Histidine-Proline Rich Glycoprotein (HPRG) binds and transduces anti-angiogenic signals through cell surface tropomyosin on endothelial cells

2004 ◽  
Vol 92 (08) ◽  
pp. 403-412 ◽  
Author(s):  
Xiaojun Guan ◽  
Jose Juarez ◽  
Xiaoping Qi ◽  
Natalya Shipulina ◽  
David Shaw ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Surface ◽  
High Affinity ◽  
P Domain ◽  
Umbilical Vein Endothelial Cells

SummaryThe anti-angiogenic properties of the histidine-proline–rich (H/P) domain of HPRG have recently been described (Juarez JC, et al. Cancer Research 2002; 62: 5344-50). However, the binding site that mediates these properties is unknown. HPRG is evolutionarily, functionally and structurally related to cleaved high molecular weight kininogen (HKa), an anti-angiogenic polypeptide that stimulates apoptosis of proliferating endothelial cells through binding to cell-surface tropomyosin (Zhang J-C, et al. Proc Natl Acad Sci USA 2002; 99: 12224-9). In this study, we demonstrate that HPRG binds with high affinity to FGF-2–stimulated human umbilical vein endothelial cells (HUVEC) and immobilized tropomyosin in a Zn2+ or pH-dependent manner, and that this interaction is mediated by the H/P domain of HPRG. At least two binding sites for HPRG, tropomyosin and heparan sulfate proteoglycans (HSPs), were identified on the surface of FGF-2–activated endothelial cells. Translocation of tropomyosin to the surface of HUVEC occurred in response to FGF-2, and the anti-angiogenic activity of HPRG in a Matrigel plug model was partially inhibited by soluble tropomyosin. These results suggest that HPRG binds to endothelial cell surface tropomyosin which at least partially mediates the antiangiogenic effects of HPRG.

scholarly journals Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1306-1311 ◽  
Author(s):  
SR Reddigari ◽  
P Kuna ◽  
G Miragliotta ◽  
Y Shibayama ◽  
K Nishikawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Light Chains ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells

Abstract High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. Excision of the vasoactive peptide bradykinin by plasma kallikrein results in kinin-free HK that consists of a 65-Kd N-terminal heavy chain (HK-HC) linked to the C-terminal 45- Kd light chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor of SH-proteases and HK-LC contains the binding sites for coagulation cofactors prekallikrein and factor XI. HK has previously been shown to bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc(2+)-dependent manner by a single class of high-affinity binding sites. We have further characterized that interaction in order to determine the cell-binding regions of HK. Competition binding experiments have indicated that either HK-LC or HK-HC was able to inhibit the binding of labeled HK with a 50% inhibitory concentration (IC50) of 77 nmol/L and 89 nmol/L, respectively. Cleaved two-chain HK (HKa) had an IC50 of 73 nmol/L, whereas uncleaved HK had an IC50 of 335 nmol/L. Direct binding experiments have indicated that HUVEC bind both purified [125I]HK-HC and [125I]HK-LC in a zinc(2+)-dependent manner and that HK-LC did not displace bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit the binding of HK to HUVEC. Our results, therefore, indicate that (1) HK is capable of binding to endothelial cells via both heavy and light chain moieties, (2) HKa has a higher affinity to HUVEC, and (3) purified heavy and light chains are capable of directly binding to HUVEC. The data are consistent with the presence of a single high-affinity site for HK on endothelial cells within which are subsites that bind to heavy and light chains.

scholarly journals Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1306-1311
Author(s):  
SR Reddigari ◽  
P Kuna ◽  
G Miragliotta ◽  
Y Shibayama ◽  
K Nishikawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Light Chains ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. Excision of the vasoactive peptide bradykinin by plasma kallikrein results in kinin-free HK that consists of a 65-Kd N-terminal heavy chain (HK-HC) linked to the C-terminal 45- Kd light chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor of SH-proteases and HK-LC contains the binding sites for coagulation cofactors prekallikrein and factor XI. HK has previously been shown to bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc(2+)-dependent manner by a single class of high-affinity binding sites. We have further characterized that interaction in order to determine the cell-binding regions of HK. Competition binding experiments have indicated that either HK-LC or HK-HC was able to inhibit the binding of labeled HK with a 50% inhibitory concentration (IC50) of 77 nmol/L and 89 nmol/L, respectively. Cleaved two-chain HK (HKa) had an IC50 of 73 nmol/L, whereas uncleaved HK had an IC50 of 335 nmol/L. Direct binding experiments have indicated that HUVEC bind both purified [125I]HK-HC and [125I]HK-LC in a zinc(2+)-dependent manner and that HK-LC did not displace bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit the binding of HK to HUVEC. Our results, therefore, indicate that (1) HK is capable of binding to endothelial cells via both heavy and light chain moieties, (2) HKa has a higher affinity to HUVEC, and (3) purified heavy and light chains are capable of directly binding to HUVEC. The data are consistent with the presence of a single high-affinity site for HK on endothelial cells within which are subsites that bind to heavy and light chains.

