activity of polymorphonuclear leukocytes testified to inflammatory character of Sch. The treatment with psychotropic drugs have led to decrease of polymorphonuclear elastase (PMN-E) activity in patient's plasma. Our in vitro study indicates that Haloperidol causes the lowering of PMN-E activity in the dose-dependent fashion.