High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β through the Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Mononuclear Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3800-3800
Author(s):  
Mohammad M. Khan ◽  
Harlan N. Bradford ◽  
Irma Isordia-Salas ◽  
Ricardo Espinola ◽  
Robert W. Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Blood ◽  
Mononuclear Cells ◽  
Inflammatory Diseases ◽  
Interleukin 1Β ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Blood Mononuclear Cells ◽  
Domain 3

Abstract High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.

High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin-1β through the NFκ B, JNK and p38 MAP Kinase Signaling Pathways in Monocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1644-1644
Author(s):  
Mohammad M. Khan ◽  
Harland N. Bradford ◽  
Irma Isordia-Salas ◽  
Yuchuan Liu ◽  
Yi Wu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Signaling Pathways ◽  
High Molecular Weight ◽  
Intracellular Signaling ◽  
Inflammatory Diseases ◽  
Specific Inhibitor ◽  
P38 Map Kinase ◽  
High Molecular Weight Kininogen ◽  
Recombinant Peptide ◽  
Domain 3

Abstract Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.

Mechanisms of Human Blood VII Activation in Plasma During Contact Activation, Clotting and Exposure to Cold

1979 ◽  
Author(s):  
U. Seligsohn ◽  
B. østerud ◽  
S.F. Brown ◽  
J.H. Griffin ◽  
S.I. Rapaport
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
High Molecular Weight ◽  
Human Blood ◽  
Factor Vii ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Partial Activation ◽  
No Activation ◽  
Fold Activation

Factor VII(VII) is activated, giving shorter clotting times with tissue factor, when plasma is exposed to kaolin, is clotted or exposed to cold. The mechanisms involved were studied. Incubation of plasma with kaolin resulted in: No activation in XII deficiency plasma (dp), partial activation (2.5 fold) in Prekallikrein (PK) dp and High Molecular Weight Kininogen (HMWK) dp, and 4.5-9 fold activation in normal or other dp. Clotting plasma by recalcification resulted in: No activation with XII dp, HMWK dp, XI dp and IX dp, and 4-5 fold activation with VIII dp, X dp and V dp. The mechanism of cold promoted activation of VII in plasma was studied by adding purified 125-XII or 125I-IX to plasma before storage at 4° and observing the extent of their proteolysis (a measure of activation) from their radioactivity profiles on reduced Polyacrylamide gels following electrophoresis in the presence of SDS. Significantly greater 125I-IX and 125I-XII proteolysis was observed in plasma from 4 subjects whose VII activated in the cold, than in plasma from 5 subjects whose VII was not activated in the cold. Addition of anti-IX antiserum inhibited 50% of the observed cold activation of VII. Thus, with kaolin XIIa was the principal activator of VII; after clotting IXa was the principal activator and in cold activation both XIIa and IXa played roles.

Commercial Immunodepleted Deficient Plasmas Contain Cleaved High Molecular Weight Kininogen (HK)

1988 ◽  
Vol 60 (03) ◽  
pp. 447-452
Author(s):  
C Mannhalter ◽  
H Lang
Keyword(s):  
Molecular Weight ◽  
Quality Control ◽  
Comparative Analysis ◽  
High Molecular Weight ◽  
Control Method ◽  
Immunoblot Analysis ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Quality Control Method ◽  
Chain Form

SummaryComparative analysis of high molecular weight kininogen (HK) in various commercial congenital and immunodepleted deficiency plasmas was performed by immunoblotting of HK. It was found, that some artificially depleted deficiency plasmas contained proteolytically cleaved, kinin-free kininogen. In contrast, in all congenitally deficient plasmas, HK was present in the intact, single chain form. Thus, cleavage of kininogen could have been triggered by or during the immunodepletion procedure. It was seen, that the degree of proteolytic cleavage and degradation of HK in depleted plasmas differed among various manufacturers. E.g. depleted products of one company contained only trace amounts of cleaved HK, in contrast to products of another one, in which HK was completely degraded. The immunoblot analysis of HK reflects the occurrence of proteolytic events during the production of artificially deficient plasmas and can therefore serve as a quality control method.

scholarly journals Evidence for the Modulation of the Immune Response in Peripheral Blood Mononuclear Cells after Stimulation with a High Molecular Weight β-glucan Isolated fromLactobacillus fermentumLf2

