High affinity binding sites for proinsulin on human umbilical vein endothelial cells (HUVEC)

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation

Blood ◽

10.1182/blood.v75.12.2335.2335 ◽

1990 ◽

Vol 75 (12) ◽

pp. 2335-2342 ◽

Cited By ~ 35

Author(s):

AP Kowalczyk ◽

RH Tulloh ◽

PJ McKeown-Longo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Binding Sites ◽

Affinity Binding ◽

Apical Surface ◽

Matrix Assembly ◽

High Affinity Binding ◽

High Affinity ◽

Fibronectin Matrix ◽

Subendothelial Matrix

Abstract Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

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Human Umbilical Vein Endothelial Cells Express High Affinity Neurotensin Receptors Coupled to Intracellular Calcium Release

Journal of Biological Chemistry ◽

10.1074/jbc.270.7.3409 ◽

1995 ◽

Vol 270 (7) ◽

pp. 3409-3413 ◽

Cited By ~ 27

Author(s):

Paul Schaeffer ◽

Marie-Claude Laplace ◽

Pierre Savi ◽

Anne-Marie Pflieger ◽

Danielle Gully ◽

...

Keyword(s):

Endothelial Cells ◽

Intracellular Calcium ◽

Calcium Release ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

High Affinity ◽

Neurotensin Receptors ◽

Intracellular Calcium Release ◽

Umbilical Vein Endothelial Cells

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Mechanism of thrombin binding to endothelial cells

Blood ◽

10.1182/blood.v61.2.368.368 ◽

1983 ◽

Vol 61 (2) ◽

pp. 368-372 ◽

Cited By ~ 36

Author(s):

PT Bauer ◽

R Machovich ◽

P Aranyi ◽

KG Buki ◽

E Csonka ◽

...

Keyword(s):

Endothelial Cells ◽

Binding Sites ◽

Antithrombin Iii ◽

Affinity Binding ◽

Heparin Binding ◽

High Affinity Binding ◽

Aortic Endothelial Cells ◽

High Affinity ◽

Lysine Residues ◽

Two Populations

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

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

Blood ◽

10.1182/blood.v81.5.1306.1306 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1306-1311 ◽

Cited By ~ 46

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.

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

Blood ◽

10.1182/blood.v81.5.1306.bloodjournal8151306 ◽

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.

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Mechanism of thrombin binding to endothelial cells

Blood ◽

10.1182/blood.v61.2.368.bloodjournal612368 ◽

1983 ◽

Vol 61 (2) ◽

pp. 368-372

Author(s):

PT Bauer ◽

R Machovich ◽

P Aranyi ◽

KG Buki ◽

E Csonka ◽

...

Keyword(s):

Endothelial Cells ◽

Binding Sites ◽

Antithrombin Iii ◽

Affinity Binding ◽

Heparin Binding ◽

High Affinity Binding ◽

Aortic Endothelial Cells ◽

High Affinity ◽

Lysine Residues ◽

Two Populations

The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

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Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation

Blood ◽

10.1182/blood.v75.12.2335.bloodjournal75122335 ◽

1990 ◽

Vol 75 (12) ◽

pp. 2335-2342

Author(s):

AP Kowalczyk ◽

RH Tulloh ◽

PJ McKeown-Longo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Binding Sites ◽

Affinity Binding ◽

Apical Surface ◽

Matrix Assembly ◽

High Affinity Binding ◽

High Affinity ◽

Fibronectin Matrix ◽

Subendothelial Matrix

Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

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

Thrombosis and Haemostasis ◽

10.1160/th04-02-0073 ◽

2004 ◽

Vol 92 (08) ◽

pp. 403-412 ◽

Cited By ~ 34

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.

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