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 Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4173-4182 ◽  
Author(s):  
LV Rao ◽  
AD Hoang ◽  
SI Rapaport
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Binding Sites ◽  
Lupus Anticoagulant ◽  
Umbilical Vein ◽  
Protein A ◽  
Thrombotic Risk ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

Abstract We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

scholarly journals Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4173-4182 ◽  
Author(s):  
LV Rao ◽  
AD Hoang ◽  
SI Rapaport
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Binding Sites ◽  
Lupus Anticoagulant ◽  
Umbilical Vein ◽  
Protein A ◽  
Thrombotic Risk ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

Verotoxin-1 Induces Tissue Factor Expression in Human Umbilical Vein Endothelial Cells through Activation of NF-κB/Rel and AP-1

2000 ◽  
Vol 84 (10) ◽  
pp. 712-721 ◽  
Author(s):  
Kimihiko Takada ◽  
Toshiyuki Higuchi ◽  
Junichi Sugiyama ◽  
Hidemi Ishii
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Nuclear Proteins ◽  
Binding Motif ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

SummaryThis study examined the effect of verotoxin-1 (VT-1), which is released from Escherichia coli O157:H7, on endothelial expression of tissue factor (TF), a cofactor required to initiate blood coagulation. In order to elucidate the molecular basis for development of hemolytic uremic syndrome (HUS) in patients infected with E. coli O157:H7, human umbilical vein endothelial cells (HUVECs) were exposed to purified VT-1. VT-1 increased both TF activity and TF mRNA in HUVECs without loss of cell viability in a time-and dose-dependent manner from 0.1 to 10 ng/ml VT-1. Nuclear proteins extracted from VT-1-stimulated HUVECs bound to the consensus NF-κB/Rel and AP-1 binding oligonucleotides in a dose-dependent manner within 2 h after the stimulation in electrophoretic mobility shift assays (EMSA). Nuclear proteins from VT-1-stimulated HUVECs formed two complexes with the NF-κB/Rel binding motif in the human TF promoter (TF-κB motif). The supershift assays, using antibodies for human p65, p50 or c-Rel, indicated that the lower complex was composed of p65/p50 and the higher complex was a p65 homo-or hetero-dimer with the Rel family, except c-Rel. The human TF promoter contains two AP-1 binding sites, the proximal and distal AP-1 binding sites. The supershift assays indicated that AP-1 containing mainly c-Jun and JunD, positively bound to the proximal AP-1 motif of TF (TF-AP-1). The distal TF-AP-1 motif did not show positive binding with nuclear proteins from VT-1-stimulated HUVECs. Pretreatment of HUVECs with curcumin, an inhibitor of NF-κB/Rel activation, synthesis of c-Jun mRNA and binding of activated AP-1 with AP-binding oligonucleotide, prevented the VT-1 induced increase in TF mRNA and activity in VT-1-stimulated HUVECs. Curcumin also inhibited NF-κB and AP-1 binding to TF-κB and proximal TF-AP-1 oligonucleotides, respectively, in a dose-dependent manner. The present work suggests that both the NF-κB/Rel and AP-1 activated in endothelial cells by stimulation with VT-1 binds to the TF-κB and proximal AP-1 binding sites, respectively, of the TF promoter.

scholarly journals Interleukin 4 or oncostatin M induces a prolonged increase in P-selectin mRNA and protein in human endothelial cells.

1996 ◽  
Vol 184 (1) ◽  
pp. 81-92 ◽  
Author(s):  
L Yao ◽  
J Pan ◽  
H Setiadi ◽  
K D Patel ◽  
R P McEver
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Messenger Rna ◽  
Umbilical Vein ◽  
Allergic Inflammation ◽  
Oncostatin M ◽  
Interleukin 4 ◽  
Necrosis Factor Alpha ◽  
Endothelial Cell Surface ◽  
Umbilical Vein Endothelial Cells

During acute inflammation, P-selectin is transiently mobilized from Weibel-Palade bodies to the surface of histamine-activated endothelial cells, where it mediates rolling adhesion of neutrophils under hydrodynamic flow. During chronic or allergic inflammation, sustained expression of P-selectin on the endothelial cell surface has been observed. We found that the cytokines interleukin 4 (IL-4) or oncostatin M (OSM) induced a five- to ninefold increase in P-selectin messenger RNA (mRNA) in human umbilical vein endothelial cells (HUVEC) that persisted as long as 72 h. IL-4 elevated P-selectin mRNA by increasing its transcription rate rather than by prolonging its already long half-life. Stimulation of P-selectin transcription by IL-4 or OSM required new protein synthesis and tyrosine phosphorylation of cellular proteins. Tumor necrosis factor alpha, IL-1 beta, lipopolysaccharide, or IL-3 did not increase P-selectin mRNA in HUVEC, and did not augment the IL-4-induced increase in P-selectin transcripts. IL-4 or OSM increased P-selectin protein on the cell surface as well as in Weibel-Palade bodies. Under flow conditions, neutrophils rolled on P-selectin expressed by IL-4-treated HUVEC, and even more neutrophils rolled on P-selectin after IL-4-treated HUVEC were stimulated with histamine. These data demonstrate that IL-4 or OSM stimulates endothelial cells to synthesize more P-selectin over prolonged periods. The increased expression of P-selectin may facilitate the emigration of leukocytes into sites of chronic or allergic inflammation.

Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

1989 ◽  
Vol 62 (02) ◽  
pp. 699-703 ◽  
Author(s):  
Rob J Aerts ◽  
Karin Gillis ◽  
Hans Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

1996 ◽  
Vol 75 (03) ◽  
pp. 497-502 ◽  
Author(s):  
Hadewijch L M Pekelharing ◽  
Henne A Kleinveld ◽  
Pieter F C.C.M Duif ◽  
Bonno N Bouma ◽  
Herman J M van Rijn
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Single Step ◽  
Plasminogen Activation ◽  
Structural Homology ◽  
Tissue Plasminogen ◽  
Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

Sign in / Sign up

Export Citation Format

Share Document