The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

1992 ◽  
Vol 67 (06) ◽  
pp. 679-685 ◽  
Author(s):  
Peter Wildgoose ◽  
Tony Jørgensen ◽  
Yutaka Komiyama ◽  
Tomohiro Nakagaki ◽  
Anders Pedersen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Tissue Factor ◽  
Calcium Binding ◽  
Factor Vii ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Fab Fragment ◽  
Calcium Dependent ◽  
Gla Domain

SummaryWhether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.

Complete Inhibition of Endotoxin-Induced Coagulation Activation in Chimpanzees with a Monoclonal Fab Fragment against Factor VII/VIIa

1995 ◽  
Vol 73 (02) ◽  
pp. 223-230 ◽  
Author(s):  
Bart J Biemond ◽  
Marcel Levi ◽  
Hugo ten Cate ◽  
Howard R Soule ◽  
Lori D Morris ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tissue Factor ◽  
Bolus Injection ◽  
Therapeutic Option ◽  
Factor Vii ◽  
Rapid Decline ◽  
Fab Fragment ◽  
Gram Negative ◽  
Fab Fragments

SummaryGram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin- induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees.Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 μg/kg (n = 2) or 100 μfig/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1+2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels. Endotoxin administration resulted in activation of the fibrinolytic system as reflected by a rapid increase in plasma plasmin- α2-antiplasmin complexes. Administration of 12D10 was without effect on the endotoxin-induced fibrinolytic activation.In conclusion, this study confirmed the importance of the TF: VII complex in the initial, endotoxin-induced activation of coagulation which was completely blocked by neutralizing all free and tissue factor-complexed factor VII/VIIa by a specific monoclonal Fab fragment. Activation of fibrinolysis was not influenced. Therefore, neutralization of factor VII/VIIa might be a promising therapeutic option in preventing endotoxin-induced microthrombosis during Gram-negative sepsis.

Purification and Characterization of Factor VII Inhibitor Found in a Patient with Life Threatening Bleeding

2000 ◽  
Vol 83 (01) ◽  
pp. 60-64 ◽  
Author(s):  
Seiji Miyamoto ◽  
Atsushi Iwasa ◽  
Masao Ishii ◽  
Kenji Okajima ◽  
Yu-ichi Kamikubo
Keyword(s):  
Light Chain ◽  
Calcium Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Vii ◽  
Immunoblotting Analysis ◽  
Calcium Dependent ◽  
Sds Page ◽  
Carboxyglutamic Acid ◽  
Life Threatening ◽  
Gla Domain

SummaryWe recently observed a patient with acquired inhibitor-induced F.VII deficiency whose plasma level of F.VII was < 1.0%. However, the biochemical nature of the inhibitor has not yet been clarified. In the present study, we purified the F.VII inhibitor from the patient’s plasma by using activated F.VII (F.VIIa)-conjugated gel and characterized the inhibitor. The results showed that the inhibitor comprised two kinds of antibodies: one was eluted with EDTA (antibody 1) and the other with glycine-HCl buffer (pH 2.3) (antibody 2) from the F.VIIa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of these inhibitors demonstrated that both antibodies had features of immunoglobulin G1 (IgG1) with κ and λ-light chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity in the presence of calcium ion, while antibody 2 bound to the F.VIIa very weakly and the binding was independent of calcium ion. Immunoblotting analysis demonstrated that antibody 1 bound to the light chain of F.VIIa after reduction with 2-mercaptoethanol, while it did not react with either the γ carboxyglutamic acid (Gla)-domainless light chain of F.VIIa or the heavy chain with the protease domain. Antibody 1 markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody 1) recognizes the calcium-dependent conformation within or near the Gla domain and inhibits F.VIIa activity by interacting with the light chain.

scholarly journals Membrane-mediated Synthesis of Tissue Factor (Thromboplastin) in Cultured Fibroblasts

Blood ◽  
1973 ◽  
Vol 41 (5) ◽  
pp. 679-685 ◽  
Author(s):  
Leo R. Zacharski ◽  
O. Ross McIntyre
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Tissue Factor ◽  
Human Skin ◽  
Actinomycin D ◽  
Shape Change ◽  
Factor Vii ◽  
Culture Vessel ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts

