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Thrombin Cleavage (thrombin + cleavage)

Distribution by Scientific Domains

Medical Sciences	50%
Chemistry	50%

Selected Abstracts

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides

FEBS JOURNAL, Issue 20 2000
Jocelyne Guay
The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S -transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S -transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6,6.3 nm for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nm) and cathepsin L (Ki = 0.12 nm) compared with cathepsin S (Ki = 65 nm). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nm) than cathepsin S (Ki = 7.6 nm) itself or cathepsin K (Ki = 7.0 nm). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J.E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745,750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained. [source]

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes ,a',

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007
N. OKUMURA
Summary. Background:,Fibrin polymerization is mediated by interactions between knobs ,A' and ,B' exposed by thrombin cleavage, and holes ,a' and ,b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous.Objectives:,To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ,a': ,364Ala, ,364His or ,364Val.Methods:,We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis.Results:,Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of ,364Val and ,364His were more delayed than ,364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ,A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes ,a' and ,b', respectively. FXIIIa-catalyzed crosslinking between ,-chains was markedly delayed for all the variants.Conclusion:,These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ,a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation. [source]

Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010
Lu Lu
Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the ,-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289,K. The crystal diffracted to 1.6,Å resolution using synchrotron radiation and belonged to space group I41, with unit-cell parameters a = 55.0, b = 55.0, c = 67.4,Å. [source]

Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability

BRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2007
Veronica H. Flood
Summary Previous studies have shown that a heterozygous mutation in the fibrinogen A, chain gene, which results in an A, R16C substitution, causes fibrinolytic resistance in the fibrin clot. This mutation prevents thrombin cleavage of fibrinopeptide A from mutant A, R16C chains, but not from wild-type A, chains. However, the mechanism underlying the fibrinolytic resistance is unclear. Therefore, this study investigated the biophysical properties of the mutant fibrin that contribute to fibrinolytic resistance. Fibrin clots made from the mutant fibrinogen incorporated molecules containing fibrinopeptide A into the polymerised clot, which resulted in a ,spiky' clot ultrastructure with barbed fibrin strands. The clots were less stiff than normal fibrin and were cross-linked slower by activated FXIII, but had an increased average fiber diameter, were more dense, had smaller pores and were less permeable. Protein sequencing showed that unclottable fibrinogen remaining in the supernatant consisted entirely of homodimeric A, R16C fibrinogen, whereas both cleaved wild-type , chains and uncleaved A, R16C chains were in the fibrin clot. Therefore, fibrinolytic resistance of the mutant clots is probably a result of altered clot ultrastructure caused by the incorporation of fibrin molecules containing fibrinopeptide A, resulting in larger diameter fibers and decreased permeability to fibrinolytic enzymes. [source]