Biomedical subjects

T S Edgington

Publications and source records attributed to T S Edgington.

At least 37 records · Page 2Linked to original sources

PubMed1995

Tissue factor mediates prolonged procoagulant activity on the luminal surface of balloon-injured aortas in rabbits.

BACKGROUND: Activation of coagulation has been implicated in both acute thrombotic occlusion and restenosis after balloon angioplasty. However, concomitant administration of antithrombotic agents has thus far failed to prevent these complications. Importantly, the factors contributing to procoagulant activity of balloon-injured arteries over time have not been defined. This study was designed to determine the duration of procoagulant activity on the luminal surface of balloon-injured arteries and the relative roles of tissue factor and thrombin in this response. METHODS AND RESULTS: Abdominal aortas in rabbits were subjected to repetitive balloon hyperinflations sufficient to disrupt the internal elastic lamina. Aortas were excised at < 1, 2, 4, 8, 16, 24, 48, and 72 hours and 1, 2, and 4 weeks after injury; divided into segments; and perfused with recalcified human pooled plasma (n = 58) or plasma depleted of vitamin K-dependent coagulation factors (n = 27) or first incubated with a monoclonal antibody to rabbit tissue factor (n = 33) followed by perfusion with human plasma. Samples of the effluent and plasma perfusate were collected over 10 minutes and assayed for fibrinopeptide A (FPA) as an index of the rate of thrombin-induced fibrin formation. FPA in the effluent from segments perfused with recalcified plasma, expressed as a percentage of FPA in the perfusate, was elevated for 16 hours after balloon-induced injury and exhibited two distinct increases occurring < 1 hour (1297 +/- 473%, mean +/- SD, n = 5) and 8 hours (1052 +/- 330%, n = 6) after injury (P < or = .000001 versus uninjured vessels). Preincubation of segments at these intervals with an antibody to tissue factor markedly attenuated the increases in FPA, as did perfusion of segments with plasma depleted of vitamin K-dependent coagulation factors, indicating that the observed increases in FPA in whole plasma did not result from performed thrombin bound to the injured vessel wall. CONCLUSIONS: Tissue factor-mediated coagulation appears to be primarily responsible for prolonged procoagulant activity of balloon-injured arteries.

Angioplasty, Balloon↗

PubMed1995

Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization.

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

Amino Acid Sequence↗

PubMed1995

Energetic contributions and topographical organization of ligand binding residues of tissue factor.

Tissue factor is the cellular receptor and macromolecular enzymatic cofactor for the serine protease coagulation factor VIIa. The ligand binding extracellular domain of tissue factor consists of two structural modules which fold similar to fibronectin type III modules, consistent with the classification of tissue factor as a member of the class 2 cytokine receptor family. On the basis of the three-dimensional structure, we here analyze the importance of tissue factor residues for binding of ligand by scanning alanine mutagenesis. The identified significant binding contacts account for as much as 80% of the calculated total free energy of ligand binding. Most residues with energetic contributions to ligand binding are well exposed to solvent, and the area for ligand interaction extends from the cleft formed by the two structural modules (residues Lys20, Ile22, Lys48, Asp58, Arg135, Phe140) to the convex-shaped edge of the three- and four-stranded sheets characterized by a patch of surface-exposed hydrophobic side chains in the amino-terminal module (residues Gln37, Asp44, Trp45, Phe76, Tyr78). The binding residues are dispersed over an extended surface area, indicating adaptation to the recognition of specific structural modules of the macromolecular ligand factor VIIa. This analysis provides detailed insight into the three-dimensional organization of the ligand docking structure of the initiating cofactor for the coagulation pathways.

Aspartic Acid↗

PubMed1995

Integrin regulation of an inflammatory effector gene. Direct induction of the tissue factor promoter by engagement of beta 1 or alpha 4 integrin chains.

