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T S Edgington

Publications and source records attributed to T S Edgington.

At least 19 recordsLinked to original sources
PubMed1992

Role of the membrane surface in the activation of human coagulation factor X.

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.

Animals↗
PubMed1992

Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis.

Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher level of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence in insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

Animals↗
PubMed1992

Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.

Amino Acid Sequence↗
PubMed1992

Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex.

High affinity binding of factor VIIa (VIIa) to its cellular receptor tissue factor (TF), as well as association of factor X with phospholipid are required for optimal assembly of the extrinsic activation complex. In addition to the interactions of substrate with phospholipid and enzyme, we here provide evidence that cofactor residues Lys-165 and Lys-166 specifically contribute to the recognition of macromolecular substrate. Ala for Lys replacement in TFA165A166 was compatible with high affinity binding of VIIa when analyzed on cell surfaces as well as in the absence of phospholipid. Dissociation of TFA165A166.VIIa did not occur with a faster rate compared to TF.VIIa, further supporting unaltered VIIa binding function of TFA165A166. Cleavage of chromogenic peptidyl substrate by TFA165A166.VIIa complexes was not diminished, demonstrating that TFA165A166 supported enhancement of catalytic function of the VIIa protease domain. In contrast, factor X activation was reduced in the presence and absence of phospholipid. Further, TFA165A166 effectively competed with wild-type TF in the cleavage of factor X at limited VIIa concentrations. Selective reduction in macromolecular substrate hydrolysis combined with normal VIIa binding by TFA165A166 indicates that the cofactor TF does contribute, either directly or indirectly via specific interactions with VIIa, to factor X recognition.

Amino Acid Sequence↗
PubMed1992

The third Trp-Lys-Ser (WKS) tripeptide motif in tissue factor is associated with a function site.

The tripeptide sequence Trp-Lys-Ser (WKS) is repeated three times in the extracellular ligand binding domain of human Tissue Factor (TF). Using site-directed mutagenesis, we replaced each of the WKS motifs in human TF by Arg-Lys-Gly (RKG), the least conserved replacement for the motif found in murine TF. This substitution in the first repeat W14KS, as well as a Trp14----Arg substitution, resulted in a structurally altered protein, whereas a conservative hydrophobic Trp14----Phe substitution resulted in a functionally normal protein. This suggests that Trp14 may contribute to a hydrophobic core rather than involvement of this motif in function. Replacement of the W45KS and W158KS motifs was associated with no detectable structural alterations; however, function was diminished with the RKG replacement of the third repeat. Mutant proteins with Lys159----Ala and Tyr157----Ala substitutions exhibited loss of function, whereas Tyr156----Ala and Ser160----Ala substitutions flanking the YWK sequence resulted in functional proteins. These data demonstrate that the W158KS motif in human TF is associated with a functional site and identify Lys159 in this motif as a functionally important residue.

Amino Acid Sequence↗
PubMed1992

Glomerular tissue factor stimulates thromboxane synthesis in human platelets via thrombin generation.

We have investigated whether or not tissue factor (TF) which is present in the supernatant of isolated glomeruli, is responsible for the stimulatory activity of TXB2 production by isolated human platelets. Reconstituted TF stimulated TXB2 synthesis in platelets in a dose-dependent manner. This effect was potentiated in the presence of a mixture of the major fatty acids found in glomerular supernatants. Addition of a neutralizing anti-TF monoclonal antibody abolished both the procoagulant activity and the platelet-TXB2 stimulatory activity of reconstituted TF and of glomerular supernatants. Anti-factor VII/VIIa (F VII/VIIa) Fab inhibited in a dose-dependent manner the platelet-TXB2 stimulatory activity of an identical dilution of reconstituted TF and of glomerular supernatants, providing evidence that the functional complex TF. VIIa and not TF itself was the active agent. Pretreatment of platelets, TF or glomerular supernatant by hirudin, an inhibitor of thrombin, as well as by antithrombin III heparin, which inhibits both activated factor X and thrombin also markedly inhibited the synthesis of TXB2 by platelets in the presence of either TF or glomerular supernatant. Taken together, these results demonstrate that the stimulatory activity for TXB2 production by platelets which is released by the glomerular cells is attributable to TF. TF does not act directly. Its effect is mediated by thrombin which is formed de novo at the platelet surface in the presence of even traces of the plasma coagulation proteins associated with platelets. TXB2 formation in platelets correlates well with TF concentration in the glomerular supernatant. The possibility of a similar set of mechanisms associated with glomerular injury may require consideration.

