Helmut Hörmann

Max Planck Institute of Biochemistry, München, Bavaria, Germany

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Publications (34)69.52 Total impact

  • Eva Schlosser · Ralph Simler · H Hörmann
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    ABSTRACT: Beads of polytetrafluoroethylene were used to investigate adsorption of thrombin and the influence of the adsorbed protease on a subsequent deposition of fibrinogen. Adsorption of active thrombin was not detected by a specific fluorogenic substrate unless > 0.1 units/ml had been applied. Adsorption was considerably improved by albumin, which protected soluble thrombin from inactivation by hydrophobic surfaces. Retention of active thrombin was optimal at ca. 0.1% albumin and decreased at higher concentrations. After incubation with plasma, negligible thrombin activity was detected at the polytetrafluoroethylene beads by the fluorogenic substrate. However, repeated incubation with fresh plasma samples resulted in adsorbed activity rising with each step. This result suggested that thrombin activity should also accumulate at a polytetrafluoroethylene surface in vivo if fresh blood is permanently flowing past. Adsorbed thrombin improved the subsequent retention of fibrinogen, monitored by an antibody technique. Concomitantly, fibrinopeptides A, AP and AY were slowly released whilst fibrinopeptide B was not detectable before 24 h.
    No preview · Article · Apr 1993 · Biomaterials
  • H Hörmann · H Richter · V Jelinić
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    ABSTRACT: Previous experiments had shown that the free N-terminal fibronectin 30-kDa-domain mediates binding of soluble 125I-fibrin to transamidase-coated polystyrene beads (Hörmann et al., Biol. Chem. Hoppe-Seyler 368, 669-674, 1987). Now, the formation of covalent adducts of the N-terminal fragment with fibrin peptide chains is demonstrated. Binding of soluble 125I-fibrin was performed in presence of N-terminal fibronectin 30-kDa or 70-kDa fragments. The material adsorbed was removed from the beads under reducing conditions and analysed by dodecylsulfate gel electrophoresis followed by autoradiography. The 30-kDa fragment gave rise to bands of 80 kDa and 180-200 kDa which were lacking in the products of the 70-kDa compound. Instead, they showed bands at 120 kDa and ca. 280 kDa. Evidently, those bands represented covalent adducts of fibrin peptide chains or their dimers with the 30-kDa or the 70-kDa fragment, respectively. In addition, dimeric gamma-chains and alpha-chain polymers of fibrin were present indicating partial polymerization of bead-attached fibrin.
    No preview · Article · Jul 1991 · Biological chemistry Hoppe-Seyler
  • H Hörmann · V Jelinić · H Richter
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    ABSTRACT: Centrifuged human platelets bound soluble 125I-labelled fibrin, mediated by a plasma factor. Binding was inhibited by D-phenylalanyl-L-prolyl-L-arginyl- chloromethane (PPACK), which specifically blocks thrombin. As the binding-promoting principle was adsorbed to barium citrate, it was tentatively characterized as prothrombin, suggesting that it might be converted to thrombin at the cell surface. The peptide GRGDSP failed to inhibit binding, thus eliminating the glycoprotein IIb/IIIa complex as a receptor. Most likely, a thrombin - fibrin complex is recognized by a cell receptor, possibly protease-nexin I. In a platelet concentrate, the cells also internalized 125I-labelled fibrin, providing evidence that platelets are involved in the clearance of circulating fibrin - monomer complexes. Engulfment was again inhibited by PPACK or hirudin but not by an antibody against the glycoprotein IIb/IIIa complex.
    No preview · Article · Nov 1990 · Blood Coagulation and Fibrinolysis
  • J Skrha · I Vacková · J Kvasnicka · V Stibor · P Stolba · H Richter · H Hörmann
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    ABSTRACT: The plasma free N-terminal fibronectin 30-kDa domain was measured in 44 type 1 diabetic patients and in 20 healthy subjects. A significantly raised mean concentration of a free N-terminal fibronectin 30-kDa domain was found in plasma of diabetic patients with proliferative retinopathy as compared with healthy persons (P less than 0.001). A positive correlation was observed between free N-terminal fibronectin 30-kDa domain and von Willebrand factor in plasma of all examined subjects (r = 0.62, P less than 0.01). A similar correlation was present between 30-kDa domain and albuminuria (r = 0.56, P less than 0.01). However, no relationship was found between fibronectin 30-kDa domain and control of diabetes as assessed by fructosamine concentration. The free N-terminal fibronectin 30-kDa domain may be used as a marker of actual endothelial cell dysfunction in diabetes.
