Quantitative Analysis of Angiogenesis Inhibition by Cleaved High Molecular Weight Kininogen Using VESGEN.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3907-3907
Author(s):  
Daniela Ribita ◽  
Benjamin Zlotoff ◽  
Terri L. McKay ◽  
Daniel J. Gedeon ◽  
Alan G. Hylton ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Transforming Growth Factor ◽  
Chorioallantoic Membrane ◽  
High Molecular Weight Kininogen ◽  
Vascular Tree ◽  
Angiogenesis Inhibition ◽  
Major Vessel ◽  
Vascular Trees

Abstract Angiogenesis requires specific molecular regulation of complex vascular branching morphology. The cleaved form of high molecular weight kininogen (HKa) has been shown to cause apoptosis of proliferating endothelial cells, and inhibit angiogenesis in vivo. However, critical morphological effects of HKa on the complex branching morphology of angiogenic vascular trees have not been defined. We used the quail chorioallantoic membrane (CAM) angiogenesis assay, in which angiogenesis agonists or antagonists cause uniform perturbation of angiogenesis throughout the entire vascular tree, to assess the effects of HKa on branching morphology. HKa (1.56 to 50 micrograms) was applied uniformly to the CAM, and its effect on angiogenesis determined after 24 hours of incubation. VESGEN vascular analysis software, currently under development at NASA Glenn Research Center, was used to quantify major vessel parameters in low-magnification images of vascular trees automatically extracted by VESGEN. Results derived from qualitative observation and initial quantification of two experiments demonstrates that HKa significantly inhibits vascular growth. Vessel length density (Lv) and branch point density (Br) in CAM specimens treated with 25 micrograms of HKa decreased by 23% and 31% respectively (24 ± 1 cm/cm2 and 441 ± 43 /cm2, compared to 31 ± 0 cm/cm2 and 677 ± 51 /cm2 in vehicle-treated controls; mean +/− S.E. for n = 3). The fractal dimension (Df) of skeletonized vascular images is a highly sensitive indicator of vessel density that ranges from 1.35 for strong angiogenesis inhibition, to 1.40 and 1.48 for controls and strong angiogenesis stimulation, respectively. Df decreased to 1.35 ± 0.01 in the HKa-treated specimens, relative to 1.39 ± 0.00 in controls. However, thickening of large vessels in response to HKa resulted in a virtually equivalent vessel area density (Av) of 0.167 ± 0.012 cm2/cm2, relative to 0.174 ± 0.015 cm2/cm2 in controls. Further studies of the site-specific effects of HKa on branching morphology within the vascular tree are in progress. These results demonstrate that the antiangiogenic effects of HKa result in a unique antiangiogenic ’fingerprint’ vascular morphology in comparison to other angiogenesis inhibitors that include transforming growth factor-β1 and angiostatin.

Inhibition of tumor angiogenesis in vivo by a monoclonal antibody targeted to domain 5 of high molecular weight kininogen

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2065-2072 ◽  
Author(s):  
James S. Song ◽  
Irma M. Sainz ◽  
Stephen C. Cosenza ◽  
Irma Isordia-Salas ◽  
Abdel Bior ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Cell Growth ◽  
Tumor Cell ◽  
High Molecular Weight ◽  
Control Group ◽  
Dependent Manner ◽  
Tumor Cell Growth ◽  
High Molecular Weight Kininogen

Abstract We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

scholarly journals High Molecular Weight Kininogen Regulates Prekallikrein Assembly and Activation on Endothelial Cells: A Novel Mechanism for Contact Activation

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Guacyara Motta ◽  
Rasmus Rojkjaer ◽  
Ahmed A.K. Hasan ◽  
Douglas B. Cines ◽  
Alvin H. Schmaier
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Factor Xii ◽  
Plasminogen Activation ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Human Endothelial Cells ◽  
Exogenous Factor

The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 ± 11 nmol/L,Bmax of 1.7 ± 0.5 × 107 sites per cell [mean ± SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L,Vmax = 12 ± 3 pmol/L/min in the absencevKm = 20 nmol/L,Vmax = 9.2 ± 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

DETECTION AND QUANTITATION OF CLEAVED AND UNCLEAVED HIGH MOLECULAR WEIGHT KININOGEN IN PLASMA BY LIGAND BLOTTING WITH RADIOLABELED PLASMA PREKALLIKREIN OR FACTOR XI

