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Biomedical subjects

J Stürzebecher

Publications and source records attributed to J Stürzebecher.

At least 19 recordsLinked to original sources
PubMed2003

Blood coagulation and fibrinolysis after extreme short-term exercise.

INTRODUCTION: Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s. METHODS: A total of 15 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of 15, 45, and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, 15 min, and 1 h after completion of exercise. For the investigation of blood coagulation, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complex (TAT), intrinsic and extrinsic total (TTPin+ex), and endogenous thrombin potential (ETPin+ex) were measured. For testing fibrinolysis, determinations of plasmin-alpha(2)-antiplasmin complex (PAP), tissue-type plasminogen activator (tPA)-antigen, plasminogen activator inhibitor (PAI)-1-antigen and D-dimer were used. RESULTS: Immediately after the exercise tests, only F1+2 (15- and 90-s test) and TTPin (45 and 90 s) showed a moderate increase (p<0.05), while TAT and ETP was unchanged. In contrast, a clear increase in PAP and tPA-antigen already after 15 s maximal exercise in relation to the exercise duration time could be investigated. These effects were not totally reversed to baseline 15 min after exercise; D-dimer and PAI-1-antigen still remained unchanged after these types of exercise. CONCLUSIONS: Maximal short-term exercise does not lead to a relevant activation of blood coagulation in healthy young subjects, it is only slightly altered within the normal range. In contrast, fibrinolysis is clearly activated, and the increase is directly dependent on exercise duration. Additionally, it could be shown for the first time that fibrinolysis is already activated after 15 s maximal exercise duration.

Adult↗
PubMed2002

Thrombin potential and thrombin generation after exhaustive exercise.

Exhaustive exercise leads to an activation of blood coagulation, but the implications of this activation are still unclear. The aim of this study was to investigate if a hypercoagulant stage exists after exhaustive treadmill- or cycle exercise; intrinsic and extrinsic endogenous thrombin potential (ETP) were measured by using the method of Hemker et al. Thirteen healthy male subjects underwent an exhaustive treadmill (TR) or cycle (CY) ergometer test and a control-day in random order. Blood samples were taken, repeatedly, after a 30 min rest, immediately before and after, and 1 h after exercise for measuring intrinsic and extrinsic total thrombin potential (TTPin, TTPex) (including free and alpha 2 -macroglobulin-bound thrombin) and endogenous thrombin potential (ETPin, ETPex), aPTT, PT, F1 + 2 and TAT. In comparison to the pre-value taken immediately before the exercise, the intrinsic TTP was significantly (p < 0.05) increased directly after exercise (TR-TTPin, + 11.6 %; CY-TTPin, + 11.5 %). In contrast, ETPin remained unchanged after both exercises. Additionally for TTPex and ETPex, no changes after exercise were detectable. aPTT was significantly (p < 0.05) shorter after exercise (TR-aPTT, - 16.2 %; CY-aPTT - 17.5 %), F1 + 2-concentrations were higher (p < 0.05) (TR-F1 + 2, + 21.2 %; CY-F1 + 2, + 9.8 %), but TAT remained unchanged. Differences between TR or CY could not be determined. These results show the expected shortening of aPTT and the increase of F1 + 2 indicating an activation of the coagulation system during exercise. However, the unchanged intrinsic and extrinsic ETP lead to the conclusion that in healthy young male subjects the potential for thrombin generation is insignificant, is directly counterbalanced by alpha 2-macroglobulin and is independent of the type of exhaustive exercise done.

Adult↗
PubMed2002

[Inhibitors of factor XIIIa].

