Kininogen Levels Research Articles

Levels of endothelin-1 and high molecular weight kininogen in blood of breast cancer patients after SARS-coV-2 infection

The aim. To analyze the blood levels of endothelin-1 (ET -1) and high molecular weight kininogen (HMWK) in patients with breast cancer (BC) previously infected with the new coronavirus.Material and methods. The study group included 20 patients with stage II -IV BC (invasive carcinoma). All patients were receiving chemotherapy at the time of their SA RS-CoV-2 infection. The comparison group included 19 women without breast cancer, who were matched for age. All women of both groups had an RT-PC R confirmed SA RS-Cov-2 infection. Blood levels of ET -1 and HMWK were measured by ELISA 3–10 weeks after the positive antigen test results. The control group included 10 women of the same age without cancer and without CO VID -19 symptoms and anti-SA RS-CoV-2 antibodies.Results. The ET -1 levels in the comparison group were within the reference range, while HMWK levels were significantly higher than those in breast cancer patients. In BC patients with lung metastases, the ET -1 levels were higher than those in the comparison group patients, while in others (no history of lung metastases, with mild infection course or pneumonia), the ET -1 levels were similar to those in the comparison and control groups. The HMWK levels in the study and comparison groups were significantly higher than those in controls. Among BC patients, there were women who had significantly higher ET -1 and HMWK levels compared to the reference levels, and the majority of these patients had lung metastases and previous CO VID -19 pneumonia.Conclusion. The measurement of HMWK blood levels demonstrated that the plasma contact activation system and the kallikrein-kinin system were active for a long period after the infection both in BC patients and in women without cancer. A high level of ET -1, the endothelial dysfunction marker, persisted for a long time in some BC patients. Our results were consistent with results of other studies supporting the hypothesis that SA RS-CoV-2 virus infection is a systemic vascular disease with long-term consequences, and its mechanisms require further study.

A Novel Immunoblot Assay to Detect Contact Activation in Patients with Cancer

Introduction:The risk of thrombosis is markedly increased in patients with cancer and associated with increased morbidity and mortality. Clinical scoring systems are helpful to stratify cancer patients at a highest risk for thrombotic events. However, more precise risk prediction is needed, particularly for patients with cancers associated with intermediate thrombotic risk. In some, but not all studies, evidence of activation of coagulation proteases as measured using assays such as thrombin generation times, D-dimer levels, etc., has correlated with thrombotic risk, though these correlations are generally not robust. Recent studies have demonstrated that the contact activation pathway plays an important role in the development of pathologic thrombosis, though its role in cancer-associated thrombosis has not been clearly defined. However, unlike assays that measure prothrombin activation, limited options for assessing contact activation are available. To examine this question, we have developed a quantitative immunoassay for contact activation that assesses cleavage of high molecular weight kininogen (HK), a marker of contact system activation, in patient plasma. Circulating cleaved HK (HKa) has been found in plasma in disorders such as hereditary angioedema, and our studies in mice demonstrate that HK is quantitatively cleaved within murine tumors.Methods:To detect circulating HKa, one microliter of plasma was analyzed using SDS-PAGE on a 4-12% gel under reduced conditions. Following transfer to low-fluorescence PVDF membranes, the sample was immunoblotted using an affinity-purified rabbit polyclonal antibody raised to a peptide encompassed by the C-terminal region of the HK heavy chain (CQPLGMISLMK). Bound antibody was detected using an IRDye® 800CW-labeled goat anti-Rabbit secondary antibody followed by band quantification using a LI-COR Odyssey imaging system and Image StudioTM Lite Software. This assay detects both intact kininogen (MW ~ 120 kD) and the cleaved kininogen heavy chain (MW ~ 50 kD). To control for different levels of kininogen in individual patients, cleaved kininogen was quantified by estimating the relative amount of kininogen heavy chain as a percentage of uncleaved kininogen using LI-COR technology.Results:To date, we have used this assay to assess kininogen cleavage in 11 patients with several different cancers including breast cancer (N = 3), bladder cancer (N = 1), pancreatic cancer (N = 5), acute myeloid leukemia (N = 1), and renal cancer (N = 1). Patients with cancer demonstrated a greater percentage of cleaved HK in plasma than normal donor controls, in whom less than 1% of kininogen was present in the cleaved form. The greatest amount of cleavage was observed in pancreatic cancer, and was estimated at 33.4% (see Table).Conclusion:These studies provide evidence that the contact activation system is activated in many patients with cancer. Current effort is focused on identifying causes of variation in contact activation between cancers, and determining whether the extent of contact activation as determined by this quantitative assay correlates with or predicts the development of thrombosis in cancer patients. [Display omitted] DisclosuresKhorana:Halozyme: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Leo: Consultancy, Honoraria, Research Funding; Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria.

