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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↗
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↗
PubMed1993

High PAI activity with correlation to triglyceride and HDL cholesterol values in patients with coronary artery disease with no difference in survivors of myocardial infarction.

The fibrinolytic capacity of blood depends mainly on the amount of tissue-type plasminogen activator (t-PA) activity and plasminogen activator inhibitor type-1 (PAI-1) activity. Previous studies linked high PAI activity or low t-PA activity with the development of atherosclerosis and thromboembolic diseases. Yet, there are conflicting reports in the literature as to whether there is higher PAI activity in patients with myocardial infarction (MI) than in patients with coronary artery disease (CAD) without previous MI. In this retrospective study, t-PA activity, t-PA antigen, and PAI activity before and after a venous occlusion test (VOT) of 10 min were assessed in 109 patients with angiographically documented CAD, in two subgroups of CAD patients with (n = 66) or without (n = 43) previous MI, and in subgroups of CAD patients according to their triglyceride levels and other risk factors. The mean values of t-PA activity in the whole patient group showed a 100-fold increase and a 3.1-fold increase in t-PA antigen after VOT (0.03 +/- 0.03 to 3.0 +/- 6.8 U/ml and 16.5 +/- 6.9 to 51.0 +/- 25.4 ng/ml, p < 0.05). PAI activity was 24.4 +/- 11.0 before and 19.6 +/- 13.2 U/ml after VOT. Within the CAD group, no difference was found between patients without MI and survivors of previous MI in PAI activity before VOT (24.6 +/- 10.7 vs. 24.3 +/- 11.3 U/ml) and after VOT (19.0 +/- 12.1 vs 20.0 +/- 14.0 U/ml), or t-PA activity before (0.03 +/- 0.01 vs. 0.04 +/- 0.04 U/ml) and after VOT (2.8 +/- 7.0 vs. 3.2 +/- 6.7 U/ml). In 39.4% of CAD patients elevated plasma PAI activity before VOT (> 25 U/ml) was found. This subgroup of patients represented the highest PAI activity after VOT (p < 0.05), the lowest t-PA activity after VOT (p < 0.001), and the highest triglyceride levels (p < 0.05). In 11% of the patients, a small increase in t-PA activity (less than 0.5 U/ml) after VOT was seen. This group showed the lowest t-PA antigen after VOT (p < 0.001) and the highest fibrinogen level (p < 0.05). Both subgroups showed the same distribution among patients with and without MI. CAD patients with triglyceride levels over 200 mg/dl had the highest PAI activity values before VOT (28.3 +/- 11.8 U/ml; p < 0.01) and after VOT (24.9 +/- 13.2 U/ml; p < 0.01), resulting in low t-PA activity after VOT (p < 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Cholesterol, HDL↗
PubMed1993

Homologous desensitization of calcitonin receptors and calcitonin-dependent adenylate cyclase in T47D cells.

Prolonged use of calcitonin (CT) in vivo leads to a loss of biological response (escape). To understand the molecular basis of this phenomenon, we examined desensitization of calcitonin receptors and down-regulation of adenylate cyclase response to CT in T47CD cells after a preincubation with CT. Preincubation with salmon or human CT (sCT, hCT) for 3 h led to a concentration-dependent loss of [125I]-sCT binding and a similar loss of adenylate cyclase response to a maximal stimulatory dose of sCT. At the same time there was an increased basal activity of the adenylate cyclase. After 24 h preincubation with sCT, basal cAMP levels fell considerably but not to basal levels. Time course experiments showed a delayed decline of maximally sCT-stimulated cAMP levels, which started after 1 h, while binding values declined over the first 60 min to one-third of the original values. Upon removal of CT from the medium, recovery of hormone binding occurred in parallel with the recovery of the adenylate cyclase response to sCT. T47D cells incubated with 25 mumol/l monensin, a lysosomal inhibitor, showed a persistent [125I]-sCT binding after removal of hormone, consistent with diminished intracellular receptor degradation. However, despite persistent binding to the cells, basal and stimulated cAMP levels dropped in the same manner as seen in controls. Our experiments support the view that tight binding of CT to its receptor stimulates adenylate cyclase in T47D cells, until receptors are removed by internalization. The physiological role of internalization of the CT receptor might be to end continuous stimulation of the adenylate cyclase, which occurs after binding.

Adenylyl Cyclases↗
PubMed1993

[Interlocking nailing of the humerus].

Within a 3-year period a total of 42 humeral fractures were treated. Nineteen of these were operated on with the Seidel intramullary interlocking nail. Most of the fractures could be classified into group A (AO classification); three type B and four type C fractures occurred. Two patients had pathological fractures due to bone metastases. Mean hospital inpatient treatment was 24 days. Besides two infections seven system-specific complications occurred. A discrepancy between the diameter of the nail and the width of medullary cavity was the reason in one case for an additional fracture and in another case for nonunion because of loosening of the nail tip in the distal fragment. Three further intraoperative fractures occurred during impaction of the nail, the reasons are also device-specific and are carefully analyzed. In two cases we were forced to remove the nail because of subacromial impingement due to an overranging proximal nail end. Finally, a normal intraoperative and postoperative course with good functional results could only be observed in approximately half the cases. Thus, our initial optimism with respect to the outstanding theoretical concept was dampened, so that the interlocking humerus nail is now just applied in pathological shaft fractures.

Adult↗
PubMed1993

[Results of treatment after different surgical procedures for management of acromioclavicular joint dislocation].

Sixty-four patients underwent surgery for acromioclavicular (AC) disruption, Tossy type III, at the Department of Surgery, University of Heidelberg, between January 1983 and May 1990. Surgery consisted of a suture of the AC and coracoclavicular ligaments. Fixation of the joint was achieved with three different techniques: tension band wire with two Kirschner wires, special hook-plate (Wolter), double tension band fixation using polydioxanon (PDS) cordula. The early postoperative complication rate was higher following tension band wires (42.9%) and hook plate (58.3%) than after tension band PDS cordula (16.7%). The patients were re-examined after an average of 35.3 months. An instability of the AC joint was found in 31.8% of patients with tension band wire, 50.0% of patients with Wolter plate, and 23.8% of patients with tension band PDS cordula. The comparison of these results with those after conservative treatment, as reported in the literature, emphasizes the need for limiting surgery to young adults and athletes. The long term results of AC joint fixation are better using PDS cordula than tension band wire or hook plate. PDS cordula has the additional advantages, that dislocation and fracture of metal implants do not occur, and metal removal is avoided. Therefore, tension PDS cordula is associated with a marked reduction of the overall hospitalization period. Further improvements of results of AC joint fixation can be expected using the described technique of double tension band PDS cordula.

Acromioclavicular Joint↗
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↗