2018 ◽  
Author(s):  
Ana Vitlic ◽  
Sohaib Sadiq ◽  
Hafiz I. Ahmed ◽  
Elisa C. Ale ◽  
Ana G. Binetti ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Immune Response ◽  
High Molecular Weight ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Immunomodulatory Activity ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Blood Mononuclear Cells

ABSTRACTLactobacillus fermentumLf 2 produces large amounts of exopolysaccharides under optimized conditions (∼2 g/L, EPS) which have been shown to possess immunomodulatory activity. In this study, the crude EPS was fractionated to give a high molecular weight (HMw) homoglycan and a mixture of medium molecular weight heteroglycans. The HMw EPS was isolated and identified as a β-glucan.Peripheral blood mononuclear cells (PBMC) were pre-treated with purified polysaccharide to determine if the HMw β-glucan is responsible for the immunomodulatory activity. Cells were also stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA) and their effects, both with and without β-glucan pre-treatment, compared.Exposure of the cells to β-glucan increased their metabolic activity and whilst a small but statistically significant drop in CD14 expression was observed at Day 1, the levels were significantly elevated at Day 2. High levels of CD14 expression were observed in cells initially exposed to the β-glucan and subsequently stimulated with either LPS or PHA. In contrast, reduced levels of TLR-2 expression were observed for cells initially exposed to the β-glucan and subsequently stimulated with LPS.TNF-α levels were elevated in β-glucan treated cells (Day1) with the levels dropping back once the β-glucan had been removed (Day 2). The stimulants LPS and PHA both induced significant rises in TNF-α levels, however, this induction was completely (LPS) or partially blocked (PHA) in β-glucan pre-treated cells.The results indicate a role for the bacterial β-glucan in modulating the immune response following exposure to agonists such as bacterial LPS.

scholarly journals Characterization of the procoagulant chain derived from human high molecular weight kininogen (Fitzgerald factor) by human tissue kallikrein

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 457-463 ◽  
Author(s):  
M Maier ◽  
KF Austen ◽  
J Spragg
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Broad Band ◽  
Limited Proteolysis ◽  
Procoagulant Activity ◽  
Plasma Kallikrein ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Amino Terminal ◽  
A Chain

Abstract Human high molecular weight kininogen (HMWK), a single-chain protein with mol wt 120,000, is cleaved by human urinary kallikrein (HUK) to release kinin from within a disulfide loop and form a two-chain protein that retains all the procoagulant activity of the native molecule. Cleavage of HMWK by HUK is associated with a reduction in size to mol wt 115,000, as assessed by SDS-PAGE of unreduced protein, whereas the two chains of the reduced protein present together as a single broad band with mol wt 64,000. The 64,000 chain with procoagulant activity was chromatographically separated from the nonfunctional chain of similar size. The homogeneous procoagulant chain had an amino acid composition similar to that of smaller procoagulant (“light”) chains isolated by others upon cleavage of HMWK with plasma kallikrein and elicited an antiserum that was monospecific by Ouchterlony analysis and inhibited the procoagulant function of HMWK. Thus, the limited proteolysis of HMWK by HUK has permitted, for the first time, the isolation of a stable procoagulant chain that is equal in size to the nonfunctional chain. The common terminology of “heavy” and “light” chain for kinin-free kininogen obtained with plasma kallikrein reflects the continued degradation of the procoagulant carboxyterminal chain and is not appropriate for the initial two-chain product formed when kinin is released from HMWK. It is proposed that the initial cleavage products of HMWK be designated the A-chain, the B-fragment, and the C- chain, representing the amino-terminal chain, the released vasoactive peptide containing the bradykinin sequence, and the carboxy-terminal procoagulant chain, respectively. Thus, intact HMWK would contain, in sequence, A, B, and C regions.

scholarly journals Inhibition of cell adhesion by high molecular weight kininogen.

