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W Ruf

Publications and source records attributed to W Ruf.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1992

Herpes simplex virus: a possible etiologic agent in some gastroduodenal ulcer disease.

There is increasing evidence that the herpes simplex virus may account for some gastric ulcer disease. To examine this possibility, 62 tissue biopsies from 21 patients were obtained during esophagogastroduodenoscopy for gastroduodenal ulcer disease and from one operative specimen during the procedure for perforation of a gastric ulcer. The samples were collected from the base and rim of the ulcer, as well as from apparently healthy tissue adjacent to the lesion. When the DNA was extracted from these tissues and hybridized to a herpes simplex virus-specific DNA probe, positive results were obtained with 9.5 per cent (2 out of 21) of the patients with benign ulcers. Positive signals were obtained only with ulcer-associated tissues and never with healthy tissue. Hybridization also occurred with DNA from one ulcerative carcinoma in the study. These data suggest that a subset of ulcer disease may be caused by herpes simplex virus or that this virus may be secondarily associating with these lesions.

Adult↗
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↗
PubMed1991

Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function.

Membrane anchoring of tissue factor (TF), the cell receptor for coagulation factor VIIa (VIIa), exemplifies an effective mechanism to localize proteolysis at the cell surface. A recombinant TF mutant (TF1-219), deleted of membrane spanning and intracellular domains, was used to evaluate the role of phospholipid interactions for assembly of substrate with the catalytic TF.VIIa complex. TF1-219 was secreted by cells rather than expressed as a cell membrane protein. Unlike free VIIa, TF1-219 as well as the TF1-219.VIIa complex demonstrated no stable association with phospholipid. In the absence of lipid, kinetic evaluation of substrate factor X cleavage by free VIIa, TF.VIIa, and TF1-219.VIIa suggests that the catalytic function of VIIa rather than substrate recognition is enhanced by complex formation. Furthermore, compared with free factor X, factor X on phospholipid was preferentially cleaved as a substrate by TF1-219.VIIa. TF-dependent initiation of the coagulation protease cascades thus involves an enhancement of the activation of factor X on the cell surface by a crucial role of the TF transmembrane domain to membrane anchor the reaction, by the TF extracellular domain to provide protein-protein interactions with VIIa to enhance the activity of the catalytic domain of VIIa, and the preferential presentation of factor X as a substrate when associated with phospholipid surfaces.

Amino Acid Sequence↗
PubMed1991

Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody.

Gram-negative bacteremia poses a major health problem, causing one-half of cases of lethal septic shock acquired during hospitalization. Bacterial lipopolysaccharide (LPS) and the inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), have been shown to be essential mediators of septic shock. Among the effects of these mediators is a coagulopathy that may be triggered by induced expression of tissue factor (TF) on macrophages and endothelial cells. We now report that 500 micrograms/kg of either immunoglobulin G (IgG) or Fab fragments of a monoclonal antibody against TF administered to baboons as a pretreatment attenuates the coagulopathy and protects against LD100 Escherichia coli. This study provides direct evidence of an essential effector role for TF in septic shock.

Adrenal Glands↗
PubMed1990

[The stabilization of malleolar fractures and flake injuries using absorbable polyglycolide pins (Biofix)].

In a one year period 15 ankle fractures (13 B-type, two fractures of the medial malleolus) and six flake fractures were operated using biodegradable pins and documented prospectively. These are made of polyglycol acid fibrils compressed by use of a melting technique. They are available in different lengths and strengths. The shear strength is decreasing about half of the initial value within four weeks. The ankle fractures were stabilized with two systems of PGS-pins and PDS or Maxon figure-of-0 tension bands. Postoperative functional treatment was applied. The ankle fractures had healed after six weeks in twelve patients, after seven weeks in two, and in one patient only after nine weeks. Osseous healing of the flake fractures took more time. After six weeks was only one, after seven weeks were two and after nine weeks three fractures healed. Full load bearing was achieved after 8.9 weeks in the malleolar fracture group and after 10.8 weeks in the flake fracture group. Total duration of treatment was 10.2 weeks, respectively 12.4 weeks. It could be shown with this study that osteosynthesis of the ankle with biodegradable pins and functional postoperative treatment is basically possible. Furthermore these pins are apted ideally for fixation of flake fractures due to their high stiffness and simple application technique.

Adult↗
PubMed1990

[Does sonographic evidence of blood in the abdomen following blunt abdominal trauma present an indication for surgery in every case?].

In a comparative study based on the diagnosis of blunt abdominal trauma, the accuracy of ultrasound (US) proved inferior, with 82-91%, to that of diagnostic peritoneal lavage, with 97-100%. The sensitivity of US, i.e. the proportion of patients with blood in the abdomen who had an abnormal test result (positive sonography) was 94%. The reasons for this may be either patient-related (severe obesity, intestinal gas superposition) or examiner-related (differing previous experience). The specificity for correct elimination of abdominal lesions was 100%. When no intra-abdominal liquid was present none appeared in the US picture; however, 3-13% of cases where intra-abdominal liquid was present this was not revealed by US. If only a small amount of intra-abdominal liquid is demonstrated after blunt trauma, the adoption of a wait-and-see attitude is justified. In intensive care conditions US can be repeated several times if necessary. In this study US showed deterioration in these circumstances in 25%, and in 21% it must be expected that an operation will be necessary.

Abdominal Injuries↗
PubMed1989

Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line.

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.

Antibodies, Monoclonal↗
PubMed1988

Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization.

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.

Blood Coagulation Disorders↗