    No preview · Article · May 1990 · European Journal of Clinical Investigation
  • Helmut Hörmann · Hartmut Richter · V Jelinić
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    ABSTRACT: Binding of soluble 125I-fibrin to platelets was investigated with thrombocytes separated by gelfiltration or by sedimentation as well as in a thrombocyte concentrate. Gelfiltered platelets failed to retain 125I-fibrin within 16 hours unless they had been pretreated with factor XIIIa. In addition, a 30 kDa-component derived from the N-terminal fibronectin domain was required as a mediator. Platelets isolated by sedimentation bound some 125I-fibrin even in the absence of those cofactors. The 30 kDa-component improved binding and only this increase was sensitive to putrescine inhibition. Evidently, centrifuged platelets unlike gelfiltered ones express two pathways of fibrin binding. In a thrombocyte concentrate with platelets in their plasmatic environment 125I-fibrin was partially internalized. Engulfed radioactivity was detectable only for a limited period between 4-6 hours after substrate application suggesting that 125I-fibrin was intracellularly degraded followed by release of the fragments. The 30 kDa-component promoted internalization, while factor XIIIa improved the capacity. Thrombin inhibitors suppressed the uptake.
    No preview · Article · May 1990 · Thrombosis Research
  • Helmut HÖRMANN · V Jelinić · Hartmut RICHTER
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    ABSTRACT: Previous experiments (Hörmann, H. & Jelinić, V. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 379-387) had shown that heparin promoted the binding of plasma fibronectin to peritoneal macrophages of guinea pigs. The present data reveal that this effect only takes place at higher fibronectin concentrations indicating cooperative processes, most likely association of fibronectin at the cell surface. An unspecific precipitation of fibronectin by heparin was prevented by calcium in the medium. The accumulation at the cell surface was inhibited by the following fibronectin fragments: N-terminal 30 kDa and 70 kDa containing a potential self-association site and a transamidase-reactive site; central 95 kDa which comprised a negatively charged region possibly involved in self-association as well as the so-called alternative cell-binding site, but was lacking the cell-binding Arg-Gly-Asp sequence; heparin-binding 37-kDa and 60-kDa fragments. All these domains and sites, therefore, were potentially important in the assembly process at the cell surface. A peptide comprising the sequence Arg-Gly-Asp was ineffective pointing against an involvement of this fibronectin cell-binding site in the overall process. Macrophages of older animals were less capable of accumulating fibronectin under the reaction conditions. Their capability was improved after preincubation with activated plasma transglutaminase (coagulation factor XIIIa) suggesting that a cell-attached transamidase might be important for the assembly process.
    No preview · Article · Jul 1989 · Biological chemistry Hoppe-Seyler
  • J Skrha · Hartmut Richter · Helmut Hörmann
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    ABSTRACT: The free N-terminal 30-kDa domain of the fibronectin subunit chains had previously been shown to mediate binding of soluble fibrin to phagocytic cells. In order to demonstrate whether the fragment is available in plasma in a suitable concentration, an indirect immunoassay procedure for its quantitative evaluation was developed. The free form of the 30-kDa domain was separated from fibronectin and the bulk of the plasma proteins by two-step affinity chromatography on gelatin- and heparin-Sepharose. In the eluate of the heparin-Sepharose the 30-kDa fragment was determined by its capacity to inhibit the immune reaction between a specific antiserum and the 30-kDa fragment immobilized on microtiter wells. The procedure offered reproductibility comparable with other immunoassays (coefficient of variation 4.0 to 8.0%); the lowest amount of detectable 30-kDa fragment was 0.1 microgram/ml. In human plasma this method detected for the first time ca. 5 micrograms/ml 30-kDa fragment. This concentration is in the range required for binding of fibrin to cells.