Inflammatory genes are regulated in cells of monocyte (Mo) lineage by a variety of cellular encounters, including adhesion mediated by integrins. The role of the beta 1 family of integrins in the direct induction of immediate early gene expression was analyzed by using the tissue factor (TF) gene. Engagement of alpha 4 or beta 1 on Mo, but not members of the beta 2 integrin family, with specific mAbs as surrogate ligands immediately and directly induced high level surface expression of TF, and accumulation of TF mRNA, as well as production of TNF-alpha and HIV-1 virus. The mechanism responsible for induction of TF gene transcription mediated by the engagement of alpha 4 or beta 1 was elucidated by using THP-1 monoblastic leukemia cells. Functional analysis of plasmids containing the TF promoter expressing the luciferase reporter gene identified a cis-acting integrin-responsive element (InRE), which contained two AP-1 sites as well as a single kappa B-like site. Mutation of either the AP-1 sites or kappa B-like site greatly diminished responsiveness to integrin engagement. This InRE also conferred responsiveness to a heterologous promoter in the same reporter plasmid. Binding of mAbs to either alpha 4 or beta 1 led to nuclear translocation of the c-Rel/p65 heterodimer that preferentially bound to the TF kappa B-like site. In contrast, constitutive binding of AP-1 proteins to the two AP-1 sites was not increased by alpha 4 or beta 1 integrin engagement. These studies expand knowledge of integrin regulation of immediate early gene expression in Mo and molecular encounters that are inferred to play an active role in Mo effector functions.

Base Sequence↗

PubMed1995

Vascular biology: integrative molecular cell biology.

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Blood Vessels↗

PubMed1995

The other AIDS crisis.

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Acquired Immunodeficiency Syndrome↗

PubMed1994

Upregulation of human immunodeficiency virus-1 in chronically infected monocytic cell line by both contact with endothelial cells and cytokines.

Cells of monocytic lineage (Mo) persistently infected with human immunodeficiency virus (HIV) have been suspected to be a major reservoir for in vivo transmission of virus to susceptible target cells. Cellular events and mechanisms that upregulate viral gene expression in such cells are important issues. Because the traffic of such cells is central to biodistribution of HIV, we have explored the impact of interaction of endothelium with HIV-1-infected U1 promonocytic cells. Coculturing of U1 with human umbilical endothelial cells (HUVEC) for 24 to 72 hours in the absence of stimulation induced HIV-1 p24 biosynthesis significantly. Antibody-blocking experiments indicated that CD11/CD18 integrins play a role in upregulation of HIV expression elicited by interaction with HUVEC. Engagement of CD11b/CD18 by adherence of U1 to surfaces coated with either the cognate ligand fibrinogen or monoclonal antibody specific for CD11b/CD18 also enhanced p24 biosynthesis. Furthermore, endothelial cells were found to constitutively synthesize and secrete soluble factors that enhanced HIV-1 synthesis. The enhancing factors, of estimated size 10 to 45 kD, were induced in HUVEC to high levels by monokines or by lipopolysaccharide, resulting in markedly enhanced HIV-1 expression by U1. These endothelial cell-derived HIV-1-enhancing factors consist of, among others, interleukin-6 (IL-6), IL-1 beta, and granulocyte-macrophage CSF (GM-CSF). Our results suggest that activation of HIV biosynthesis in infected Mo via interaction with endothelium may impact significantly on the tissue distribution and pathogenesis of HIV infections.

Antibodies, Monoclonal↗

PubMed1994

High molecular weight kininogen binds to Mac-1 on neutrophils by its heavy chain (domain 3) and its light chain (domain 5).