A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

Biomarkers of Hypercoagulability and Endothelial Cell Activation in Patients with Severe COVID-19 Admitted at the Intensive Care Unit. the Roadmap-COVID-19 Prospective Observational Study

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) characterized by acute pneumonia which may progress to respiratory failure and life-threatening complications, including acute respiratory distress syndrome (ARDS) and multisystem organ failure with fatal outcome. COVID-19 should be regarded as a systemic disease involving multiple systems. COVID-19 is associated with excessive inflammation, platelet activation, endothelial dysfunction, blood coagulation activation and fibrin formation. The development of a screening algorithm for the evaluation of endothelial and blood coagulation activation is an urgent need for the optimisation of the management of the patients with severe COVID-19. To this aim biomarkers of endothelial cell activation and hypercoagulability have central placeAim: The prospective observational monocentric cohort study ROADMAP-COVID-19 study aimed to evaluate the biomarkers of hypercoagulability and endothelial cell activation which they could be used in the development of a screening algorithm in patients with COVID-19Methods: The ROADMAP-COVID-19 study enrolled 90 consecutive patients hospitalized at Tenon University Hospital (AP-HP, Paris) between March 18th and April 30th, 2020 with confirmed SARS-CoV-2 infection admitted to the ICU with clinically deteriorated severe COVID-19 (ICU- group). Patients were assessed upon the first day to the ICU admission. The control group consisted 30 healthy individuals (C-group). All patients and controls were evaluated for, clotting times (PT and aPTT), fibrinogen, D-Dimers, antithrombin (AT), protein C (PC) and protein S (Ps) activity, clotting factors V (FV), VIII (FVIII) and XII (FXII), high molecular weight kininogen (HK), von Willebrand factor (FvW), thrombin generation assay (TG) using TF 20 pM-PPP-Reagent® (Thrombogram-Thrombinoscope from Stago), fibrin monomers (FM), free TFPI and TF activity, thrombomodulin activity (TM), TF expressing microparticles (the ratio TF/TFPI was also calculated) and Heparanase. The IL-6 levels were also measured. All patients hospitalized in conventional medical department or in ICU routinely received thromboprophylaxis with body weight adapted enoxaparin. The ratio of the mean values of each studied biomarker in the ICU and the C-group was calculated. Biomarkers were classified in four clusters (Decrease, Stable, Slight Increase, Moderate Increase and High Increase).Results :The ICU-group consisted of 102 patients; 87% of these patients were admitted to the ICU directly from the emergency department. Males were 76 out of 102 patients. Age ranged from 30 to 93 years in the W-group. The changes of the biomarkers in patients with COVID-19 admitted at the ICU as compared to healthy individuals with are shown in Table 1. The ICU patients with COVID-19 were characterized by marked increase of IL-6 followed by increase of heparanase and D-dimers. The PPL-ct showed the most important decrease. Patients showed a significant decrease in FXII levels which was associated with a moderate increase of kininogen levels. The levels of the natural coagulation inhibitors as well as the clotting times (PT and aPTT) did not significantly modify in patients as compared to the controls. The rate of the propagation phase of thrombin generation (MRI) and the Peak of thrombin were significantly decrease and the lag-time was prolonged reflecting the antithrombotic effect of treatment with LMWH. Heparanse, free TFPI, and TM showed moderate or high increase. The decreased Ratio of TF/TFPI indicated that the free TFPI release dominated over the release of soluble TF activity.Conclusion: Patients with severe COVID-19 requiring ICU admission showed inhaled inflammatory reaction, hyperocoagulability and endothelial cell activation whereas platelet activation appeared to be secondary. Among the studied biomarkers the PPL-ct D-dimers and heparanase followed by TFPI, FvW, FM and TM levels appear to be mandatory for hypercoagulability of cellular origin. Interestingly patients with severe COVID-19 showed consumption of FXII indicating intrinsic clotting pathway activation and high levels of HK which also indicates endothelial cell activation. The clinical relevance of these biomarkers for early detection of hypercoagulability of cellular origin in patients with COVID-19 is being evaluated in prospective study.DisclosuresNo relevant conflicts of interest to declare.

