Antithrombin III, the Major Modulator of Intravascular Coagulation, Is Synthesized by Human Endothelial Cells

1981 ◽  
Vol 46 (02) ◽  
pp. 504-506 ◽  
Author(s):  
T K Chan ◽  
Vivian Chan
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Antithrombin Iii ◽  
Heparin Binding ◽  
Human Endothelial Cells ◽  
Intravascular Coagulation ◽  
Cells In Culture ◽  
Crossed Immunoelectrophoresis ◽  
At Iii

SummaryHuman endothelial cells in culture is shown to synthesize antithrombin III (At-III). The endothelial cell At-III(EC-At-III) consists of a small fraction similar to plasma At-III and a larger fraction with decreased heparin-binding as tested by crossed immunoelectrophoresis. However, both the anti-Xa and thrombin-neutralizing activities of the EC-At-III were rapid and active even in the absence of added heparin. It is concluded that the major portion was probably bound to endogenous heparin-like substance, thus accounting for its decreased exogenous heparin binding. The presence of At-III and other antithrombotic factors in the vascular endothelium offer protection against thrombosis and possibly atherosclerosis.

Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 303-311
Author(s):  
Tone Børsum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Rabbit Antiserum ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoelectrophoretic Analysis ◽  
Glycoprotein Complex ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180 ◽  
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

Abstract We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

Formation of Activated Protein C and Inactivation of Cell-Bound Thrombin by Antithrombin III at the Surface of Cultured Vascular Endothelial Cells - A Comparative Study of Two Anticoagulant Mechanisms

1987 ◽  
Vol 57 (01) ◽  
pp. 087-091 ◽  
Author(s):  
Ulrich Delvos ◽  
Petra Meusel ◽  
Klaus T Preissner ◽  
Gert Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Protein C ◽  
Antithrombin Iii ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
At Iii ◽  
Inhibition Of Thrombin ◽  
Aortic Endothelial

SummaryIntact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (AT III) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by AT III and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 μM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 μM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII. Preincubation of BAEC with blocking antibodies against ATIII excluded a possible influence of BAEC-bound ATIII on the capacity of cell-bound thrombin to activate PC. The results suggest that an enhancing mechanism for the inactivation of thrombin by ATIII was not operative at the surfaces of cultured bovine aortic endothelial cells, but instead, appreciable amounts of activated PC become generated in the presence of an excess of ATIII.

Studies of Heparin Binding to Antithrombin III by Crossed Immunoelectrophoresis

1979 ◽  
Vol 42 (05) ◽  
pp. 1434-1445 ◽  
Author(s):  
Trevor W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Heparin Binding ◽  
Binding Ability ◽  
Crossed Immunoelectrophoresis ◽  
Binding Material ◽  
At Iii ◽  
Heparin Fractions

SummaryThe technique of crossed immunoelectrophoresis has been used to study the binding to purified antithrombin III (At III) of heparin and other mucopolysaccharides. The technique was unable to detect differences among samples of whole heparin from various manufacturers, but proved useful in studying the binding of heparin fractions; at the same molarities, low and high molecular weight heparin fractions displayed equal binding ability to At III. A semi-synthetic heparin analogue showed no evidence of binding to At III, but a sample of heparan sulphate did interact with At III at a concentration 3 times that of heparin. Samples of purified At III from four different manufacturers all displayed heterogeneity with respect to heparin binding. A proportion of the total At III did not bind to heparin and, in one sample, this non-binding material constituted about 40% of the total. An antiserum made against purified At III contained antibodies with different cross-reactivities against heparin bound and non-heparin bound At III.

Antithrombin III inhibits nuclear factor κB activation in human monocytes and vascular endothelial cells

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4015-4020 ◽  
Author(s):  
Christian Oelschläger ◽  
Jürgen Römisch ◽  
Anne Staubitz ◽  
Harald Stauss ◽  
Boris Leithäuser ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antithrombin Iii ◽  
Binding Capacity ◽  
Nuclear Factor Κb ◽  
Early Gene ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Human Monocytes ◽  
Heparin Binding ◽  
At Iii

The serpin antithrombin III (AT III), the most important natural inhibitor of thrombin activity, has been shown to exert marked anti-inflammatory properties and proven to be efficacious in experimental models of sepsis, septic shock, and disseminated intravascular coagulation. Moreover, clinical observations suggest a possible therapeutic role for AT III in septic disorders. The molecular mechanism, however, by which AT III attenuates inflammatory events is not yet entirely understood. We show here that AT III potently blocks the activation of nuclear factor κB (NF-κB), a transcription factor involved in immediate early gene activation during inflammation. AT III inhibited agonist-induced DNA binding of NF-κB in cultured human monocytes and endothelial cells in a dose-dependent manner, suggesting that AT III interferes with signal transduction leading to NF-κB activation. This idea was supported by demonstrating that AT III prevents the phosphorylation and proteolytic degradation of the inhibitor protein IκBα. In parallel to reducing NF-κB activity, AT III inhibited the expression of interleukin-6, tumor necrosis factor-α, and tissue factor, genes known to be under the control of NF-κB. The observation that chemically modified AT III that lacks heparin-binding capacity had no effect on NF-κB activation supports the current understanding that the inhibitory potency of AT III depends on the interaction of AT III with heparinlike cell surface glycosaminoglycans. This hypothesis was underscored by the finding that the AT III β-isoform, known to have higher affinity for glycosaminoglycans, is more effective in preventing NF-κB transactivation than α–AT III. These data indicate that AT III can alter inflammatory processes via inhibition of NF-κB activation.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

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