126 Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2+3 of the urokinase receptor

1997 ◽  
Vol 11 ◽  
pp. 36
Author(s):  
Robert W. Colman ◽  
Robin A. Pixley ◽  
Syeda Najamunnisa ◽  
Wuyi Yan ◽  
Jieyi Wang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Urokinase Receptor ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
A Site

CULTURED HUMAN ENDOTHELIAL CELLS BIND AND INTERNALIZE HIGH MOLECULAR WEIGHT KININOGEN

1987 ◽  
Author(s):  
Freek van Iwaarden ◽  
G Philip ◽  
de Groot ◽  
Bonno N Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Binding Sites ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Coagulation Pathway

The presence of High Molecular Weight kininogen (HMWK) was demonstrated in cultured human endothelial cells (EC) by immunofluorescence techniques. Using an enzyme linked immunosorbent assay a concentration of 58 ng HMWK/10 cells was determined. Immunoprecipitation studies performed with lysed metabolically labelled endothelial cells and mono-specific antisera directed against HMWK suggested that HMWK is not synthesized by the endothelial cells. Endothelial cells cultured in the presence of HMWK-depleted serum did not contain HMWK. This, suggests that endothelial cells can internalize HMWK. Using 125I-HMWK it was demonstrated that cultured endothelial cells bind HMWK in a time-dependent, specific and saturable.way. The cells were found to internalize 125I-HMWK, since I-HMWK was detected in solubilized endothelial cells after the cell bound 125I-HMWK had been eluted with dextran sulphate.The binding of I-HMWK required the presence of zinc ions. Optimal binding of 125I-HMWK was observed at 50 μM Zn++ . Calcium ions inhibited the Zn++ dependent binding of 125I-HMWK |25EC. In the presence of 3 mM CaCl2 the total binding of 125I-HMWK was significantly decreased, and a .concentration of 200 μM Zn++ was Required for the binding of 125I-HMWK to thecells. Higher,. Ca concentrations did not further decrease the binding of 125I-HMWK. Analysis of tl^e binding data by the ligand computer program indicated 3.2 x 10 binding sites per cell for HMWK with a Kd of 35 nM at 50 μM ZnCl2 and 1 mM CaCl2. Specify binding of HMWK did also occur at physiological plasma Zn++ concentrations. Half maximal binding was observed at HMWK concentrations of ± 105 nM at 10 μM ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 pM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 16 molecules of HMWK bound per cell and at 80 nM with 2.8 x 106 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.

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.

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.

Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor–dependent interactions

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 514-522 ◽  
Author(s):  
Triantafyllos Chavakis ◽  
Sandip M. Kanse ◽  
Florea Lupu ◽  
Hans-Peter Hammes ◽  
Werner Müller-Esterl ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Urokinase Receptor ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Terminal Portion ◽  
Single Chain ◽  
Amino Terminal

Abstract Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent vβ3 integrin–mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with vβ3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn2+-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN (“somatomedin B region”). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn2+-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys–rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions.

Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor–dependent interactions

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 514-522 ◽  
Author(s):  
Triantafyllos Chavakis ◽  
Sandip M. Kanse ◽  
Florea Lupu ◽  
Hans-Peter Hammes ◽  
Werner Müller-Esterl ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Urokinase Receptor ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Terminal Portion ◽  
Single Chain ◽  
Amino Terminal

Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent vβ3 integrin–mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with vβ3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn2+-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN (“somatomedin B region”). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn2+-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys–rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions.

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor

2013 ◽  
Vol 394 (3) ◽  
pp. 385-391 ◽  
Author(s):  
Thomas Kryza ◽  
Gilles Lalmanach ◽  
Marion Lavergne ◽  
Fabien Lecaille ◽  
Pascale Reverdiau ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Kinin Receptor ◽  
Angiogenic Effect ◽  
Lung Endothelial Cells ◽  
Carboxy Terminal

Abstract Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

scholarly journals Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (11) ◽  
pp. 2177-2184 ◽  
Author(s):  
SH Tannenbaum ◽  
HR Gralnick
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Inflammatory Mediators ◽  
Interleukin 1 ◽  
Gamma Interferon ◽  
Human Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

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