Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When Na+ binds to thrombin, a conformational change is induced that renders the enzyme kinetically faster and more specific in the activation of fibrinogen. Two Na+ binding sites have here been identified crystallographically by exchanging Na+ with Rb+. One is intermolecular, found on the surface between two symmetry-related thrombin molecules. Since it is not present in thrombin crystal structures having different crystal systems, the other Na+ site is the functionally relevant one. The second site has octahedral coordination with the carbonyl oxygen atoms of Arg221A and Lys224 and four conserved water molecules. It is located near Asp189 of the S1 specificity site in an elongated solvent channel (8 x 18 A) formed by four antiparallel beta-strands between Cys182-Cys191 and Val213-Tyr228. This channel, extending from the active site to the opposite surface of the enzyme, was first noted in the hirudin-thrombin structure and contains about 20 conserved water molecules linked together by a hydrogen bonding network that connects to the main chain of thrombin. Although the antiparallel beta-strand interactions of the functional Na+ binding site are the same in prethrombin2, the loops between the strands are very different, so that Asp189 and Arg221A are not positioned properly for either substrate or Na+ binding in prethrombin2. A water molecule with octahedral coordination has also been identified in factor Xa at the topologically equivalent Na+ site position of thrombin. Since activated protein C shows enhanced activity with monovalant cation binding, the same position is probably utilized by Na+. Since thrombin crystals could not be grown in the absence of Na+, the cation was leached from Na(+)-bound thrombin crystals by diffusion/exchange. Although both Na+ and their coordinating water molecules were removed from the Na+ binding sites, the remainder of the thrombin structure was, unexpectedly, the same. The lack of an allosteric change is most likely attributable to crystal packing effects. Thus, the structure of the slow form remains to be established crystallographically.
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PMID:The molecular environment of the Na+ binding site of thrombin. 910 91

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.
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PMID:An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli. 930 71

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.
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PMID:Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study. 951 42

In this report, we describe a new approach for the production of lipid antigens that elicit specific immune responses against phosphatidylserine (PS). Because phospholipids are small nonimmunogenic haptens, PS analogs containing activated coupling groups were synthesized and covalently attached to carrier proteins. Sulfhydryl-reactive PS was generated by acylation of 1-oleoyl-2-(aminocaproyl)-phosphatidylcholine with N-succinimidyl-3-(2-pyridyldithio) propionate, converted to PS by phospholipase D-catalyzed base exchange with L-serine, and conjugated to carrier proteins by thiol-disulfide exchange. Antisera to these lipid hapten-protein carrier conjugates were developed in rabbits. Antibodies bound PS but not phosphatidylcholine (PC), phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine (PE) when presented together with PC. Inhibition studies using water-soluble lipid analogs and sonicated vesicles indicated that antibody specificity was directed toward the lipid's polar head group. These antibodies also inhibited the PS-dependent prothrombinase activity assay by approximately 60%. These data show that the covalent coupling of phospholipid haptens to protein carriers via the lipid's fatty acyl side chains preserves its primary head group moiety for the production of specific lipid antibodies.
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PMID:Synthesis of disulfide-containing phospholipid analogs for the preparation of head group-specific lipid antigens: generation of phosphatidylserine antibodies. 954 41