3:27 PM Abstract No. 193 Identification of mRNA-encoded prognostic biomarkers for hepatocellular carcinoma

Hepatocellular Carcinoma (HCC) has become the fastest growing lethal cancer in the United States. Current selection of a specific locoregional treatment for HCC is based on imaging findings and operator experience. There remains a clear lack of reliable objective criteria for selection of an appropriate loco regional treatment. Non-invasive serological biomarkers combined with imaging techniques can be a valuable tool for determining appropriate locoregional therapies. The purpose of this study is to identify mRNA encoded prognostic biomarkers for precision treatment and outcome prediction. We have studied mRNA expression and overall survival (OS) of 364 HCC patients downloaded from The Cancer Genome Atlas (TCGA) using the Kaplan-Meier plotter database (1). The cohorts of patients’ OS were analyzed by multivariate analysis. Hazard ratio, false discovery rate, and log rank P values were subsequently determined. A P value < 0.01 was considered statistically significant. Transcriptome of those selected mRNAs in HCC tissues were analyzed by RNA-seq. Validation of those mRNA encoded proteins were performed using patients’ plasma by ELISA assays. High mRNA expression of ANXA2, FUBP1 and CDK1 are significantly correlated with lower OS, whereas high mRNA expression of KNG1 and CD274 and TRIM72 were associated with higher OS in all combined stages. RNA-seq analysis for transcriptome indicated only ANXA2, FUBP1, CDK1 and KNG1 are prognostic for liver cancer survival. High expression of ANXA2, FUBP1, CDK1 are unfavorable, whereas high expression of KNG1 is favorable for liver cancer survival. Analysis of those mRNA encoded proteins in patients’ plasma demonstrated high level of Annexin A2, but low level of Kininogen 1 expression in liver cancer patients comparing to healthy donors. This study identified a panel of 4 mRNA molecules (ANXA2, FUBP1, CDK1 and KNG1) and their encoded proteins which can be used as potential prognostic biomarkers for HCC patients. Future studies with a larger sample size are needed to validate our findings. Applications of those prognostic biomarkers will improve precision treatment and outcome prediction of hepatocellular carcinoma.

Plasma Concentrations of High Molecular Weight Kininogen and Prekallikrein and Venous Thromboembolism Incidence in the General Population.

The kallikrein/kinin system, an intravascular biochemical pathway that includes several proteins involved in the contact activation system of coagulation, renin-angiotensin activation and inflammation, may or may not play a role in venous thromboembolism (VTE) occurrence. Within a large prospective population-based study in the United States, we conducted a nested case-cohort study to test the hypothesis that higher plasma levels of high molecular weight kininogen (HK) or prekallikrein are associated with greater VTE incidence. We related baseline enzyme-linked immunosorbent assay measures of HK and prekallikrein in 1993 to 1995 to incidence VTE of the lower extremity (n = 612) through 2015 (mean follow-up = 18 years). We found no evidence that plasma HK or prekallikrein was associated positively with incident VTE. HK, in fact, was associated inversely and significantly with VTE in most proportional hazards regression models. For example, the hazard ratio of VTE per standard deviation higher HK concentration was 0.88 (95% confidence interval = 0.81, 0.97), after adjustment for several VTE risk factors. Our findings suggest that plasma levels of these factors do not determine the risk of VTE in the general population.

Kininogen Level in the Cerebrospinal Fluid May Be a Potential Biomarker for Predicting Epileptogenesis.