    No preview · Article · Oct 1988 · Analytical Biochemistry
  • Helmut Hörmann · Hartmut Richter · Viktorija Jelinić
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    ABSTRACT: Gelfiltered unstimulated human platelets neither bound 125-I-fibrinogen nor 125-I-fibrin. Fibrin-binding was, however, stimulated by N-terminal fibronectin 30 kD-and 70 kD-fragments while fibronectin was ineffective. The 30 kD-fragment also stimulated some platelet preparations to bind fibrinogen which, however, was suppressed by minute amounts of the thrombin inhibitor PPACK. PPACK hardly influenced fibrin-binding. Fragment-promoted fibrinogen-binding was also inhibited by a monoclonal antibody recognizing the membrane glycoprotein IIb/IIIa complex known to act as fibrinogen receptor. This antibody failed to influence fragment-stimulated fibrin-binding giving evidence that fibrinogen and fibrin were retained by different receptors. In contrast to 125-I-fibrin its plasmin-derived and 125-I-labelled fragment X was not recognized by the platelets in presence of the fibronectin 30 kD-fragment. Fragment-stimulated binding of 125-I-fibrin showed a lag phase and was completely inhibited by 0.25 mM putrescine as well as by 1 mM EDTA or 0.1 mM N-ethylmaleinimide. Evidently, a cell-attached transamidase was involved in fibrin-binding possibly by forming a ternary complex with fibrin and the fibronectin fragment.
    No preview · Article · Sep 1988 · Thrombosis Research
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    G Schmidt · H Robenek · B Harrach · J Glössl · V Nolte · H Hörmann · H Richter · H Kresse
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    ABSTRACT: Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.
    Full-text · Article · Jul 1987 · The Journal of Cell Biology
  • Helmut HÖRMANN · Hartmut RICHTER · V Jelinić · Christa WENDT
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    ABSTRACT: Polystyrene beads coated with thrombin-activated Factor XIII (plasma transglutaminase, plasma transamidase) retained soluble 125I-fibrin, if a 30-kDa fragment of fibronectin was present. The fragment was obtained by proteolytic cleavage of plasma fibronectin with trypsin, but was also available with plasmin or thrombin. It represented a fibrin-binding domain at the N-terminus of each of the two subunit chains and contained close to its amino end a transamidase-reactive site. Fibronectin was unable to mediate the binding of 125I-fibrin to Factor XIIIa-coated beads. 125I-fibrinogen was hardly recognized by the beads even in presence of the fibronectin fragment. The relatively slow binding of 125I-fibrin was inhibited by 0.15 mM putrescine or by a pretreatment of the coated beads with EDTA or N-ethylmaleinimide indicating the involvement of a transamidation in the binding reaction. Immobilization of 125I-fibrin in presence of the fibronectin fragment is assumed to require a covalent cross-linking of the two ligands at the immobilized transamidase giving rise to a product which is retained strongly. The possibility is discussed that a surface-attached transamidase might act as a fibrin receptor which requires the fibronectin fragment as a cofactor.
    No preview · Article · Jul 1987 · Biological chemistry Hoppe-Seyler
  • Helmut Hörmann · Hartmut Richter · Viktorija Jelinić
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    ABSTRACT: Proteolytic fragments from the N-terminus of the fibronectin subunit chains were shown to mediate the binding of 125-I-fibrin to macrophages. With increasing molecular weight of the fragments, binding activity decreased and intact plasma fibronectin was inactive. Fibrin binding to macrophages was a time dependent reaction and proceeded considerably faster than binding of fibrinogen. The binding reaction was inhibited by putrescine suggesting the involvement of a transamidase. Pericellular transamidase was demonstrated on macrophages by incorporation of 14-C-putrescine into fibronectin 30 kD-fragment. Expression of this enzyme appeared to be rate-limiting for the binding reaction which was accelerated after loading the cells with placental transamidase.
