The covalent nature of the human antithrombin III--thrombin bond.
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ABSTRACT:
SUBMITTER: Longas MO
PROVIDER: S-EPMC1162028 | biostudies-other | 1980 Sep
REPOSITORIES: biostudies-other
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Project description:The inactivation of thrombin by heat and by its physiological inhibitor, antithrombin-III, shows quite different dependence on heparin concentration. Heparin at 250 microgram/ml protects thrombin against heat inactivation, and thrombin behaves heterogeneously in this reaction. In the absence of heparin, the thermodynamic activation parameters change with temperature (deltaH+ = 733 kJ/mol and 210 kJ/mol at 50 and 58 degrees C respectively). When heparin is present, heat inactivation of the protected thrombin species proceeds with deltaH+ = 88 kJ/mol and is independent of temperature in the same range. On the other hand, heparin at 0.125-2.5 microgram/ml accelerates the thrombin-antithrombin-III reaction. Thrombin does not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0 and 37 degrees C appear to follow first-order kinetics. Activation enthalpy is independent of heparin concentration or temperature, deltaH+ = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin specific activity. Heparin seems to exert its effect by increasing activation entropy. On the basis of these data we suggest a mechanism of action of heparin in the thrombin-antithrombin-III reaction which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.
| S-EPMC1185853 | biostudies-other
Project description:Complexes of the form [U(η6-arene)(BH4)3] where arene = C6H6; C6H5Me; C6H3-1,3,5-R3 (R = Et, iPr, tBu, Ph); C6Me6; and triphenylene (C6H4)3 were investigated towards an understanding of the nature of the uranium-arene interaction. Density functional theory (DFT) shows the interaction energy reflects the interplay between higher energy electron rich π-systems which drive electrostatic contributions, and lower energy electron poor π-systems which give rise to larger orbital contributions. The interaction is weak in all cases, which is consistent with the picture that emerges from a topological analysis of the electron density where metrics indicative of covalency show limited dependence on the nature of the ligand - the interaction is predominantly electrostatic in nature. Complete active space natural orbital analyses reveal low occupancy U-arene π-bonding interactions dominate in all cases, while δ-bonding interactions are only found with high-symmetry and electron-rich C6Me6. Finally, both DFT and multireference calculations on a reduced, formally U(ii), congener, [U(C6Me6)(BH4)3]-, suggests the electronic structure (S = 1 or 2), and hence metal oxidation state, of such a species cannot be deduced from structural features such as arene distortion alone. We show that arene geometry strongly depends on the spin-state of the complex, but that in both spin-states the complex is best described as U(iii) with an arene-centred radical.
| S-EPMC10829017 | biostudies-literature
Project description:As an important intermolecular interaction, halogen bonding has been studied extensively, but its nature still suffers from controversy without one uniform essence. Electrostatics, charge transfer, polarization and dispersion are emphasized, but the covalent nature is usually overlooked except for the strong halogen bonding species I3 -, which is widely accepted as a result of a three-center four-electron (3c-4e) interaction. In our study, the potential energy surface of I3 - has been evaluated to explore the dissociation from I3 - to I2⋯I-. We found that different from an equivalent 3c-4e bond in I3 -, I2⋯I- can be rationalized by a polarized one. In addition, when the orbitals are polarized, it is exactly what traditional charge transfer or the popular σ-hole picture describes. I3 - can be described by the Lewis theory model with the middle I+ cation serving as the Lewis acid and two terminal I- anions acting as Lewis base. Therefore, we further extended this model to a series of I-containing species with chemical composition of L-I+-L, F--I+-L and H3P-I+-L (L = OH-, F-, Cl-, Br-, I-, PH3, NH3, H2S, HI, H2O, HBr and HCl) to explore the nature of halogen bonding. When the forces of two bases around I+ are the same, it corresponds to an equivalent 3c-4e bond, such as I3 -. Otherwise, it is a polarized multicenter bond, such as I2⋯I-. This work gives a new insight into the nature of halogen bonding compounds: besides the well-known I3 -, the nature of the other species is also a multicenter bond, existing as equivalent and polarized 3c-4e bonds, respectively.
| S-EPMC9042191 | biostudies-literature
Project description:IntroductionIn recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.MethodsWe have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.ResultsWe demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.ConclusionCompound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.
| S-EPMC6072017 | biostudies-literature
Project description:Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.
| S-EPMC4207992 | biostudies-literature
Project description:The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.
| S-EPMC1144217 | biostudies-other
Project description:Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.
| S-EPMC7924598 | biostudies-literature
Project description:The factors responsible for the enhancement of the halogen bond by an adjacent hydrogen bond have been quantitatively explored by means of state-of-the-art computational methods. It is found that the strength of a halogen bond is enhanced by ca. 3 kcal/mol when the halogen donor simultaneously operates as a halogen bond donor and a hydrogen bond acceptor. This enhancement is the result of both stronger electrostatic and orbital interactions between the XB donor and the XB acceptor, which indicates a significant degree of covalency in these halogen bonds. In addition, the halogen bond strength can be easily tuned by modifying the electron density of the aryl group of the XB donor as well as the acidity of the hydrogen atoms responsible for the hydrogen bond.
| S-EPMC8036253 | biostudies-literature
Project description:Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.
| S-EPMC6829041 | biostudies-literature
Project description:AimsAntithrombin III (AT-III) concentrates have been used to prevent critical thrombosis in the immediate post-liver transplantation period without clear evidence regarding the optimal dose or administration scheme. The relationship between the AT-III dosage and the plasma activity levels during the period was evaluated in this study.MethodsThe plasma AT-III activity levels and clinical data obtained from patients who received liver transplantation from January 2017 to September 2018 were retrospectively analysed. A population pharmacokinetic (PK) model was developed using nonlinear mixed-effects method and externally validated thereafter. Several dosing scenarios were simulated to maintain the plasma AT-III activity level within the normal range using the developed PK model to search for an optimal AT-III dosing regimen.ResultsThe plasma AT-III activity levels were best described by a single compartment model with first order elimination kinetics. The recovery of endogenous AT-III level during the postoperative days was modelled using an Emax model. The typical values (95% confidence interval) of volume of distribution and clearance were 3.86 (3.40-4.32) L, and 0.129 (0.111-0.147) L h-1 , respectively. Serum albumin and body weight had significant effect on clearance and were included in the model. External validation of the proposed model demonstrated adequate prediction performance. Furthermore, simulation of previously suggested or modified dosing scenarios showed successful maintenance of AT-III activity level within the normal range.ConclusionA population PK model of AT-III concentrate was developed using data from liver recipients. Dosing scenarios simulated in our study may help establish a practical guide for AT-III concentrate titration after liver transplantation.
| S-EPMC7163384 | biostudies-literature