High molecular weight kininogen (HK) binds specifically, saturably, and reversibly to neutrophils and also reciprocally inhibits the binding of fibrinogen to neutrophils. Since fibrinogen binds to the leukocyte integrin CD11b/18 (Mac-1, alpha M beta 2), we investigated whether HK bound to Mac-1 and whether the binding site was similar to that for factor X. We also examined whether one or both chains of cleaved HK (HKa) were involved. Two monoclonal antibodies, 2B5 (0.29 microM) to HK heavy chain domains 2 (D2) and 3 (D3), and C11C1 (0.26 microM) to HK light chain domain 5 (D5), inhibited by 99 and 93% the binding, respectively, of 125I-HK (8.3 nM) to neutrophils. To minimize steric hindrance, we further demonstrated that the Fab' fragments of 2B5 and C11C1 were able to inhibit the binding of this ligand to virtually the same extent as the intact antibody, indicating that, as in binding of HK to platelets and endothelial cells, both chains are involved. To directly demonstrate the involvement of each chain, we showed that the reduced alkylated light chain derived from HK and low molecular weight kininogen, which contains the same heavy chain as HK, each markedly inhibited the binding of HK to neutrophils. We localized the domain responsible for the binding in each chain by showing that recombinant D3 and D5 decreased the binding of HK to neutrophils. To define the receptor for HK, we employed three monoclonal antibodies to Mac-1: OKM1 and OKM10 to epitopes on the alpha M subunit and IB4 to an epitope on the beta 2 chain. OKM1, which can inhibit fibrinogen binding to neutrophils, inhibited HK binding by 79%, whereas the other antibodies inhibited HK binding less than 25%. Coagulation factor X also binds to Mac-1 on monocytes at a similar site to C3bi. Synthetic peptides which define noncontiguous surface loops in factor X that interact with Mac-1, failed to inhibit 125I-HK binding to neutrophils. We conclude that HK binds, via domains on its heavy chain, D3, and light chain, D5, to Mac-1 on the neutrophil surface, and HK occupies a site overlapping with fibrinogen and different from factor X.

Amino Acid Sequence↗

PubMed1994

Key ligand interface residues in tissue factor contribute independently to factor VIIa binding.

Scanning alanine mutagenesis of the cell surface protease receptor tissue factor suggested importance of residues Lys20, Ile22, Asp58, Arg135, and Phe140 for binding of ligand, the serine protease coagulation factor VIIa. Ligand binding by single alanine replacement mutants was characterized by functional assays which concordantly demonstrated a calculated 1-2.5 kcal/mol reduction in free energy of binding as a result of each of the mutations. Catalytic and proteolytic function appeared to be not impaired by the residue replacements, indicating that these residues are not specifically required for the catalytic enhancement of VIIa produced by the assembly with tissue factor. Multiple mutations were further combined in one mutant protein to assess whether these residues provide independent contacts with the ligand VIIa. The Lys20/Asp58 and the Arg135/Phe140 residue pairs did not independently contribute to the binding of ligand. In contrast, the combination with Ile22 consistently produced a further decrease in affinity for VIIa, demonstrating that this residue acts as an independent contact site for the ligand VIIa. The total contribution of the five residues to the free energy of binding of VIIa at 37 degrees C was calculated to be 5.4 kcal/mol representing approximately one-third of the total binding energy.

Alanine↗

PubMed1994

Monoclonal antibody to an activation neoepitope of alpha M beta 2 inhibits multiple alpha M beta 2 functions.

alpha M beta 2, a beta 2 integrin expressed on cells of myelomonocytic differentiation, functions as a receptor for factor X (X) and fibrinogen (Fg) and participates in leukocyte adhesion to vascular endothelium. Acquisition of high affinity ligand binding has been suggested to result from a conformational change of alpha M beta 2 in response to agonist-induced leukocyte stimulation. We now describe mAb 7A10, which preferentially binds to alpha M beta 2 on activated cells and reports the functional activation of this integrin. Agonist-stimulated monocytes and monocytic cells, but not resting cells, maximally expressed the 7A10 neoepitope, whereas expression of other selected alpha M beta 2 epitopes remained unchanged. The neoepitope was elicited equally by exposure of cells to either ADP or FMLP and did not require divalent cations for expression. Saturation of the 7A10 neoepitope by this Ab on stimulated THP-1 cells inhibited both X and Fg binding and abolished the alpha M beta 2-driven cellular coagulant response. Stimulated monocytic cells, which bound X and/or Fg, exhibited a sustained adhesion to unstimulated endothelial cell monolayers and Ab 7A10 inhibited this sustained adhesion. We conclude that activated conformers of alpha M beta 2 mediate X and Fg binding, that assembly of either X or Fg or both on alpha M beta 2 mediates leukocyte adhesion to unstimulated endothelium through sparse ICAM-1, and that mAb 7A10 can report and functionally inhibit this pathway of leukocyte adhesion.

