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Molecular Aspects of Medicine Aug 2008Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the... (Review)
Review
Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarizes our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+ -free slow from E and the Na+ -bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+ -free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na + and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes.
Topics: Allosteric Regulation; Animals; Anticoagulants; Blood Coagulation; Humans; Thrombin
PubMed: 18329094
DOI: 10.1016/j.mam.2008.01.001 -
BioMed Research International 2013
Topics: Blood Coagulation; Clinical Laboratory Techniques; Humans; Molecular Diagnostic Techniques; Thrombin; Translational Research, Biomedical
PubMed: 23762833
DOI: 10.1155/2013/269194 -
Journal of Thrombosis and Haemostasis :... Oct 2016Thrombin is a multifunctional serine protease produced from prothrombin, and is a key regulator in hemostatic and non-hemostatic processes. It is the main effector... (Review)
Review
Thrombin is a multifunctional serine protease produced from prothrombin, and is a key regulator in hemostatic and non-hemostatic processes. It is the main effector protease in primary hemostasis by activating platelets, and plays a key role in secondary hemostasis. Besides its well-known functions in hemostasis, thrombin also plays a role in various non-hemostatic biological and pathophysiologic processes, predominantly mediated through activation of protease-activated receptors (PARs). Depending on several factors, such as the concentration of thrombin, the duration of activation, the location of PARs, the presence of coreceptors, and the formation of PAR heterodimers, activation of the receptor by thrombin can induce different cellular responses. Moreover, thrombin can have opposing effects in the same cell; it can induce both inflammatory and anti-inflammatory signals. Owing to the complexity of thrombin's signal transduction pathways, the exact mechanism behind the dichotomy of thrombin is yet still unknown. In this review, we highlight the hemostatic and non-hemostatic functions of thrombin, and specifically focus on the non-hemostatic dual role of thrombin under various conditions and in relation to cardiovascular disease.
Topics: Animals; Blood Coagulation; Blood Platelets; Cardiovascular Diseases; Hemostasis; Humans; Inflammation; Platelet Activation; Protein Multimerization; Receptors, Proteinase-Activated; Receptors, Thrombin; Serine Endopeptidases; Serine Proteases; Signal Transduction; Thrombin; Transcriptional Activation
PubMed: 27513692
DOI: 10.1111/jth.13441 -
The Journal of Biological Chemistry Jun 2020The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms....
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.
Topics: Crystallography, X-Ray; Fluorine; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation; Protein Precursors; Prothrombin; Thrombin
PubMed: 32358061
DOI: 10.1074/jbc.RA120.013419 -
Journal of Thrombosis and Haemostasis :... Jul 2007Thrombin is a Na(+)-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na(+) is the major driving force behind... (Review)
Review
Thrombin is a Na(+)-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na(+) is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. This basic regulatory feature of thrombin has fostered the rational engineering of mutants with selectively compromised fibrinogen and PAR1 cleavage. The discovery of the Na(+) effect on thrombin interaction with substrates and the mapping of functional epitopes by Ala scanning mutagenesis have provided a rational and effective strategy for dissociating the procoagulant and anticoagulant activities of the enzyme. Thrombin mutants with selectively compromised activity toward fibrinogen and PAR1 are effective in vivo as anticoagulant and antithrombotic agents.
Topics: Animals; Anticoagulants; Coagulants; Humans; Thrombin
PubMed: 17635727
DOI: 10.1111/j.1538-7836.2007.02485.x -
Protein Science : a Publication of the... Dec 2023Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy...
From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).
Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (K = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a K of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (K = 1.9 μM) and its potency was enhanced by 10-fold after Phe3 → β-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (K = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (K = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.
Topics: Hirudins; Thrombin; Amino Acid Sequence; Peptides; Heme
PubMed: 37924304
DOI: 10.1002/pro.4825 -
Journal of Biomedicine & Biotechnology 2010Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors... (Review)
Review
Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor X(a). Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.
Topics: Animals; Blood Coagulation; Fibrinolytic Agents; Humans; Thrombin
PubMed: 20976270
DOI: 10.1155/2010/641025 -
Blood Oct 2005Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of... (Review)
Review
Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.
Topics: Allosteric Regulation; Animals; Coenzymes; Hemostasis; Humans; Substrate Specificity; Thrombin
PubMed: 15994286
DOI: 10.1182/blood-2005-04-1710 -
Nucleic Acids Research Jul 2020While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases...
While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Humans; Protein Binding; Thrombin; Vascular Endothelial Growth Factor A
PubMed: 32442276
DOI: 10.1093/nar/gkaa405 -
Journal of Thrombosis and Haemostasis :... Jul 2003The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiation... (Review)
Review
The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiation phase, small amounts of factor (F)Xa, FIXa and thrombin are generated. The latter activates the procofactors FV and FVIII to the activated cofactors which together with their companion serine proteases form the intrinsic FX activator (FVIIIa-FIXa) and prothrombinase (FVa-FXa) which generate the bulk of FXa and thrombin during a propagation phase. The clotting process (fibrin formation) occurs at the inception of the propagation phase when only 5-10 nM thrombin has been produced. Consequently, the vast majority (greater than 95%) of thrombin is produced after clotting during the propagation phase of thrombin generation. The blood of individuals with either hemophilia A or hemophilia B has no ability to generate the intrinsic FXase, and hence is unable to support the propagation phase of the reaction. Since clot based assays conclude before the propagation phase they are not sensitive to hemophilia A and B. The inception and magnitude of the propagation phase of thrombin generation is influenced by genetic polymorphisms associated with thrombotic and hemorrhagic disease, by the natural abundance of pro- and anticoagulants in healthy individuals and by pharmacologic interventions which influence thrombotic pathology. Therefore, it is our suspicion that the performance of the entire process of thrombin generation from initiation through propagation and termination phases of the reaction are relevant with respect to both hemorrhagic and thrombotic pathology.
Topics: Animals; Blood Platelets; Factor IXa; Factor Xa; Hemophilia A; Hemophilia B; Hemostasis; Homozygote; Humans; Immunoblotting; Models, Biological; Thrombin; Time Factors
PubMed: 12871286
DOI: 10.1046/j.1538-7836.2003.00298.x