1992 ◽  
Vol 116 (2) ◽  
pp. 465-476 ◽  
Author(s):  
S Asakura ◽  
R W Hurley ◽  
K Skorstengaard ◽  
I Ohkubo ◽  
D F Mosher
Keyword(s):  
Molecular Weight ◽  
Cell Adhesion ◽  
High Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Adhesive Protein ◽  
Rich Domain ◽  
Growth Promoting ◽  
Heparin Affinity ◽  
Mononuclear Blood Cells

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

scholarly journals Upregulation of tissue factor in monocytes by cleaved high molecular weight kininogen is dependent on TNF-α and IL-1β

2010 ◽  
Vol 298 (2) ◽  
pp. H652-H658 ◽  
Author(s):  
Mohammad M. Khan ◽  
Yuchuan Liu ◽  
Munir E. Khan ◽  
Megan L. Gilman ◽  
Sabina T. Khan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
High Molecular Weight ◽  
Inflammatory Diseases ◽  
Human Monocytes ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Tnf Α ◽  
Chemical Inhibitors ◽  
Inflammatory Bowel

Inflammatory bowel disease and arthritis are associated with contact activation that results in cleavage of kininogen to form high molecular weight kininogen (HKa) and bradykinin. We have previously demonstrated that HKa can stimulate inflammatory cytokine and chemokine secretion from human monocytes. We now show that HKa can upregulate tissue factor antigen and procoagulant activity on human monocytes as a function of time (1–4 h) and HKa concentration (75–900 nM). The amino acid sequence responsible to block HKa effects is G440–H455. The HKa receptor macrophage-1 (Mac-1; CD11b18) is the binding site as shown by inhibition by a monoclonal antibody to CD11b/18. Chemical inhibitors of JNK, ERK, and p38 signaling pathways block cell signaling, as does an inhibitor to the transcription factor NF-κB. A combination of monoclonal antibodies to TNF-α and IL-1β but neither alone inhibited the HKa induction of tissue factor. These results suggest that HKa mimics LPS by triggering a paracrine pathway in monocytes that depends on TNF-α and IL-1β. Antibodies to kininogen or peptidomimetics might be a useful and safe therapy in inflammatory diseases or sepsis involving cytokines.

scholarly journals Thrombin-induced Platelet Aggregation Is Inhibited by the Heptapeptide LeuAla of Domain 3 in the Heavy Chain of High Molecular Weight Kininogen

1996 ◽  
Vol 271 (19) ◽  
pp. 11228-11235 ◽  
Author(s):  
Satya P. Kunapuli ◽  
Harlan N. Bradford ◽  
Bradford A. Jameson ◽  
Raul A. DeLa Cadena ◽  
Leonard Rick ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
High Molecular Weight ◽  
Heavy Chain ◽  
High Molecular Weight Kininogen ◽  
Domain 3

Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3305-3305
Author(s):  
Venkaiah Betapudi ◽  
Keith R. McCrae
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Negative Regulator ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Single Chain

Abstract Abstract 3305 Background and objective: High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis. Results: We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid. Conclusion: These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis. Disclosures: No relevant conflicts of interest to declare.

Functional Characterization of a Variant Prekallikrein (PK Zürich)

1993 ◽  
Vol 70 (03) ◽  
pp. 427-432 ◽  
Author(s):  
W A Wuillemin ◽  
M Furlan ◽  
A von Felten ◽  
B Lämmle
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
High Molecular Weight ◽  
Dextran Sulfate ◽  
Functional Characterization ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Normal Plasma ◽  
Sulfate Activation

SummaryThe plasma of a 68-year-old man with cross reacting material (CRM)-positive prekallikrein (PK) deficiency was studied. PK clotting activity was <0.01 U/ml, and PK antigen was 0.1 U/ml. No circulating anticoagulant against PK was detectable. The abnormal PK molecule, denoted as prekallikrein Zürich, was partially characterized by immunological and functional studies on the propositus’ plasma. Immunobiotting analysis showed the abnormal PK being a single chain molecule of the same M r (80 kDa) as normal PK. Dextran sulfate activation of the propositus’ plasma did not lead to proteolytic cleavage of the variant PK molecule, in contrast to dextran sulfate activation of a mixture of 1 volume normal plasma and 9 volumes CRM-negative PK deficient plasma. Agarose gel electrophoresis followed by immunoblotting demonstrated that PK Zürich was complexed with high molecular weight kininogen similarly to PK in normal plasma. Incubation of the propositus’ plasma with purified β-FXIIa resulted in impaired cleavage of PK Zürich when compared with PK hydrolysis in a mixture of 10% normal plasma and 90% CRM-negative PK deficient plasma. Moreover, proteolytically cleaved PK Zürich showed no enzymatic activity against factor XII and high molecular weight kininogen.These studies show that the functional defect of prekallikrein Zürich is due to an impaired cleavage by activated factor XII and probably the lack of enzymatic activity of the cleaved variant molecule.

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