1987 ◽  
Author(s):  
B Lämmle ◽  
B L Zuraw ◽  
M J Heeb ◽  
H P Schwarz ◽  
J G Curd ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Factor Xi ◽  
Sds Page ◽  
Normal Human

A method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight kininogen (HMWK) in plasma has been developed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electroblotting of the electropherogram to nitrocellulose membranes and detection of the inmobilized HMWK with its physiologic ligands, plasma prekallikrein or factor XI. Using 1251-prekallikrein or 125I-F.XI overlay nitrocellulose bound HMWK can be visualized by autoradiography.Using unreduced SDS-PAGE cleaved two-chain HMWK (Mr 107,000 and 95,000) is electrophoretically separated from uncleaved single chain HMWK (Mr 150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMWK permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMWK is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards.This technique is highly specific and sensitive to ˜ 50 ng of either cleaved or uncleaved HMWK. Varying concentrations of cleaved HMWK were found in plasmas from patients suffering from various systemic inflanmatory conditions. Higher levels of in vivo cleaved HMWK were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency.This technique may be useful for the assessment of the degree of in vitro or in vivo activation of the contact system of plasma.

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

2008 ◽  
Vol 294 (3) ◽  
pp. L505-L515 ◽  
Author(s):  
Lan G. Coffman ◽  
Julie C. Brown ◽  
David A. Johnson ◽  
Narayanan Parthasarathy ◽  
Ralph B. D'Agostino ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Solid Phase ◽  
Direct Interaction ◽  
Human Monocyte ◽  
Biologically Active ◽  
High Molecular Weight Kininogen ◽  
Binding Studies ◽  
Iron Storage

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45–75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kdof 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

Recombinant α1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep

2002 ◽  
Vol 93 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
Mario Scuri ◽  
Yelena Botvinnikova ◽  
Isabel T. Lauredo ◽  
William M. Abraham
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteinase Inhibitor ◽  
Serine Protease Inhibitor ◽  
Tissue Kallikrein ◽  
Oxygen Species ◽  
High Molecular Weight Kininogen ◽  
Airway Responses ◽  
Stimulation Of

α1-Proteinase inhibitor (α1-PI) is a natural serine protease inhibitor. Although mainly thought to protect the airways from neutrophil elastase, α1-PI may also regulate the development of airway hyperresponsiveness (AHR), as indicated by our previous findings of an inverse relationship between lung α1-PI activity and the severity of antigen-induced AHR. Because allergic stimulation of the airways causes release of elastase, tissue kallikrein, and reactive oxygen species (ROS), all of which can reduce α1-PI activity and contribute to AHR, we hypothesized that administration of exogenous α1-PI should protect against pathophysiological airway responses caused by these agents. In untreated allergic sheep, airway challenge with elastase, xanthine/xanthine oxidase (which generates ROS), high-molecular-weight kininogen, the substrate for tissue kallikrein, and antigen resulted in bronchoconstriction. ROS and antigen also induced AHR to inhaled carbachol. Treatment with 10 mg of recombinant α1-PI (rα1-PI) blocked the bronchoconstriction caused by elastase, high-molecular-weight kininogen, and ROS, and the AHR induced by ROS and antigen. One milligram of rα1-PI was ineffective. These are the first in vivo data demonstrating the effects of rα1-PI. Our results are consistent with and extend findings obtained with human plasma-derived α1-PI and suggest that α1-PI may be important in the regulation of airway responsiveness.

scholarly journals High Molecular Weight Kininogen Regulates Prekallikrein Assembly and Activation on Endothelial Cells: A Novel Mechanism for Contact Activation

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Guacyara Motta ◽  
Rasmus Rojkjaer ◽  
Ahmed A.K. Hasan ◽  
Douglas B. Cines ◽  
Alvin H. Schmaier
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Factor Xii ◽  
Plasminogen Activation ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Human Endothelial Cells ◽  
Exogenous Factor

Abstract The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 ± 11 nmol/L,Bmax of 1.7 ± 0.5 × 107 sites per cell [mean ± SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L,Vmax = 12 ± 3 pmol/L/min in the absencevKm = 20 nmol/L,Vmax = 9.2 ± 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

Fibrinolysis and the Contact System: A Role for Factor XI in the Down-Regulation of Fibrinolysis

1999 ◽  
Vol 82 (08) ◽  
pp. 243-250 ◽  
Author(s):  
Joost Meijers ◽  
Bonno Bouma
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Fibrinolytic System ◽  
Contact System ◽  
Factor Xii ◽  
Charged Surface ◽  
High Molecular Weight Kininogen ◽  
Factor Xi ◽  
Factor Xiia