Antibodies↗
PubMed1992

Cellular immune and cytokine pathways resulting in tissue factor expression and relevance to septic shock.

Cells of monocyte lineage serve as effector cells in the cellular immune response. In addition, they respond to LPS and cytokines with activation and expression of inflammatory effector gene products similar to those elicited by the antigen driven response. The response to antigen proceeds at the T helper cell level through two independent forms of cellular collaboration, contact and lymphokine. We review the control of expression of the Tissue Factor (TF) gene and the function of the TF protein. The enhanced initiation of transcription of the TF gene appears to require engagement of a 56 bp LPS Response Element, an enhancer that is engaged by both AP-1 type heterodimeric complexes as well as NF kappa B like heterodimeric complexes. Dissociation of NF kappa B from Ig kappa B by cytokine and LPS stimulation, and possibly activated T cells, may represent a common pathway to induction of the TF and other inflammatory genes. Enhancement of expression of TF is observed upon adhesion of Mo to endothelial cells and extracellular matrix proteins, as well as upon engagement of leukocyte integrins. The biological effects that follow from expression of TF by vascular cells have been resolved by analysis of function aided by the use of recombinant full length TF and truncated surface domain of TF. The rules of assembly of the cognate ligands of TF, namely the zymogen plasma factors VII and the serine protease factor VIIa, with the soluble surface domain of TF in free solution, in the presence of phospholipid surfaces and cell surface and of the anchored TF molecule have been described. It is evident that assembly of the surface domain of TF with VIIa to form the binary TF.VIIa complex induces a significant increase in the Kcat of the catalytic domain of VIIa for small peptidyl substrates and more profoundly for protein substrate. This provides substantial evidence for an allosteric effect on the catalytic cleft of VIIa that is imparted by binding to TF, its cognate catalytic cofactor. It is also evident that the TF.VIIa complex is proteolytically active and can activate the zymogen plasma factor X to the serine protease Xa in free solution, inferring that extended substrate recognition by induced structural loci of the TF.VIIa complex are created from either or both proteins to constitute a new recognition structure. It is also evident that association of X with charged phospholipid surfaces enhances the proteolytic activation of this zymogen by increasing recognition and susceptibility of the sessile peptide bond deduced from the markedly decreased Km and increased Kcat.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites.

Lipopolysaccharide (LPS) activation of cells of monocytic lineage leads to rapid and transient expression of a set of inflammatory gene products, including tissue factor (TF). This transmembrane receptor is the major cellular initiator of the blood coagulation cascades, and induced expression of TF is postulated to play a role in inflammation. Functional studies using transfected THP-1 monocytic cells revealed the presence of a 56-bp LPS response element (LRE) within the TF promoter that conferred LPS responsiveness to a heterologous promoter. LPS stimulation of these cells activated proteins that bound to nucleotide sequences within the LRE resembling consensus binding sites for activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). Induction of the TF gene may represent a prototypic example of gene activation in monocytic cells by assembly of transcription factor complexes, and may clarify the role of AP-1 and NF-kappa B in the regulation of other LPS-responsive genes.

Base Sequence↗
PubMed1991

Structurally homologous ligand binding of integrin Mac-1 and viral glycoprotein C receptors.

Three spatially distant surface loops were found to mediate the interaction of the coagulation protein factor X with the leukocyte integrin Mac-1. This interacting region, which by computational modeling defines a three-dimensional macromotif in the catalytic domain, was also recognized by glycoprotein C (gC), a factor X receptor expressed on herpes simplex virus (HSV)-infected endothelial cells. Peptidyl mimicry of each loop inhibited factor X binding to Mac-1 and gC, blocked monocyte generation of thrombin, and prevented monocyte adhesion to HSV-infected endothelium. These data link the ligand recognition of Mac-1 to established mechanisms of receptor-mediated vascular injury.

Amino Acid Sequence↗
PubMed1991

An anti-tissue factor monoclonal antibody which inhibits TF.VIIa complex is a potent anticoagulant in plasma.

Tissue factor (TF) functions as the receptor and cofactor for factor VIIa (VIIa) to form a proteolytically active TF.VIIa complex on cell surfaces. We here demonstrate that most MAbs against human TF were poor inhibitors of TF function in plasma and that they inhibited preformed TF.VIIa complex at a slow rate which was dependent on dissociation of VIIa from the cell surface TF. An exception was defined by one MAb (TF8-5G9) which was an effective immediate anticoagulant in plasma. Binding of TF8-5G9 to TF.VIIa inhibited catalytic function prior to dissociation of the TF.VIIa complex. This analysis thus establishes two distinct mechanisms by which MAbs interfere with TF function. The MAb TF8-5G9 introduces a therapeutic principle for rapid arrest of inappropriate triggering of coagulation by TF as well as the TF.VIIa complex in vivo.