    No preview · Article · May 1987 · Thrombosis Research
  • Helmut Hörmann · Hartmut Richter
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    ABSTRACT: Fibronectin, consisting of two threadlike subunits connected close to their C-terminal ends, exists in a soluble and a fibrous form. Models are presented that propose distinct foldings of the rodlike subunits in the soluble dimer and an extended arrangement in the aggregates. The proposed conformations are based on the analysis of electrostatic attractions and noncovalent affinities between domains. Electrofocusing of proteolytic fragments revealed a sequence of four domains with alternating charges in the N-terminal half of each subunit and two domains with opposite charges close to the C-terminus. Complementary sites with affinity to each other were localized by radioimmuno-binding assay in the gelatin-binding and in the subsequent DNA-binding domain of the N-terminal tetra-domain sequence. Claiming that in soluble fibronectin electrostatic attractions and noncovalent affinities should be neutralized within the molecule resulted in the construction of a conformation with backfolded subunits, each containing an extra loop in which domains with complementary affinity sites are saturated by each other. The model is in accord with hydrodynamic and electronmicroscopic data. There is, however, an alternative folding in which electrostatic and noncovalent affinity sites in the N-terminal half of each subunit are saturated by an interchain interaction within the molecule. Consequently, a rearrangement of the molecule without significant shape change cannot be excluded. In the aggregated form, the N-terminal tetra-domain sequence gives rise to an intermolecular interaction while the C-terminal domains become available for binding ligands.
    No preview · Article · Jun 1986 · Biopolymers
  • H Hörmann

    No preview · Article · Dec 1985 · Blut
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    ABSTRACT: The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)
    No preview · Article · Nov 1985 · Biological chemistry Hoppe-Seyler
  • Helmut Hörmann

    No preview · Article · Oct 1985 · Annals of Hematology
  • Hartmut RICHTER · Christa WENDT · Helmut HÖRMANN
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    ABSTRACT: The precipitation of plasma fibronectin by heparin in dependence on various parameters was investigated. Rising heparin concentration augmented the precipitates up to a maximum beyond which precipitation decreased. Yields close to 80% were obtained at low temperatures, but some precipitation was observed at 37 degrees C as well. Insolubilization was considerably dependent on the ionic strength, indicating that electrostatic forces play a major role in the aggregation of fibronectin. Calcium already prevented precipitation by heparin at low concentrations. If precipitation was performed on hydrophobized glass cover slides, the formation of fibrils visible by phase-contrast microscopy was observed. On hydrophilic surfaces amorphous precipitates were generally obtained, most likely due to trapping of aggregates by adsorption prior to their arrangement to fibrils. The results are discussed on the basis of a model assuming that heparin induces a conformational rearrangement of plasma fibronectin so that masked binding sites responsible for self-association become exposed.
    No preview · Article · Jun 1985 · Biological chemistry Hoppe-Seyler
  • Helmut HÖRMANN · Hartmut RICHTER · V Jelinić
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    ABSTRACT: Various proteolytic fragments from the central region of the fibronectin subunit chains containing the main cell-affinity site were applied in cell binding studies using peritoneal macrophages of guinea pigs. A 125I-labelled 23-kDa peptide was relatively well bound by the cells. Attachment to cells was partially inhibited by wheat germ lectin, suggesting a lectin-like site in the cell-binding domain which recognizes oligosaccharide groups with terminal N-acetylglucosamine or N-acetylneuraminic acid. Binding was inhibited by N-acetylneuraminic acid with half-maximal effect at 2 X 10(-3) M. Other inhibitors were a sialic acid rich ganglioside preparation and fetuin, a sialic acid-containing glycoprotein. In contrast to the 23-kDa peptide a 125I-labelled 125-kDa fragment was only weakly bound, although it included the sequence of the 23-kDa peptide on its C-terminus. The residual binding was weakly inhibited by low concentrations of wheat germ lectin and was remarkably improved by higher concentrations. The behavior of the peptide was explained by the presence of a sialic acid-containing oligosaccharide side chain localized outside of the 23-kDa region and interacting with the lectin-like site in the cell-binding sequence. In accord with this suggestion a 95-kDa fragment representing the oligosaccharide-containing part of the 125-kDa peptide was capable of inhibiting at least partially the cell attachment of the 23-kDa piece. The results indicate a lectin-like affinity site in the cell-binding region of fibronectin which is accessible in the 23-kDa peptide, but is masked in the 125-kDa fragment and in fibronectin by a sialic acid-containing oligosaccharide moiety.
