Biomedical subjects

W Ruf

Publications and source records attributed to W Ruf.

At least 19 recordsLinked 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

Tissue factor residues Lys165 and Lys166 are essential for rapid formation of the quaternary complex of tissue factor.VIIa with Xa.tissue factor pathway inhibitor.

The extrinsic coagulation pathway is initiated by the binding of plasma factor VII(a) (VIIa) to the cell surface receptor tissue factor (TF), which serves as the cofactor for the ligand protease VIIa in the activation of macromolecular substrate factors X and IX. The catalytic function of the TF.VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), which forms a stoichiometric complex with the serine protease factor Xa (Xa), resulting in greatly accelerated inhibition of the extrinsic initiation complex as compared to free inhibitor. In the present study we identify specific residues in the TF-VIIa complex that are involved in the factor Xa-mediated acceleration of TFPI inhibitory function. VIIa residue Arg290, which contributes to extended recognition of macromolecular substrate factor X, is not involved in the interaction with the TFPI.Xa complex. In contrast, TF residues Lys165 and Lys166, which are important for the activation of factor X, are required for the accelerated inhibition of the TF.VIIa complex by TFPI mediated by factor Xa. These data indicate that similar interactions contribute to the assembly of substrate factor X as well as of product Xa after complex formation with TFPI, suggesting a central role for the carboxyl-terminal structural module of TF in regulating the proteolytic activity of TF.VIIa.

Blood Coagulation↗

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

Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells.

The human melanoma cell line M24met expresses tissue factor, the cellular initiator of the blood coagulation cascade. Blocking of the coagulation pathways at the level of tissue factor, factor Xa, or thrombin inhibits hematogenous M24met metastasis in SCID mice, implicating a role for thrombin generation in this process. Dependent on cell surface tissue factor activity, M24met cells generate thrombin in vitro. Thrombin and the thrombin receptor agonist peptide TRP-14 activate a signaling pathway in M24met cells that involves an increase in intracellular calcium and induces cell proliferation. Immunofluorescence evidences expression of the signaling thrombin receptor on these cells. Thus, M24met melanoma cells express both the initiating cell surface receptor for the coagulation pathways and the central signaling receptor of the coagulation system, suggesting the in situ generation of proliferative signals which can contribute to the malignant phenotype.

Animals↗

PubMed1995

Tissue factor: molecular recognition and cofactor function.

One aspect of the inflammatory response is the activation of the coagulation protease cascade resulting from the expression of tissue factor (TF) on vascular cells. TF is the cell-surface receptor for the coagulation serine protease factor VIIa, providing cofactor function by "switching on" the catalytic site of the bound enzyme and by contributing to the assembly with macromolecular substrate. The recently determined crystal structure of the TF extracellular domain shows two beta-strand modules of C2 immunoglobulin-like topology that align at a 125 degrees angle with an extensive intermodule interface. Mutagenesis studies have identified residues in both modules that are important for the binding of ligand. The deduced ligand interface extends from the convex side of the molecule into the concave side of the elbow angle. Specific binding residues control the catalytic activity of the bound protease. At the lower end of the carboxyl-terminal module, basic residues form part of a region that is important for both recognition and activation of macromolecular substrate and, potentially, for modulation of proteolytic function. After combining the biochemical data with the crystal structure, a model of TF function can be proposed in which the catalytic activity of the active site of the protease and the extended recognition of macromolecular substrates are separately controlled by distinct structural sites of the cofactor.

Binding Sites↗

PubMed1994

Factor VIIa residue Arg290 is required for efficient activation of the macromolecular substrate factor X.

The serine protease factor VIIa (VIIa) in complex with tissue factor is responsible for initiating proteolytic events in the coagulation pathways. Efficient proteolysis by the extrinsic activation complex appears to depend on structural determinants in the cofactor as well as the light and heavy chain domains of VIIa. This study characterizes the functional defect resulting from alanine replacement for R290 in the VIIa protease domain. VIIa R290-->A bound both full-length and soluble tissue factor with affinities indistinguishable from wild-type VIIa, consistent with overall unaltered folding of the mutant protein. The catalytic function of VIIa R290-->A was further demonstrated to be unperturbed when analyzed with three different peptidyl p-nitroanilide substrates, indicating that the function of the catalytic triad is not affected by the mutation. However, proteolytic activation of factor X was diminished due to a 4-5-fold decreased kcat in the presence and a > 10-fold decreased rate in the absence of a negatively charged phospholipid surface. The functional defect resulting from the R290-->A replacement was observed in the presence and absence of cofactor. Within the structural framework of serine protease domains, R290 is predicted to be localized in a surface-exposed loop suggested to contribute to substrate selectivity in other serine proteases, consistent with the proposed functional role of R290 in the proteolytic activation of the natural substrate factor X.

Amides↗

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↗

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

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↗