IntroductionExposure of blood to negatively-charged surfaces, such as collagen, kaolin, or glass, results in the activation of the contact system of the intrinsic pathway of coagulation. Prekallikrein, factor XII, high molecular weight kininogen, and factor XI are the proteins involved in this contact reaction. The assembly of these components on a negatively-charged surface leads to the activation of factor XI, thereby propagating the intrinsic coagulation pathway. Simultaneously, several other reactions occur, such as the activation of factor VII and the initiation of the fibrinolytic system, kinin-forming pathway, and renin-angiotensin pathway.The first step in the contact phase is to bind factor XII to the negatively-charged surface, making it highly susceptible for proteolysis by kallikrein.1-3 Activated factor XII (α-factor XIIa) is formed in a process that may involve autoactivation.4-7 Prekallikrein is bound to high molecular weight kininogen in plasma. High molecular weight kininogen associates with a negatively-charged surface, thereby localizing prekallikrein to the surface. Limited proteolysis by α-factor XIIa converts prekallikrein to kallikrein. Kallikrein can dissociate from the surface and act on surface-bound factor XII at distant sites, thereby propagating the reciprocal cycle.7 Factor XI circulates plasma in a complex with high molcular weight kininogen. High molecular weight kininogen links factor XI to a negatively charged surface where it is activated by surface bound:α-factor XIIa. Although the in vivo, activating, negatively-charged surface is unknown, assembly and activation of the contact system on biological membranes of endothelial cells, platelets, neutrophils, and monocytes can take place, suggesting that these surfaces are the actual activating surfaces in vivo.8 The physiological significance of the contact system in blood coagulation remains unclear, however, because a deficiency of factor XII, prekallikrein, and high molecular weight kininogen does not result in a bleeding disorder. In contrast, patients deficient in factor XI, most common among Ashkenazi Jews, do suffer from variable bleeding abnormalities, especially from tissues with high local fibrinolytic activity (e.g., urinary tract, nose, oral cavity, tonsils).9,10 This suggested that there was an alternative route for the activation of factor XI, and recently, such a route was described.11,12 Thrombin was found to activate factor XI even in the absence of a negatively-charged surface,11-15 and factor XI was shown to play a role in the downregulation of fibrinolysis.16 In this article, the role of the contact system, with an emphasis on factor XI in the regulation of the fibrinolytic system, will be described.

Zinc-dependent contact system activation induces vascular leakage and hypotension in rodents

2013 ◽  
Vol 394 (9) ◽  
pp. 1195-1204 ◽  
Author(s):  
Jenny Björkqvist ◽  
Bernd Lecher ◽  
Coen Maas ◽  
Thomas Renné
Keyword(s):  
Blood Pressure ◽  
Molecular Weight ◽  
High Molecular Weight ◽  
Dextran Sulfate ◽  
Vascular Leakage ◽  
Laser Scanning Microscopy ◽  
Factor Xii ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen

Abstract Contact to polyanions induces autoactivation of the serine protease factor XII that triggers the kallikrei-kinin system. Recent studies indicate that polysaccharide-induced autoactivation of factor XII has a role in allergy-related vascular leakage, and angioedema. Here, we characterize in vivo effects of the synthetic polysaccharide dextran sulfate in human plasma and in rodent models. Minute amounts of high-molecular-weight dextran sulfate-initiated factor XII-autoactivation and triggered formation of the inflammatory mediator bradykinin via plasma kallikrein-mediated cleavage of high-molecular-weight kininogen. High-molecular-weight kininogen fragments, containing the HKH20 sequence in domain D5H, blocked dextran sulfate-initiated bradykinin-generation by depleting plasma Zn2+ ions. Topical application of high molecular weight dextran sulfate increased leakage in murine skin microvessels, in a bradykinin-dependent manner. Intravital laser scanning microscopy showed a greater than two-fold elevated and accelerated fluid extravasation in C1 esterase inhibitor deficient mice that lack the major inhibitor of factor XII, compared to wild-type controls. Intra-arterial infusion of dextran sulfate induced a rapid transient drop in arterial blood pressure in rats and preinjection of kinin B2 receptor antagonists or HKH20 peptide blunted dextran sulfate-triggered hypotensive reactions. The data characterize dextran sulfate as a potent in vivo activator of factor XII with implications for bradykinin-mediated vascular permeability and blood pressure control.

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