Factor XIIIa (FXIIIa) catalyzes the covalent crosslinking of fibrin polymers and incorporation of proteins into the fibrin network and thus confers on the thrombus additional structural stability and relative resistance to plasmin-mediated degradation. Moreover, FXIIIa is involved in other physiological and pathophysiological processes such as wound healing and arteriosclerosis. Selective FXIIIa inhibitors may be a valuable tool for evaluation of the various functions of FXIIIa and their pharmacological control. This paper presents an overview of the inhibitors of FXIIIa. Analogues of natural FXIIIa substrates - including glutamine containing peptides and low molecular weight substituted alkylamines - are incorporated into the fibrin network and thus prevent crosslinking of fibrin. Naturally occurring, direct inhibitors of FXIIIa have been isolated from a leech species and microorganisms. With effective concentrations in the nanomolar range the peptide tridegin is the most potent FXIIIa inhibitor up to now. The majority of the synthetic, low molecular Weight inhibitors bind covalently to Cys314 at the active site of FXIIIa. Besides the relatively nonspecific thiol reagents, azol derivatives, azolium salts and related substances are described as specific inhibitors of FXIIIa. They inhibit the activity of FXIIIa at nanomolar concentrations. Animal experiments have demonstrated improved thrombolysis by a plasminogen activator in combination with a FXIIIa inhibitor.

Arteriosclerosis↗
PubMed2001

Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition.

The binding of a series of low molecular weight ligands towards trypsin and thrombin has been studied by isothermal titration calorimetry and protein crystallography. In a series of congeneric ligands, surprising changes of protonation states occur and are overlaid on the binding process. They result from induced pK(a) shifts depending on the local environment experienced by the ligand and protein functional groups in the complex (induced dielectric fit). They involve additional heat effects that must be corrected before any conclusion on the binding enthalpy (DeltaH) and entropy (DeltaS) can be drawn. After correction, trends in both contributions can be interpreted in structural terms with respect to the hydrogen bond inventory or residual ligand motions. For all inhibitors studied, a strong negative heat capacity change (DeltaC(p)) is detected, thus binding becomes more exothermic and entropically less favourable with increasing temperature. Due to a mutual compensation, Gibbs free energy remains virtually unchanged. The strong negative DeltaC(p) value cannot solely be explained by the removal of hydrophobic surface portions of the protein or ligand from water exposure. Additional contributions must be considered, presumably arising from modulations of the local water structure, changes in vibrational modes or other ordering parameters. For thrombin, smaller negative DeltaC(p) values are observed for ligand binding in the presence of sodium ions compared to the other alkali ions, probably due to stabilising effects on the protein or changes in the bound water structure.

Animals↗
PubMed2001

Transport of peptidomimetic thrombin inhibitors with a 3-amidino-phenylalanine structure: permeability and efflux mechanism in monolayers of a human intestinal cell line (Caco-2).

PURPOSE: Peptidomimetic thrombin inhibitors derived from Nalpha-(2-naphthylsulfonyl)-3-amidino-phenylalanine with different basic and acidic substituents were investigated with respect to their intestinal transport behavior. METHODS: Intestinal permeability coefficients were studied using Caco-2 monolayers and a reversed-phase HPLC method for quantitation. RESULTS: Apparent permeability coefficients Papp of compounds with a free amidino group were in general low (<10 x 10(-8) cm/s) and independent of the structure of the amide part (C-terminus). Polarized efflux, however, was strongly affected by substituents in the amide moiety yielding the following efflux ratios (ER): methylpiperidide (1) (ER 45) > piperidine carboxylic acid methylester (ER 6-11) > piperidine carboxylic acids (ER 1.9-2.9) > piperazide (ER -0.17). Efflux of (1) was temperature-dependent, but independent of the enantiomeric configuration, accompanied by an increase in transepithelial electrical resistance (TEER), and could be reduced by P-gp inhibitors (PSC 833, Cremophor EL) but not by indomethacin. Replacement of the amidine group of (1) by aminomethyl-, amino-, and oxamidine- moieties drastically increased absorptive permeability (46-68 fold) with ER < 3.4. In contrast, the oxamidine with a C-terminal nipecotic acid residue (8) displayed also a temperature dependent efflux- without altering TEER (ER 22). This efflux was sensitive to PSC833/Cremophor EL and indomethacin. CONCLUSIONS: Basic and acidic residues of amidino-phenylalanine-derived thrombin inhibitors mediate affinity to intestinal efflux pumps. presumably P-gp and MRP. P-gp mediated efflux was related to a net positive charge and accompanied by an increased TEER. Among the methylpiperide (1) promoieties studied the oxamidino group seems to be very promising in overcoming both transport and efflux problems frequently encountered with peptidomimetics containing amidines.

