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Catabolism of hirudin and thrombin-hirudin complexes in the rat.

ABSTRACT: The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney.

SUBMITTER: Bichler J

PROVIDER: S-EPMC1137762 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Project description:Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range. The one-dimensional (1)H nuclear magnetic resonance spectrum recorded at 600 MHz reveals a resonance 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in complexes of human α-thrombin with hirunorm IV, hirunorm V, an Nα(Me)Arg peptide, RGD-hirudin, and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-Donor-acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from the observed signal [Kovach, I. M., et al. (2009) Biochemistry 48, 7296-7304] for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin complexes are near 1.0 within 20% error. The most likely site of the short H-bond in complexes of thrombin with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu(57') and Glu(58') are embedded and interact with Arg(75) and Arg(77) and their solvate water (on thrombin). Glu(57') and Glu(58') present in the hirudin family of inhibitors make up a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the short hydrogen bond as a binding element at the fibrinogen recognition site.

Project description:Purpose: To reveal the effects of thrombin on the proliferation, morphology, and gene expression in chondrocytes. Methods: Rat primary chondrocytes were cultured with RS, RP and RPT for 24hours, and RNA was extracted for sequencing. The chondrocytes’ morphology was investigated under inverted microscope. Chondrocyte’s proliferation was evaluated by Edu staining, CCK8 methods and PPI network of proliferation-associated genes. The differential gene expression was visualized using a volcano plot. The variance in expression levels of the genes was visualized by the heatmap. GO-based functional annotation clustering analysis was performed on the upregulated and downregulated DEGs, which included biological process, cellular component, and molecular function. The endoplasmic reticulum of chondrocytes from three groups were labeled by molecular probes. The expression levels of Col2a1 and proteoglycan synthesis were detected by cellular immunofluorescence and Safranin O staining separately. The expression levels of Lox were determined by immunocytochemistry staining. The following genes including Comp, Eln, P3H1 and Colgalt1 were examined by RT-qPCR. The expression of MMPs and Sox9 were evaluated by Western Blot. Further analysis of PPI network was performed, and the corresponding modules were selected. Results: As compared with RP group, chondrocytes proliferation was obvious and normal chondrocytes quickly transformed into fibroblast-like chondrocytes in the presence of RS and RPT. we found that 727 genes were up-regulated, and 1162 genes were down-regulated in Venn diagram through analyzing differentially expressed genes (DEGs). The results of GO analyses showed that both the expression of ECM and MMPs were significantly influenced by RS and RPT. Both the labeled endoplasmic reticulum and the expression of representative genes analyzed from the results of RNA sequencing confirmed that chondrocytes were dominated by catabolism. The expression of ECM, MMPs, inflammatory factors and chemokines were verified at the protein level or at the RNA level which agreed with PPI network and functional modules. Conclusions: Thrombin stimulated the proliferation and promoted the morphology transformation towards fibrotic chondrocytes. Thrombin improved the chondrocytes’ catabolism by regulating the expression of the key enzymes which were responsible for ECM synthesis. Meanwhile thrombin increased the expression of MMPs and chemokines that aimed at extracellular ECM degradation.

Project description:A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human alpha-thrombin and hirunorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 A resolution and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"-Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C-termninal tail and related compounds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite-directed synthetic thrombin inhibitors.

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Catabolism of hirudin and thrombin-hirudin complexes in the rat.

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