    No preview · Article · Jun 1984 · Hoppe-Seyler's Zeitschrift für physiologische Chemie
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    ABSTRACT: At low ionic strength (0.05 M) the sedimentation coefficient of monomeric plasma fibronectin was found to vary from 8S, at pH 3 and 11, to 13.5S at neutral pH. The lower s20,w value indicates a stretched arrangement of the value indicates a stretched arrangement of the two arms of the molecule which was observed in most electron microscopic studies. The higher value is consistent with a still very asymmetric but more condensed shape, which is probably brought about by back-folding and interactions between chain segments of different net charge. A model of this internal association is based on the finding that segments of rather different isoelectric points alternate along the fibronectin chains. A similar pH dependence was observed for a 140-kDa fragment from the middle region of fibronectin which carries segments of low and high isoelectric points at its ends. At high ionic strength (0.35 M) the pH dependence of the sedimentation coefficients was less pronounced and intermediate s20,w values were found. This is expected when both repulsive and attractive interactions are weakened by the electrolyte. It was verified by circular dichroism spectra that the protein was not denatured at pH 3 or 11. Thermal transition curves revealed a destabilization at pH 3 but the thermal denaturation occurred well above 20 degrees C at which the pH dependence of the solution shape was studied.
    No preview · Article · Jan 1984 · Hoppe-Seyler's Zeitschrift für physiologische Chemie
  • H Hörmann · V Jelinić · H Richter
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    ABSTRACT: A chymotrypsin-derived and 125I-labelled 125-kDa fragment of human plasma fibronectin which contained the cell binding site, was only weakly bound by peritoneal macrophages of guinea pigs and binding was not saturable. In presence of wheat germ lectin binding increased proportionally to the logarithm of the lectin concentration. Association of 125I-fragment with cells was partially prevented by non-labelled fragment indicating a saturable receptor-ligand interaction. An apparent affinity constant of about 2--4 x 10(-5) M was evaluated. A considerable fraction of the cell-bound 125I-fragment resisted removal by proteases suggesting that it was internalized. In order to investigate an influence of wheat germ lectin on the binding of 125I-fibronectin by the cells the macrophages were preincubated with the lectin followed by washing and evaluation of 125I-fibronectin binding. A simultaneous incubation of the cells with 125I-fibronectin and lectin was impractical due to partial interaction of the two proteins giving rise to some unspecific precipitates. Preincubation with wheat germ lectin considerably improved the capacity of the macrophages for binding of 125I-fibronectin. Again the binding of 125I-labelled protein could be restricted by unlabelled one. N-acetyl-glucosamine inhibited the binding of 125I-fibronectin by wheat germ lectin-treated cells if applied in the preincubation phase and more effectively, if applied in the final 125I-fibronectin binding assay. N-Acetylneuraminic acid also inhibited this step. In addition to wheat germ lectin concanavalin A was capable of generating fibronectin receptors on the cell surface. Soy bean lectin, however, was ineffective.
    No preview · Article · Sep 1983 · Hoppe-Seyler's Zeitschrift für physiologische Chemie
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    H Richter · H Hörmann
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    ABSTRACT: A mild cathepsin D digest of fibronectin only contained single-chain peptides of 200, 140 and 70 kDa and double-chain fragments of about 300 and 140 kDa containing the C-terminal disulfide link. Among the single-chain fragments the 200 kDa peptide was a precursor of the 140 kDa and 70 kDa peptides. The latter was correlated to the N-terminal and the former to the central region of the fibronectin subunit chains.
    Preview · Article · Jun 1983 · FEBS Letters