Antibodies, Monoclonal↗

PubMed1994

Structural biology of tissue factor, the initiator of thrombogenesis in vivo.

Thromboembolic disorders are commonly associated with cardiovascular, infectious, and neoplastic disease. A major link in the pathophysiology of thrombosis is the excessive triggering of the coagulation pathways by the initiating cofactor molecule termed tissue factor, an integral membrane glycoprotein. The tissue factor extracellular ligand binding domain is predicted to fold in an architecture similar to the cytokine receptor homology module. Functional sites in tissue factor have been defined by a combination of antibody, chemical cross-linking, and mutational analyses providing a model for cofactor function that involves discrete interactions with both enzyme and substrate. The understanding of the structural basis of tissue factor function promises to facilitate rational design of inhibitor molecules for defined functional sites, eventually leading to effective in vivo therapeutics.

Amino Acid Sequence↗

PubMed1994

Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain.

Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction. The tissue factor extracellular domain is suggested to adopt the folding pattern of the cytokine receptor homology unit, which is typically composed of two seven-beta-strand modules. Evaluation of the mutational analysis within this structural context suggests that functionally important residues are spatially proximate and clustered at the boundary of the predicted beta-strand modules. Residues contributing to ligand binding by tissue factor were identified in positions corresponding to ligand interactive residues in the growth hormone receptor and contact residues of other cytokine receptors, consistent with a conserved structural region for ligand interaction throughout the cytokine receptor family.

Alanine↗

PubMed1994

Microfilament reorganization is associated with functional activation of alpha M beta 2 on monocytic cells.

Selected agonists convert the leukocyte integrin alpha M beta 2 on monocytes from a low to a high affinity state competent to bind factor X and fibrinogen. Conformational changes of alpha M beta 2 re hypothesized to account for this functional transition. Here we report that cytochalasins known to interfere with actin filaments induce the alpha M beta 2 functional transition. Upon exposure to cytochalasin B, isolated human blood monocytes and cells of the monocytic cell line THP-1 bound 125I-factor X (X) or 125I-fibrinogen (Fg) in a Ca(2+)-dependent, saturable manner. Monoclonal antibodies (mAbs) to the alpha M subunit and the common beta 2 subunit of leukocyte integrins inhibited X and Fg binding, whereas mAbs to the alpha chains of the other leukocyte integrins had no effect. Anti-alpha M mAb immunoprecipitated 125I-X that had been chemically cross-linked to its cognate receptor. Specific binding was not associated with an increased surface density of beta 2 integrins consistent with conformational remodeling of the receptor. Simultaneous analysis of actin forms in viable monocytes indicated a dynamic redistribution of cellular actin. The transient increase in G actin concurrent with an agonist action such as cytochalasin or ADP was reversed by an increase in F actin coincident with X/Ca2+ binding. A potential role of actin redistribution in alpha M beta 2 functional transition is supported by the finding that cells in which cellular actin is restricted to G rather than F form bound X and initiated a rapid coagulant response. We propose that a transient disassembly of actin filaments may relieve constraints on alpha M beta 2 via the cytoplasmic domains, permitting the conformational dynamics required for recognition of ligands.

Actins↗

PubMed1994