Colloid solutions have been developed and used over the past 70 years as expanders of the intravascular space, based on an understanding of Starling's law. Increasing osmotic pressure with colloidal products has remained an attractive theoretical premise for volume resuscitation. Indeed, colloids have been shown to increase osmotic pressure in clinical practice; however, the effects are short-lived. Lower molecular weight colloids exert a larger initial osmotic effect, but are rapidly cleared from the circulation. Larger molecules exert a smaller osmotic pressure that is sustained longer. The main drawback to colloid therapy lies in pathological states with endothelial injury and capillary leak, precisely the clinical scenario where colloids are commonly given. The colloid solution may leak into the interstitium and remain there exerting an osmotic gradient, pulling additional water into the interstitium. There are 4 general types of colloid products available for clinical use. Albumin is the predominant plasma protein and remains the standard against which other colloids are compared. Albumin, pooled from human donors, is in short supply and remains expensive. Dextrans have been used to prevent deep venous thrombosis and to lower blood viscosity during surgery. Hetastarch has been widely used as a plasma volume expander. It provides equivalent plasma volume expansion to albumin, but has been shown to alter clotting parameters in studies (prolonging the activated partial thromboplastin time and prothrombin time). Although severe coagulopathies have been reported in sporadic cases, hetastarch has not been shown to increase postoperative bleeding compared with albumin therapy, even in large doses (3 L/day). Despite some theoretical advantages compared with crystalloid therapy, colloid administration has not been shown to decrease the risk of acute lung injury or to improve survival. Specific indications for colloid products include hypoproteinaemic or malnourished states, patients who require plasma volume expansion who are unable to tolerate larger amounts of fluid, orthopaedic and reconstructive procedures requiring prevention of thrombus formation and leukapheresis.
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PMID:Colloid volume expanders. Problems, pitfalls and possibilities. 958 60

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1-44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-A resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3'-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1-45] factor Xa and Des[1-44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.
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PMID:Structural basis for chemical inhibition of human blood coagulation factor Xa. 961 63

The serine protease inhibitor antithrombin undergoes extensive conformational changes during functional interaction with its target proteases. Changes include insertion of the reactive loop region into a beta-sheet structure in the protein core. We explore the possibility that these changes are linked to water transfer. Volumes of water transferred during inhibition of coagulation factor Xa are compared to water-permeable volumes in the x-ray structure of two different antithrombin conformers. In one conformer, the reactive loop is largely exposed to solvent, and in the other, the loop is inserted. Hydration fingerprints of antithrombin (that is, water-permeable pockets) are analyzed to determine their location, volume, and size of access pores, using alpha shape-based methods from computational geometry. Water transfer during reactions is calculated from changes in rate with osmotic pressure. Hydration fingerprints prove markedly different in the two conformers. There is an excess of 61-76 water molecules in loop-exposed as compared to loop-inserted conformers. Quantitatively, rate increases with osmotic pressure are consistent with the transfer of 73 +/- 7 water molecules. This study demonstrates that conformational changes of antithrombin, including loop insertion, are linked to water transfer from antithrombin to bulk solution. It also illustrates the combined use of osmotic stress and analytical geometry as a new and effective tool for structure/function studies.
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PMID:Hydration structure of antithrombin conformers and water transfer during reactive loop insertion. 967 60

A 53-year old man on long-term hemodialysis (HD) with anticoagulant therapy was scheduled for nephrectomy due to renal cell carcinoma. Two months before surgery, a coronary stent had been placed due to right coronary artery disease. One week before surgery, percutaneous transmural coronary angioplasty (PTCA) was performed for unstable angina. Aggressive oral antiplatelet therapy (aspirin and ticlopidine) was absolutely required to maintain patency. Following withdrawal of the antiplatelets, unfractionated heparin (UFH) was titrated to an activated partial thromboplastin time (APTT) of 1.5 times greater than the control value. Maintenance UFH (800 U.h-1) was continued until the time of arrival in the operation room (activated clotting time (ACT) was 166 seconds). One hour after arrival, reduced dose of UFH (200 U.h-1) was reinfused, and ACT was 121 140 seconds. Hemodynamic change was minimized using balanced general anesthesia (nitrous oxide-isoflurane, fentanyl, midazolam and vecuronium) accompanied by nitroglycerin and diltiazem. There was no ischemic change on ECG or transesophageal echocardiography. Following surgery, the UFH dose was augmented (400 U.h-1), and the maintenance dose was attained 11 hours after surgery. HD on the second postoperative day was performed uneventfully. This hemodynamic stability might be come from the no water removal. Fourteen days after surgery, the patient was discharged without hemorrhagic complications or clinical ischemic events. We conclude that perioperative UFH infusion is not contraindicated for dialysis patient if strict ACT control is maintained.
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PMID:[Perioperative management for nephrectomy in a long-term hemodialysis patient with anticoagulants for coronary stent]. 969 92