Purpose: Epilepsy is a highly disabling neurological disorder. Brain insult is the most critical cause of epilepsy in adults. This study aimed to find reliable and efficient biomarkers for predicting secondary epilepsy.Materials and methods: The LiCl-pilocarpine (LiCl-Pilo) chronic epilepsy rat model was used, and rat cerebrospinal fluid (CSF) was collected 5 days after status epilepticus (SE). The CSF was analyzed using the label-free LC-ESI-Q-TOF-MS/MS. Differential expression of proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The corresponding protein level in the CSF of patients with encephalitis in the postacute phase was determined using ELISA and compared between patients with and without symptomatic epilepsy after encephalitis during a 2-year follow-up.Results: The proteomics and ELISA results showed that the protein level of kininogen (KNG) was obviously elevated in both CSF and hippocampus, but not in serum, 5 days after the onset of SE in LiCl-Pilo chronic epilepsy model rats. In patients with encephalitis, the protein level of KNG in the CSF in the postacute phase was significantly elevated in patients with a recurrent epileptic seizure during a 2-year follow-up than in patients without a recurrent seizure.Conclusion: KNG in the CSF may serve as a potential biomarker for predicting epileptogenesis in patients with encephalitis.

Early depletion of contact system in patients with sepsis: a prospective matched control observational study.

Activation of the contact system generates bradykinin from high-molecular-weight kininogen and has been suggested to participate in the pathophysiology of sepsis. To test this, we prospectively measured bradykinin and high-molecular-weight kininogen levels in a cohort of sepsis patients requiring intensive care. From 29 patients meeting criteria for sepsis or septic shock according to Sepsis-3, blood was sampled within 24 h and on the fourth day following admittance to intensive care. Patients planned for neurosurgery served as matched controls. Sequential organ failure assessment score and 90-day mortality was registered. Bradykinin levels (median [interquartile range]) were lower in sepsis patients (79 [62-172] pg/ml) compared to controls (130 [86-255] pg/ml, p < 0.025) and did not correlate with mortality or severity of circulatory derangement. High-molecular-weight kininogen levels were lower in sepsis patients (1.6 [0.8-4.8] densitometry units) compared to controls (4.4 [2.9-7.7] densitometry units, p < 0.001), suggesting previous contact system activation. High-molecular-weight kininogen levels were lower in non-survivors than survivors (p = 0.003) and negatively correlated to severity of circulatory derangement. We conclude that a role for bradykinin in later stages of severe sepsis must be challenged. Low high-molecular-weight kininogen concentrations suggest that the decrease in bradykinin is due to substrate depletion.

LANADELUMAB EXPOSURE DURING STEADY STATE: ACHIEVEMENT OF EFFECTIVE CONCENTRATIONS IN PATIENTS IN THE HELP STUDY

Introduction In the HELP Study (NCT02586805), treatment with lanadelumab (a monoclonal antibody inhibitor of plasma kallirein) 150 mg q4wks, 300 mg q4wks, or 300 mg q2wks significantly decreased attack rates over 26 weeks. We evaluated the relationship between lanadelumab exposure and efficacy during steady state (days 70-182) among the 3 dose groups. Methods Blood samples were collected from patients prior to dosing at weeks 0, 8, 14, and 20 for measurement of lanadelumab concentrations and cleaved high molecular weight kininogen (cHMWK) levels. Results Mean observed steady state lanadelumab concentrations in plasma increased and cHMWK levels decreased with dose and dosing frequency. These were associated with decreased attack rates. Attack rates decreased to the greatest extent in patients who received lanadelumab 300 mg q2wks (86.9% reduction vs placebo). The IC90 of lanadelumab (concentration associated with 90% of the maximum inhibitory effect on cHMWK levels) was previously determined to be 18,777 ng/mL. 88.9% of patients in the 300 mg q2wks group attained a maximum concentration at steady state (Cmax,ss) ≥IC90, compared with 65.5% and 0% of patients in the 300 mg q4wks and 150 mg q4wks groups, respectively (Figure). The minimum concentration at steady state (Cmin,ss) and average concentration at steady state (Cavg,ss) was ≥IC90 in 77.8% and 85.2% of patients, respectively, in the 300 mg q2wks group. Conclusions Lanadelumab concentrations were maintained ≥IC90 in the majority of patients during steady state in the 300 mg q2wks group, correlating with the high extent of attack reduction observed. Table. Percentage of patients with lanadelumab exposure ≥IC90

Kininogen deficiency or depletion reduces enhanced pause independent of pulmonary inflammation in a house dust mite-induced murine asthma model.

