Form Of Kininogen Research Articles

Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen

CLL is an incurable disease with variable prognosis. The hyper reactivity of the B-cell receptor (BCR) to unknown antigen ligation plays a pivotal role in CLL-cell survival. We aimed to investigate the BCR signalling pathway using proteomics to identify novel proteins which may have clinical relevance in this disease. Three CLL samples were selected based upon BCR responsiveness, demonstrated by upregulation of phospho-ERK following in vitro stimulation. The differential expression of proteins, upon artificial stimulation of the BCR, was examined in these samples using two-dimensional gel electrophoresis in combination with mass spectrometry. Proteins of interest were subsequently examined using immunoblotting. Proteomic analysis revealed that kininogen, a critical protein of kinin-kallikrein system, was upregulated in all 3 clinical samples upon BCR stimulation. There are 2 forms of kininogen: HMWK and LMWK. The upregulation of LMWK upon BCR stimulation was confirmed by immunoblotting in all 3 of these samples. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases (147months versus 253months for LMWK negative cases; p=0.125). Kininogen may be a novel therapeutic target in CLL and the possible association with prognosis warrants further investigation. We have identified the upregulation of LMWK upon BCR stimulation of CLL samples. There is no previous published research to suggest a link between kininogen and normal B-cells or CLL cells. In 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases. The absence of LMWK protein expression on normal B-cells suggests that this could be a biomarker for CLL and further research should be undertaken.

The Kinin-Kallikre System in Chronic Lymphocytic Leukemia - A Potential Target for Therapy?

AbstractAbstract 3904CLL remains an incurable disease with standard therapy regimens. The hyper reactivity of the B cell Receptor (BCR) to unknown antigen ligation plays a pivotal role in B-cell survival. Recent clinical trials of BCR-targeted therapies prompted the need for a better understanding of the biology of BCR signaling. We previously employed proteomics to study protein expression changes associated with BCR ligation. Using 2-dimensional gel electrophoresis (2DE) with MALDI-TOF mass spectrometry (MS) we identified that Kininogen (KNG) was upregulated in 3/3 “poor prognosis” clinical samples upon BCR stimulation. KNG (a critical protein of Kinin-Kallikrein System) is the precursor for Kinins which act via the B1 and B2 receptors (B1R and B2R, respectively) and is known to play a critical role in cell migration, proliferation, vascular permeability, inflammation and intracellular Ca2+ influx. Consequently we hypothesized that there is a functional role for B1R and B2R, Kinins and their precursor KNG in CLL B-cell survival which may offer a potential therapeutic target.Following ethical approval, blood samples were collected from CLL patients. Time to first treatment, clinical stage, IgVh status, CD38 and ZAP-70 data were available. “Poor prognosis” samples were defined as unmutated IgVh status, high WBC and hyper-responsive BCR (p-ERK expression ≥ 2.0- fold increase upon stimulation). Immunoblotting was employed to confirm KNG upregulation and Tissue Kallikrein expression in CLL B-cells. A series of 59 CLL samples was screened for constituent KNG expression using immunoblotting. The presence of mRNA transcripts for KNG was assessed by PCR. An Automated One-stage Factor Assay on the Instrumentation Laboratory ACL-TOP analyser was used to determine the level of Plasma Kallikrein in CLL patients vs healthy controls. Fluorescence Activated Cell Sorting (FACS) was utilized for CLL B-cell enrichment according to CD20 status. Isolated CLL B-cells were subjected to immunodetection and flow cytometry for B1R and B2R cell surface expression identification.Proteomic analysis by 2DE/MALDI-TOF MS previously revealed upregulation of KNG after 5.5 hours of in vitro BCR stimulation in 3/3 clinical samples. Upregulation of KNG after stimulation was confirmed by immunoblotting in 5 samples, including all 3 which were previously analyzed using proteomics. There are 2 forms of KNG: HMWK and LMWK. The analysis of constitutive KNG by immunoblotting revealed positive expression in 72.9% (43/59) and 80% (28/35) for LMWK and HMWK, respectively. No statistical significance was found with preliminary clinical correlations, however, there was a trend towards shorter median survival in LMWK positive cases (152 months vs. 264 months for LMWK negative cases) (p=0.161). PCR confirmed the presence of KNG transcript in CLL samples. An automated one-stage factor assay revealed that 47.2% (26/55) CLL patients expressed elevated Plasma Prekallikrein (the zymogen form of Plasma Kallikrein) level. Tissue Kallikrein overexpression was confirmed in 14 CLL samples, using an anti-KLK 6 antibody. We investigated the presence of inducible B1R and constitutive B2R on CD20+ CLL B-cells and demonstrated that B1R and B2R are expressed by CLL B-cells, however, B1R expression was highly upregulated.We demonstrate for the first time that CLL B-cells express differing levels of KNG protein and that the expression can be significantly increased after BCR ligation. Consequently, high concentration of KNG is achieved in near proximity to B1R and B2R, identified on the surface of CLL B-cells. Therefore, we show that CLL B-cells comprise the complete system for the synthesis and liberation of Kinins which in turn mediate the activation of B1R and B2R via an autocrine loop. We suggest, that simultaneous and sustained activation of B1R and B2R, which is known to be advantageous for immediate triggering of ERK1/2 global phosphorylation and PLCγ2-dependent intracellular Ca2+ mobilization, play a functional role in the induction of pathologic signal transduction leading to B-cells survival and that Ca2+ influx-dependent transcriptional activation and MEK1/ERK1/2 pathway signaling may be dependent upon simultaneous complimentary stimuli input from BCR, B1R and B2R and possibly other disease-associated stimuli. The inhibition of Kinin receptor function may be a potential target for combined therapy in CLL. Disclosures:No relevant conflicts of interest to declare.

Bradykinin and Kininogens in Bovine Milk

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.

High concentrations of free kinins and kinin system components in abdominal transudate of a patient with nephrotic syndrome

High levels of bradykinin (60–80 ng/ml) were found in abdominal transudate from a patient with nephrotic syndrome caused by chronic glomerulonephritis. The abdominal transudate contained neutral kininogenase and its precursor, identified with plasma kallikrein and prekallikrein, respectively, as well as both forms of kininogen, the low-molecular-weight form predominating. The abdominal transudate was characterized also by very low kininase activity and low levels of α 1-antitrypsin (0.46 g/1) and α 2-macroglobulin. Large amounts of very low density lipoproteins were present in the transudate. Despite the difference in total protein content between the abdominal transudate and the patient's serum (4.3 g/1 and 48 g/1, respectively) their protein fraction composition was similar. The data obtained suggest that bradykinin is important in maintenance of long-lasting blood vessel hyperpermeability, which, in turn, is a driving force in the pathogenesis of refractory nephrotic edema.