Amidines↗
PubMed2001

Structure-activity relationships of new NAPAP-analogs.

Several new analogs of the known thrombin inhibitor NAPAP were synthesized, in which the P2 glycine residue was substituted by natural and unnatural amino acids. The thrombin inhibitory potency was comparable to that of NAPAP. Several of the compounds had inhibition constants lower than 10 nM and a very high selectivity compared to trypsin, factor Xa and plasmin. In addition, analogs were prepared by alkylation of the N alpha-atom of the 4-amidinophenylalanine in P1 position, which showed a more than 10-fold lower thrombin inhibition. Furthermore, azaglycine was introduced instead of P2 glycine. For most of the inhibitors similar fast elimination rates were seen in rats after intravenous dosing, as found previously for NAPAP. Only some compounds, which contained a second basic group showed a slightly decreased cumulative biliary clearance.

Animals↗
PubMed2001

Synthetic urokinase inhibitors as potential anti-invasive drugs.

In carcinogenesis, proteinases play an important role in metastasis of solid malignant tumors. Aside from several matrix metalloproteinases and cathepsins, the urokinase-plasmin system seems to be one of the main players within the cell surface-associated proteolytic activity, facilitating tumor cell migration and invasion. Upon binding to a specific receptor, the enzymatic activity of urokinase is focused to the cell surface. The significance of the plasminogen activator system in malignancy is underlined by the finding of elevated levels of urokinase and its receptor in tumor tissues. Therefore, the possible use of urokinase inhibitors in the therapeutic treatment of cancer and metastasis has been the subject of intensive investigations. This review covers synthetic inhibitors of urokinase described up to December 2000, although the number of lead structures is still relatively small.

Journal Article↗
PubMed2000

Crystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Amiloride↗
PubMed2000

(4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase.

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure-activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1'/S2' subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Amino Acid Sequence↗
PubMed2000

Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds.

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.

Chronic Disease↗
PubMed2000

Urethanyl-3-amidinophenylalanine derivatives as inhibitors of factor Xa. X-ray crystal structure of a trypsin/inhibitor complex and modeling studies.

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the arginine-mimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the X-ray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R)- and (S)-derivatives, combined with modeling experiments, led to an hypothetical non-substrate like binding mode, which was fully confirmed by the remarkably enhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl-(R)-3-amidinophenylalanine-phenethylamide+ ++ a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.

Animals↗
PubMed2000

New bivalent thrombin inhibitors with N(alpha)(methyl)arginine at the P1-position.

A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.

Amino Acid Sequence↗
PubMed1999

3-Amidinophenylalanine-based inhibitors of urokinase.

Synthesis and anti-uPA activity of a series of Nalpha-triisopropyl-phenylsulfonyl-protected 3-amidinophenylalanine amides are described. We have explored SAR around the C-terminal amide part for inhibition of uPA, plasmin and trypsin. Modification of the amide part has been found to affect potency but not selectivity. With a Ki of 0.41 microM 2r-L is one of the most potent uPA inhibitors described so far. The X-ray crystal structure of 2r-L was solved in complex with trypsin, superimposed with uPA and the results suggest an unique binding mode of this inhibitor type.

Benzamidines↗
PubMed1999

The structure of the human betaII-tryptase tetramer: fo(u)r better or worse.

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-A crystal structure of human betaII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 A x 15 A govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

Amino Acid Sequence↗
PubMed1999

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.

Amino Acid Sequence↗