Brownian computations were directed at Ribonuclease A (RNase A) and variants in folded states so as to quantify information of the statistical type at the atom/covalent bond level. This advanced the research reported in this journal last year on the information properties of enzyme primary structure. Brownian computation data are ...

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two-step strategy, a target amino acid validation using mechanism-based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be ...

Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, ...

Prothrombin is the zymogen precursor of the clotting enzyme thrombin, which is generated by two sequential cleavages at R271 and R320 by the prothrombinase complex. The structure of prothrombin is currently unknown. Prethrombin-1 differs from prothrombin for the absence of 155 residues in the N-terminal domain and is composed of ...

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite ...

 Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues ...

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with ...

A series of natural flavonoids has been evaluated as potential inhibitors of thrombin using the optimized method of thrombin time. Myricetin and quercetin have shown to be the best thrombin inhibitors tested. In order to investigate the thrombin recognition of the most active and selective compounds, a molecular modeling study ...

The irreversible thrombin inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was covalently immobilized to PEGylated polymer thin films at its primary alpha-amino group. Activity assays and capture of radioconjugated thrombin reveal that the PPACK-decorated surfaces could bind thrombin forming up to 30% of a monolayer density. Back-calculation of this high thrombin-inhibiting capacity indicated that ...

The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend ...

Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific ...

The quest for an ideal anticoagulant is ongoing. Oral agents that do not require blood level monitoring are presently undergoing taut scrutiny for efficacies and potential side effects, and could soon be ready to transform the history of coagulation medicine. The first part of this article (published in last month's ...

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D ...

The blood clotting enzyme thrombin converts fibrinogen molecules into fibrin monomers which polymerize to form a fibrous three-dimensional gel. The concentration of thrombin affects the architecture of the resulting gel, in particular, a higher concentration of thrombin produces a gel with more branch points per unit volume and with shorter ...

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. ...

A high molecular mass, non toxic metalloprotease the NN-PF3 with the bound Ca(2+) and Zn(2+) from the Naja naja venom has been studied further for its anticoagulant property. The molecular mass by MALDI-TOF mass spectrometry was 67.81 kDa. The NN-PF3 exhibited fibrin(ogen)olytic activity. In addition to fibrinogen, NN-PF3 hydrolyzed blood ...

Dabigatran etexilate is an oral, reversible direct thrombin inhibitor that is approved in the EU and several other countries for the prevention of venous thromboembolism after elective hip and knee replacement, and is in advanced clinical development for other thromboembolic disorders. Dabigatran has a predictable pharmacokinetic profile, allowing for a ...

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this ...

Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly ...

The present study aimed to assess whether the fibrin network structure is modified by the direct thrombin-inhibitors lepirudin, argatroban or bivalirudin and by the indirect Xa-inhibitor danaparoid. Using an in vitro assay that imitates the physiological process of coagulation from thrombin generation to fibrin formation, we examined a normal plasma ...

Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to ...

As activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid-derived single-domain antibodies towards TAFI. This study reports the generation and characterization of a panel of 22 ...

A series of 27 benzamidine inhibitors covering a wide range of biological activity and chemical diversity was analysed to derive a Linear Interaction Energy in Continuum Electrostatics (LIECE) model for analysing the thrombin inhibitory activity. The main interactions occurring at the thrombin binding site and the preferred binding conformations of ...

SUMMARY: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor ...

BACKGROUND: Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin-activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial. OBJECTIVE: To evaluate the ...

SUMMARY BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro-domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half-life ...

Previous data have shown an inter-individual difference in the thrombin generating capacity in vitro as well as phenotypic bleeding pattern among patients with severe haemophilia A (FVIII:C activity below 1%). The reason for this is not known. In addition, there are no reports on how thrombin generation may correlate between ...

Although the maintenance of precise balance between coagulation and fibrinolysis is of utmost importance for normal haemostasis, until recently these two systems were considered as completely separate mechanisms involved in the process of formation and dissolution of blood clot. Thrombin activatable fibrinolysis inhibitor (TAFI) is a recently described attenuator of ...

Myocardial ischemia and other acute coronary syndromes are leading causes of death worldwide, and often result from a thrombus that blocks an atherosclerotic coronary artery. A key enzyme in thrombus formation is the serine protease thrombin, which is responsible for both the conversion of soluble fibrinogen into insoluble fibrin, as ...

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the ...

A sensitive and selective high-performance analytical method based on capillary zone electrophoresis (CZE) was developed for investigating interactions between natural products isolated from Millettia nitita var. hirsutissima and thrombin qualitatively and quantitatively for the first time. The results showed that, compared with positive and negative control, the compounds ZYY-5 (genistein-8-C-beta-d-apiofuranosyl-(1-->6)-O-beta-d-glucopyranoside), ...

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, ...

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity ...

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (TBA) is an efficient tool for the inhibition of thrombin function. We have studied conformations and thermodynamic stability of a number of modified TBA oligonucleotides containing thiophosphoryl substitution at different internucleotide sites. Using circular dichroism such modifications were found not to disrupt the antiparallel intramolecular quadruplex ...

Direct thrombin inhibitors (DTIs) are a class of anticoagulants that bind selectively to thrombin and block its interaction with its substrates. Dabigatran etexilate and AZD0837, the new generation of DTIs, are now under intense development, and are potentially of great interest for internists. Dabigatran etexilate is a potent, non-peptidic small ...

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII-thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against ...

Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 degrees ...

Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the ...

BACKGROUND: Coagulation is a highly regulated process where the ability to prevent blood loss after injury is balanced against the maintenance of blood fluidity. Thrombin is at the center of this balancing act. It is the critical enzyme for producing and stabilizing a clot, but when complexed with thrombomodulin (TM) ...

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with ...

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, ...

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and ...

Blood coagulation is the result of a cascade of zymogen activation events; however, its initiation is allosteric. Factor VIIa circulates in a zymogen-like state and is allosterically activated by binding to tissue factor. Thrombin, the final protease generated in the blood coagulation cascade, has also been shown to exist in ...

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 ...

The field of medicinal chemistry aims to design and optimize small molecule leads into drug candidates that may positively interfere with pathological disease situations in humans or combat the growth of infective pathogens. From the plethora of crystal structures of protein-inhibitor complexes we have learned how molecules recognize each other ...

Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized ...

A solid-state electrochemiluminescence (ECL) biosensing switch system based on special ferrocene-labeled molecular beacon aptamer (Fc-MBA) has been developed successfully for thrombin detections. Such special switch system includes two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle ...

Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug ...

The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to ...

Direct inhibition of blood coagulation factors such as thrombin, factor VIIa, and factor Xa has shown great promise for treating thrombosis disorders. Inhibitors of these serine proteases have been designed, synthesized, and evaluated. Small molecule thrombin inhibitors typically contain P(1)-P(2)-P(3) components targeting the active binding site. Structure optimizations on the ...