High-molecular-weight kininogen is an important substrate of the kallikrein-kinin system. Activation of this system has been associated with aggravation of hallmark features in asthma. We aimed to determine the role of kininogen in enhanced pause (Penh) measurements and lung inflammation in a house dust mite (HDM)-induced murine asthma model. Normal wild-type mice and mice with a genetic deficiency of kininogen were subjected to repeated HDM exposure (sensitization on days 0, 1, and 2; challenge on days 14, 15, 18, and 19) via the airways to induce allergic lung inflammation. Alternatively, kininogen was depleted after HDM sensitization by twice-weekly injections of a specific antisense oligonucleotide (kininogen ASO) starting at day 3. In kininogen-deficient mice HDM induced in Penh was completely prevented. Remarkably, kininogen deficiency did not modify HDM-induced eosinophil/neutrophil influx, T helper 2 responses, mucus production, or lung pathology. kininogen ASO treatment started after HDM sensitization reduced plasma kininogen levels by 75% and reproduced the phenotype of kininogen deficiency: kininogen ASO administration prevented the HDM-induced increase in Penh without influencing leukocyte influx, Th2 responses, mucus production, or lung pathology. This study suggests that kininogen could contribute to HDM-induced rise in Penh independently of allergic lung inflammation. Further research is warranted to confirm these data using invasive measurements of airway responsiveness.

The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats

PurposeThis study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B1 and B2 receptors on LPS- induced fever and the POA cells involved in this response. Material and methodsMale Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca++ signaling in POA cells was performed in rat pup brain tissue microcultures. ResultsIcatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE2concentration. Effect abolished by indomethacin (i.p.). ConclusionsLPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B2-receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B2-receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever.

Urine proteome analysis to evaluate protein biomarkers in children with autism

BackgroundAutism is a complex developmental disability for which no specific diagnostic markers have been identified so far. The present study aimed to evaluate whether there is any abnormal protein(s) excreted in the urine of autistic children by proteome analysis which may act as diagnostic marker. MethodsUrine proteome analysis was carried out in first void urine samples of autistic and normal children (n=30) in the age group of 4–12years by 2D-PAGE followed by MALDI–TOF–MS analysis. ResultsComparison of 2D-PAGE gels revealed that many urinary proteins are expressed differentially in autistic children. Total numbers of spots observed were 250 and 159 in autism and normal samples respectively, out of which 95 matches were observed. In addition, 3 spots of abnormally expressed peptides were selected, excised and analyzed. Peptide sequence with significant match score was for kininogen-1 (KNG-1)–50 (spot-1), IgG1 heavy chain variable region-35(spot-2) and mannan-binding lectin serine protease-2 isoform-2 precursor-45(spot-3). All the autistic children showed significant increase (p<0.001) in urinary kininogen level measured quantitatively by ELISA, when compared to normal children. ConclusionIncreased urinary kininogen-1 level in all the autistic children and the possibility of this protein as a diagnostic marker need further investigation.

Screening and identification of potential predictive biomarkers for Down’s syndrome from second trimester maternal serum

Objective: In this study, we aimed to search for noninvasive predictive biomarkers for prenatal diagnosis of Down’s syndrome (DS). Methods: Maternal serum samples from five DS-affected pregnant women and five DS-unaffected women were analyzed by 2D gel electrophoresis and MALDI-TOF mass spectrometry to screen for potential predictive biomarkers of DS. Then, differential levels of dGTPase, β2-glycoprotein I (β2-GPI), complement factor H-related protein 1 precursor (CFHR1) and kininogen 1 isoform 2 were further verified by western blotting tests in another independent group. Results: Statistical analysis results revealed 29 protein spots whose levels differed significantly in the DS-affected pregnancies group. Of these, the eight most differentially expressed in DP were identified successfully. Among these, levels of dGTPase, CFHR1 and kininogen 1 were elevated significantly, whereas β2-GPI was reduced in DP. Discussion: These preliminarily verified proteins might serve as potential predictive biomarkers for DS-affected pregnancies.

