Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.
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PMID:Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin. 138 67

We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain alpha-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn.HCl). The denatured E2c monomers (apparent M(r) = 27,000) were diluted 100-fold at 25 degrees C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn.HCl at 25 degrees C. The E2c trimers with an apparent M(r) of 84,000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn.HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex: requirement for chaperonins GroEL and GroES. 791 32

5' GTGGTGGGTGGGTGGGT-3' (AR177) is a 17-mer oligonucleotide with anti-human immunodeficiency virus (HIV) activity that is composed of a phosphodiester backbone and single phosphorothioate linkages at the 3' and 5' ends. A hemodynamic toxicity study was conducted in which cynomolgus monkeys were infused i.v. over a 10-minute period with single doses of 5, 20 or 50 mg AR177/kg or saline. Blood pressure, ECG, clinical chemistry, hematology, complement factors, coagulation parameters and the AR177 plasma concentration were determined. AR177 did not cause any mortality in this study, nor did it cause changes in blood pressure, ECG, clinical chemistry or hematology parameters at any dose. There was a minimal, dose-dependent increase in the levels of complement split product Bb and total hemolytic complement. There was a significant dose-dependent and reversible inhibition of coagulation with the 20- and 50-mg/kg doses that lasted up to several hours after infusion. The time course of the inhibition of coagulation closely matched the plasma levels of AR177. There was a no-effect plasma AR177 concentration vs. activated partial thromboplastin time of approximately 60 to 100 micrograms AR177/ml, above which there was prolongation of activated partial thromboplastin time. These data demonstrate that AR177 does not cause significant hemodynamic toxicity at the doses studied and that this drug could be administered as a rapid infusion without any acute, life-threatening effects at doses that produce plasma concentrations that have shown anti-HIV activity in vitro.
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PMID:Single-dose hemodynamic toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after intravenous infusion to cynomolgus monkeys. 881 16

5'GTGGTGGGTGGGTGGGT-3' (AR177) is a partial phosphorothioate, 17-mer oligonucleotide that has been shown to have anti-human immunodeficiency virus (HIV) activity in vitro and to be a potent inhibitor of HIV-1 integrase. A repeat-dose toxicity and pharmacokinetic study was conducted in which cynomolgus monkeys were given bolus i.v. injections of 2.5, 10 or 40 mg AR177/kg/day every other day for a total of 12 doses. Control monkeys received saline. ECG, clinical chemistry, hematology, coagulation parameters, histopathology and the AR177 plasma concentration were evaluated. AR177 did not cause any mortality in this study, nor did it cause changes in ECG, clinical chemistry, hematology values or histology. However, there was a dose-dependent inhibition of coagulation measured by a prolongation of activated partial thromboplastin time; this inhibition was reversible with drug washout. Analysis of plasma samples by HPLC demonstrated that there was no difference between the AR177 plasma concentrations that were achieved after the 1st and 12th (last) doses of 2.5, 10 or 40 mg/kg. There was a direct relationship between the AR177 plasma concentration and activated partial thromboplastin time. These results indicate that repeated bolus i.v. administration of AR177 to cynomolgus monkeys at doses as high as 40 mg/kg was well tolerated and was not associated with the serious cardiovascular responses previously observed with other oligonucleotides administered i.v.
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PMID:Repeat-dose toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after bolus intravenous administration to cynomolgus monkeys. 881 17

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.
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PMID:Binding site for blood coagulation factor Xa involving residues 311-325 in factor Va. 961 93

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.
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PMID:Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex. 971 89

Trecovirsen, a 25-mer antisense phosphorothioate oligonucleotide targeted at the gag site of the HIV gene, was administered to HIV-positive volunteers as an i.v. infusion. Single doses ranged from 0.1 to 2.5 mg/kg in an ascending escalation in cohorts of 6 to 12 subjects. Plasma trecovirsen concentrations and pharmacokinetic parameters could be assessed at doses > or = 0.3 mg/kg. Peak plasma concentrations and AUC values increased disproportionately with increasing dose while elimination half-life increased and plasma clearance decreased, indicating a saturable process over this dose range. The only significant adverse event observed was an isolated, transitory increase in activated partial thromboplastin time at doses > or = 2.0 mg/kg that was related to plasma trecovirsen concentrations and is attributed to the polyanionic character of the molecule. Thus, trecovirsen administration was well tolerated in single i.v. doses up to 2.5 mg/kg.
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PMID:Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. 998

Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.
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PMID:Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism. 1130 14

A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.
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PMID:Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys. 1144 91

ISIS 104838 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that binds tumor necrosis factor-alpha (TNF-alpha) mRNA. It carries a 2'-methoxyethyl modification on the five 3' and 5' nucleotide sugars, with 10 central unmodified deoxynucleotides. ISIS 104838 was identified from a 264 ASO screen in phorbol myristate acetate-activated keratinocytes, and the dose response was assessed in lipopolysaccharide (LPS)-activated monocytes. Healthy males received multiple intravenous (i.v.) ISIS 104838 infusions in a placebo-controlled dose escalation trial (0.1-6 mg/kg). Additional volunteers received single or multiple subcutaneous (s.c.) injections. ISIS 104838 suppressed TNF-alpha protein by 85% in stimulated keratinocytes. The IC50 for TNF-alpha mRNA inhibition in stimulated monocytes was <1 microM. For i.v., C(max) occurred at the end of infusion. The effective plasma half-life was 15 to 45 min at 0.1 to 0.5 mg/kg and 1 to 1.8 h for higher doses. The apparent terminal plasma elimination half-life approximated 25 days. Obese subjects had higher plasma levels following equivalent mg/kg doses. For s.c. injections, C(max) occurred at 2 to 4 h and was lower than with equivalent i.v. dosing. Plasma bioavailability compared with i.v. was 82% following a 200 mg/ml s.c. injection. Transient activated partial thromboplastin time prolongation occurred after i.v. infusions and minimally after s.c. injections. Two subjects experienced rash, one a reversible platelet decrease, and mild injection site tenderness was noted. TNF-alpha production by peripheral blood leukocytes, induced ex vivo by LPS, was decreased by ISIS 104838 (p < 0.01). ISIS 104838, a second-generation antisense oligonucleotide, was generally well tolerated intravenously and subcutaneously. The pharmacokinetics support an infrequent dosing interval. Inhibition of TNF-alpha production ex vivo was demonstrated.
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PMID:Phase I trial of ISIS 104838, a 2'-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor-alpha. 1243 59


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