A small, 257 g centrifugal pump was tested as a left ventricular assist device (LVAD) in sheep given a myocardial infarction. Pump performance, hemolysis, end organ function, weaning, explant procedure, and the incidence of thromboemboli at autopsy were studied over intervals of 1 to 44 days. Twelve sheep were given acute myocardial infarction by ligation of the anterior descending coronary artery and 11 had insertion of the AB-180 Circulatory Support System (CSS). One sheep served as a control for the space occupying effects of the pump in the left chest. Inflow was from the left atrium and outflow was to the descending thoracic aorta. Heparin (57-83 U/ml) in sterile water was infused into the pump at a rate of 10 ml/hr. Pump flows of 1-5.7 L/min were tested. The AB-180 CSS supported 73.5% of the total cardiac output (pump + heart) of 3.89 L/min, with a mean arterial pressure of 86 +/- 7 mmHg at a pump speed of 4,162 +/- 276 rpm immediately after implant. Hemolysis was <10 mg/dl and activated partial thromboplastin time (aPTT) values were in the normal range for sheep (<52 sec) after 48 hr of pumping. Liver enzyme concentrations returned to normal within 2 weeks. There was no evidence of thrombocytopenia. No signs of infection were present during assist and none was found at autopsy. The device was successfully removed three times without the use of pressor agents or blood transfusion. Alarm systems performed appropriately. During the 106 days of cumulative pumping, two sheep showed small (<1.5 cm) renal infarcts. Both were associated with intervals of pump stasis. The AB-180 CSS pump was easily implanted into the left chest without the use of cardiopulmonary bypass. It appears to have a low thromboembolic potential in sheep, without the need for large doses of heparin to elevate aPTT values. This characteristic may ameliorate the excessive bleeding seen clinically with current LVAD systems used for post cardiotomy cardiogenic shock, which require anticoagulation with heparin. The small size and weight of the device permit implantation within the chest and allow chest closure. This may reduce the incidence of infection associated with temporary left ventricular assist and an open sternum.
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PMID:Left ventricular support with the implantable AB-180 centrifugal pump in sheep with acute myocardial infarction. 983 Oct 89

As in adults, desmopressin (DDAVP) can be used in children for prophylaxis of bleeding and to stop bleeding in many hereditary and acquired bleeding disorders. DDAVP is the treatment of choice in children with mild hemophilia and type 1 von Willebrand's disease (vWD). It is effective in some variants of vWD and in many patients with platelet function defects. It reduces the bleeding diathesis of children with uremia and drug-induced bleeding complications. In any case, a test dose of DDAVP has to be given to the patient to predict the hemostatic effect before relying on this drug for treatment. The response can be measured by shortening of the bleeding time (BT) and of partial thromboplastin time (PTT), indicating a rise of Factor (F) VIII or von Willebrand factor (vWF). Side effects such as facial flushing, transient headache, increased pulse rate, and drop in systolic blood pressure are mild and transient. They can be minimized when the dose is not exceeding 0.3 microg/kg body weight, and the infusion lasts at least 20 to 30 minutes. The strong antidiuretic action of DDAVP has some potential problems that are negligible in adults and older children when water intake is restricted. In infants and small children under the age of 18 months, however, DDAVP should be used with caution and with close surveillance in order to prevent water intoxication and electrolyte imbalance. The danger is increased when the patients receive parenteral fluid substitution. The advantages of DDAVP include the reduction in the use of plasma factor concentrates, thereby minimizing the danger of immunological or infectious complications, as well as the considerable reduction of costs realized by treatment with this form of medication. Fortunately, it can be applied successfully in the most frequent hereditary bleeding disorder, namely vWD type 1.
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PMID:Desmopressin (DDAVP) in bleeding disorders of childhood. 1006 51


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