Abstract A potent procoagulant synthesized by cultured human skin fibroblasts has been identified as tissue factor (factor III, thromboplastin), since it binds factor VII and is blocked by a specific antitissue factor antibody. Fibroblast tissue factor is, at least in part, a labile superficial, membrane-associated substance the synthesis of which is mediated by cell adhesion but is inhibited by actinomycin-D and puromycin. Tissue factor production is related to the shape change that occurs as the cells spread on the floor of the culture vessel, but tissue factor is not synonymous with the configuration on the cell surface responsible for cell adhesion. These observations suggest that cell membranes may play a significant role in hemostasis and thrombosis by virtue of their tissue factor content.

scholarly journals Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool

2015 ◽  
Vol 47 (6) ◽  
pp. 2107-2114 ◽  
Author(s):  
RYO TSUMURA ◽  
RYUTA SATO ◽  
FUMIAKI FURUYA ◽  
YOSHIKATSU KOGA ◽  
YOSHIYUKI YAMAMOTO ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tissue Factor ◽  
Feasibility Study ◽  
Diagnostic Tool ◽  
Fab Fragment

scholarly journals Two Distinct Notch1 Mutant Alleles Are Involved in the Induction of T-Cell Leukemia in c-myc Transgenic Mice

2000 ◽  
Vol 20 (11) ◽  
pp. 3831-3842 ◽  
Author(s):  
C. D. Hoemann ◽  
N. Beaulieu ◽  
L. Girard ◽  
N. Rebai ◽  
P. Jolicoeur
Keyword(s):  
Cell Surface ◽  
Transgenic Mice ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Tumor Formation ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Extracellular Medium ◽  
Calcium Dependent

ABSTRACT We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTVD/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutantNotch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)Mut] which, in contrast to the processed wild-type ectodomain [N(EC)WT], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (ΔCT). The type II Notch1ΔCT protein was expressed as a normally processed receptor [N(EC)WT and N(IC)ΔCT] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)ΔCT expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)Mut and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

scholarly journals High-mobility group box 1 protein induces tissue factor expression in vascular endothelial cells via activation of NF-κB and Egr-1

2009 ◽  
Vol 102 (08) ◽  
pp. 352-359 ◽  
Author(s):  
Haichao Wang ◽  
Yiting Tang ◽  
Zhang Fan ◽  
Ben Lv ◽  
Xianzhong Xiao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Vascular Endothelial Cells ◽  
High Mobility ◽  
High Mobility Group ◽  
Cell Surface Receptors ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Surface Receptors

SummaryHigh-mobility group box 1 protein (HMGB1), an abundant nuclear protein, was recently established as a proinflammatory mediator of experimental sepsis.Although extracellular HMGB1 has been found in atherosclerotic plaques, its potential role in the pathogenesis of atherothrombosis remains elusive. In the present study, we determined whether HMGB1 induces tissue factor (TF) expression in vascular endothelial cells (ECs) and macrophages. Our data showed that HMGB1 stimulated ECs to express TF (but not TF pathway inhibitor) mRNA and protein in a concentration and time-dependent manner. Blockade of cell surface receptors (including TLR4, TLR2, and RAGE) with specific neutralising antibodies partially reduced HMGB1-induced TF expression. Moreover, HMGB1 increased expression of Egr-1 and nuclear translocation of NF-κB (c-Rel/p65) in ECs. Taken together, our data suggest that HMGB1 induces TF expression in vascular endothelial cells via cell surface receptors (TLR4, TLR2, and RAGE), and through activation of transcription factors (NF-κB and Egr-1).

Expression and Assembly of Procoagulant Complexes by Human Pleural Mesothelial Cells

1994 ◽  
Vol 71 (05) ◽  
pp. 587-592 ◽  
Author(s):  
Anuradha Kumar ◽  
Kathleen B Koenig ◽  
Alice R Johnson ◽  
Steven Idell
Keyword(s):  
Tissue Factor ◽  
Factor Xa ◽  
Mesothelial Cells ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Specific Concentration ◽  
Pleural Mesothelial Cells ◽  
Direct Binding ◽  
Thrombin Formation

SummaryMany pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor Vila to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

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