Antibodies, Monoclonal↗
PubMed1991

Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa.

Tissue factor (TF) binds the serine protease coagulation factor VIIa and initiates the coagulation protease cascade by forming a catalytic cofactor-enzyme complex. Using a photoactivatable crosslinking reagent coupled to factor VIIa, we have identified interactive sites in the amino-terminal (residues 44-84) and the carboxyl-terminal (residues 129-169) aspect of the extracellular domain of TF. Epitopes of inhibitory antibodies have previously indicated participation of these regions in TF function. The presence of the gamma-carboxyglutamic acid domain in factor VIIa appears to facilitate the interaction with the negatively charged, amino-proximate site, whereas crosslinking of TF with VIIa or des-(1-38)-VIIa at the positively charged carboxyl-proximate site was similar. Lack of alpha-helical secondary structure in the TF extracellular domain is consistent with the proposed structural similarity of TF with the cytokine receptor family. The interactive sites identified for TF are located in sequence spans that demonstrate a low degree of sequence conservation among the members of this receptor family. Regions with highly conserved residues, such as sequences encoded by exon 2 and 5 in TF, were not implicated in ligand recognition, suggesting that conserved residues in the receptor family may maintain the common beta-strand architecture, and variable regions provide a pair of nonidentical motifs for oriented ligand recognition.

Affinity Labels↗
PubMed1991

Antibody mapping of tissue factor implicates two different exon-encoded regions in function.

Tissue Factor (TF), a small transmembrane glycoprotein, is the cellular receptor for the zymogen Factor VII and the serine protease Factor VIIa (VIIa). TF provides cofactor function for VIIa in the catalytically active (TF: VIIa) binary complex. To explore the structural loci of TF that are responsible for binding of VII and VIIa, monoclonal antibodies (MAbs) and sequence-specific polyclonal antibodies to the native TF protein were analysed for inhibition of VII binding. Two independent epitopes of MAbs were localized by reciprocal competition and by binding of the MAbs to different proteolytic fragments of TF. The epitopes were also characterized in part by progressive C-terminal deletional mutation of the TF protein. Reactivity of the anti-(locus II) MAb TF9-6G4 is consistent with epitope localization in residues Thr40-Val83, encoded by exon 3. In contrast, the anti-(locus I) MAb TF9-5G9 was reactive with fragments encompassing exon 4 (Thr106-Lys165). Antibodies to linear sequences encoded by the same two exons also inhibited VII binding. These data suggest a minimum requirement for two of the four exon-encoded regions of TF for the functional integrity of this receptor cofactor with respect to ligand recognition and high-affinity binding.

Antibodies, Monoclonal↗
PubMed1991

Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions.

Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.

Antibodies, Monoclonal↗
PubMed1991

The structural biology of expression and function of tissue factor.

Analysis of the structural biology of TF provides insights into the both the expression of the gene and the function of this cell surface receptor in the initiation of the coagulation protease cascades. The advance of information may permit inferential hypotheses for the structural biology of other cofactor regulated catalytic steps in blood coagulation.

Amino Acid Sequence↗
PubMed1991

The integrity of the cysteine 186-cysteine 209 bond of the second disulfide loop of tissue factor is required for binding of factor VII.

The structural basis of function of tissue factor (TF), the cell surface receptor and cofactor for the serine protease factor VIIa, cannot be inferred from the primary sequence. The functional significance of the two disulfide bonded loops in the surface domain of TF has been analyzed using site-directed mutagenesis to selectively preclude covalent stabilization of these loops by pairwise substitution of serine residues for cysteines. Mutant TF lacking either the amino (TFS49S57) or carboxyl (TFS186S209) disulfide bond were expressed on the surface of cells consistent with proper processing. Each reacted with a panel of monoclonal antibodies further suggesting proper global folding of the mutant proteins. TFS186S209 exhibited a selective decrease in reactivity with an antibody directed against one epitope locus in the carboxyl aspect of the surface domain of TF. Whereas TFS49S57 was functionally comparable to the wild type protein, TFS186S209 was functionally 30-40-fold less effective, and the affinity of factor VIIa binding to this mutant was indirectly estimated to be diminished 20-fold. These data suggest that the Cys186-Cys209 disulfide bond is required to maintain conformation and implicate the disulfide loop or adjacent structures in the carboxyl half of the surface domain of TF in receptor function.

Animals↗