Bradykinin-Forming Components in Kuwaiti Patients with Type 2 Diabetes

Diabetes is the most common risk factor in inducing hypertension, nephropathy and retinopathy. The bradykinin (BK)-forming system has been proposed to protect cardiovascular and renal functions. We therefore evaluated urinary active and proactive kallikrein, total kininogen, plasma tissue kallikrein, plasma creatinine, plasma glucose and plasma HbA1c in newly diagnosed untreated type 2 diabetic patients and healthy subjects. In diabetic patients, urinary and plasma tissue kallikrein concentrations were significantly increased. In addition, plasma prekallikrein levels were also significantly higher. However, urinary kininogen values were significantly reduced in diabetic patients when compared with healthy subjects. This is the first investigation among Kuwaiti Arab patients with type 2 diabetes showing abnormal activities in the BK-forming system. High levels of plasma prekallikrein may be a risk factor for developing high blood pressure as well as nephropathy. The urinary and plasma tissue kallikrein concentrations were higher in diabetic patients, which could indicate the hyperactivities of these components, and may result in increased levels of plasma glucose to induce diabetes. Furthermore, the urinary kininogen levels were reduced in diabetic patients. These alterations might reflect the utilization of urinary kininogen to form BK, a potent inflammatory agent. However, this hypothesis needs further investigation.

Identification of O-glycosylated Proteins That Are Aberrantly Excreted in the Urine of Patients with Early Stage Ovarian Cancer

Cancer is known to induce or alter the O-glycosylation of selective proteins that may eventually be excreted in the patients’ urine. The present study was performed to identify O-glycosylated proteins that are aberrantly excreted in the urine of patients with early stage ovarian cancer (OCa). These urinary glycoproteins are potential biomarkers for early detection of OCa. In this study, urinary proteins of patients with early stage OCa and age-matched OCa negative women were subjected to two-dimensional gel electrophoresis and detection using a lectin that binds to the O-glycosylated proteins. Our analysis demonstrated significant enhanced expression of clusterin and leucine-rich alpha-2-glycoprotein, but lower levels of kininogen in the urine of the OCa patients compared to the controls. The different altered levels of these urinary glycoproteins were further confirmed using competitive ELISA. Our data are suggestive of the potential use of the aberrantly excreted urinary O-glycosylated proteins as biomarkers for the early detection of OCa, although this requires further validation in a large clinically representative population.

The Effects of Captopril on Cardiac Regression, Blood Pressure and Bradykinin Components in Diabetic Wistar Kyoto Rats

The present study examined the left ventricular wall thickness (LVWT), total urinary kallikrein, total plasma kininogen and mean arterial blood pressure (MABP) in diabetic and non-diabetic Wistar Kyoto (WKY) rats. The MABP was significantly raised (P<0.01) in diabetic WKY rats compared to the respective controls. The LVWT was also significantly (P<0.01) increased in diabetic WKY rats than that of control WKY rats. The mean total urinary kallikrein level and the mean total plasma kininogen level were higher (P<0.01) in diabetic WKY rats, when these rats were treated with captopril (40 mg/kg and 80 mg/kg) against the mean value obtained from control WKY rats. In conclusion, this investigation suggests that diabetes induced in these rats can cause hypertension, increased LVWT and changes in the BK-forming components. Captopril treatment caused reduction in MABP, regression of LVWT and alterations in bradykinin (BK)-forming components. The possible significance of these observations is discussed.

Identification of a high molecular weight kininogen fragment as a marker for early gastric cancer by serum proteome analysis

Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n=81) and age- and sex-matched healthy controls (n=66). Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209Da peptide (AUC=0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC=0.593, carbohydrate antigen 19-9 AUC=0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients. Serum levels of the 2209Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.

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.

Evaluation of inflammatory kinin-forming components in experimental epilepsy in rats

The study was conducted to evaluate the total and active brain tissue kallikrein levels as well as plasma kininogen concentrations in the experimental models of epilepsy induced by maximal electroshock (MES), submaximal electroshock (SMES) and pentylenetetrazole (PTZ) in adult Sprague-Dawley rats. There was significant increase in the total brain tissue kallikrein (P < 0.001) and active tissue kallikrein (P < 0.05) after MES, SMES and PTZ induced epilepsy. Also, the total plasma kininogen levels fell significantly (P < 0.05) after production of MES, SMES and PTZ epilepsy. These results may suggest that these changes may indicate the formation of proinflammatory mediator, kinins, in the brain and peripheral circulation in experimental epilepsy.

Vasoactive mediators and hypotensive transfusion reactions

Vasoactive mediators and hypotensive transfusion reactions