Pharmacological profile of recombinant, human activated protein C (LY203638) in a canine model of coronary artery thrombosis.
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The antithrombotic activity of recombinant, human activated protein C (rh-APC, LY203638) was examined in a model of canine coronary artery thrombosis. Three doses of rh-APC (0.5, 1.0, and 2.0 mg/kg/h) were administered intravenously for 2 h. Whole blood clotting times (thrombin time, activated partial thromboplastin time), ex vivo platelet aggregation, and template bleeding times were determined. Activated partial thromboplastin time significantly increased 2- and 3.7-fold during the 2-h infusion of rh-APC (1.0 and 2.0 mg/kg/h, respectively); thrombin time did not change. Intravenous infusions of rh-APC (1.0 and 2.0 mg/kg/h) produced significant prolongations to occlusion, 186 +/- 21 and 190 +/- 22 min, respectively, compared with the vehicle and the 0.5 mg/kg/h group (86 +/- 12 and 93 +/- 17 min, respectively). Vessel patency was better at the end of the experiment in the intermediate- and high-dose groups (3 of 6 and 3 of 5 vessels, 1.0 and 2.0 mg/kg/h, respectively) compared with the vehicle and 0.5 mg/kg/h groups (0/5 and 0/6, respectively). Only the 1.0 mg/kg/h group was found to have significantly elevated template bleeding times, with peak increases seen 60 min into the drug infusion. All groups had returned to baseline values by the end of the study. There was no observed inhibition of platelet aggregation. These data demonstrate that recombinant, human activated protein C is an effective anticoagulant and antithrombotic agent in the dog. (+info)
Pharmacodynamics and pharmacokinetics of polyethylene glycol-hirudin in patients with chronic renal failure.
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BACKGROUND: Hirudin selectively inhibits thrombin without cofactors and is eliminated via the kidneys. Recombinant hirudin (r-hi) has a terminal elimination half-life (t1/2) of about 50 to 100 minutes. Coupling of polyethylene glycol (PEG) to r-hi, giving PEG-hirudin (PEG-Hi), prolongs its t1/2 while enhancing efficacy. We looked at the pharmacodynamic and pharmacokinetic behavior of PEG-Hi in patients with impaired renal function. METHODS: Anticoagulant activity and the pharmacokinetic parameters of a single intravenous bolus injection of 0.05 mg/kg body weight PEG-Hi were studied in 38 subjects. They were assigned to five groups: group IA, creatinine clearance (CCr) >/= 80 mL/min, 8 healthy volunteers; group IB, CCr >/= 80 mL/min, 8 patients with normal renal function); group II, CCr 79 to 50 mL/min, 7 patients with mild chronic renal failure (CRF); group III, CCr 49 to 20 mL/min, 10 patients with moderate CRF; and group IV, CCr +info)
Posttranslational modifications of recombinant myotube-synthesized human factor IX.
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Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX. (+info)
Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase.
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In buffer systems, heparin and low molecular weight heparin (LMWH) directly inhibit the intrinsic factor X-activating complex (intrinsic tenase) but have no effect on the prothrombin-activating complex (prothrombinase). Although chemical modification of LMWH, to lower its affinity for antithrombin (LA-LMWH) has no effect on its ability to inhibit intrinsic tenase, N-desulfation of LMWH reduces its activity 12-fold. To further explore the role of sulfation, hypersulfated LA-LMWH was synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent inhibitor of intrinsic tenase than LA-LMWH; it also acquires prothrombinase inhibitory activity. A direct correlation between the extent of sulfation of LA-LMWH and its inhibitory activity against intrinsic tenase and prothrombinase is observed. In plasma-based assays of tenase and prothrombinase, sLA-LMWH produces similar prolongation of clotting times in plasma depleted of antithrombin and/or heparin cofactor II as it does in control plasma. In contrast, heparin has no effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on various components of tenase and prothrombinase was examined, its inhibitory activity was found to be cofactor-dependent (factors Va and VIIIa) and phospholipid-independent. These studies reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase. (+info)
Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries.
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BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect. METHODS AND RESULTS: We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial ss-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3+/-8.7% (mean+/-SD, n=14) in saline-treated arteries and 33.3+/-4.2% in AdLacZ-infected arteries (P:=NS versus saline). However, it was reduced to 25.5+/-8.5% in rTFPI-irrigated arteries (P:<0.01 versus saline) and to 20.7+/-5.3% in AdTFPI-infected arteries (P:<0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6+/-4.7% (P:<0.01 versus rTFPI, P:<0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals. CONCLUSIONS: A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans. (+info)
Regulation of human coagulation factor X gene expression by GATA-4 and the Sp family of transcription factors.
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Serine protease factor Xa plays a critical role in the coagulation cascade. Zymogen factor X is synthesized and modified in the liver. To understand the mechanisms governing the liver-specific expression of factor X, the proximal promoter of human factor X was previously characterized. Two crucial cis elements at -73 and -128 and their cognate binding proteins, HNF-4 and NF-Y, respectively, were identified. In this report, studies are extended to 3 additional cis elements within the factor X promoter. Using gel mobility shift assays, the liver-enriched protein GATA-4 was identified as the protein binding to the GATA element at -96. GATA-4 transactivates the factor X promoter 28-fold in transient transfection experiments. It was also determined that the Sp family of transcription factors binds 2 DNase I-footprinted sites at -165 and -195. Disruption of Sp protein binding at either site reduces the promoter activity by half. Simultaneous disruption of both sites reduces the promoter activity 8-fold. This is the first report indicating the involvement of GATA-4 in the regulation of clotting factor expression. These observations provide novel insight into mechanisms by which the vitamin K-dependent coagulation factors are regulated. (+info)
Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation.
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A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells. (+info)
Population modelling of the effect of inogatran, at thrombin inhibitor, on ex vivo coagulation time (APTT) in healthy subjects and patients with coronary artery disease.
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AIMS: The purpose of this study was to characterize the relationship between the degree of anticoagulation, assessed by APTT, and the plasma concentration of inogatran in healthy subjects and in patients with coronary artery disease. METHODS: Data from five phase I studies in 78 healthy males and two phase II multicentre studies in 948 patients of both sexes with unstable angina pectoris or non-Q-wave myocardial infarction were evaluated. A total of 3296 pairs of concentration-APTT samples were obtained before, during, and after intravenous infusions of inogatran. Mixed effects modelling was used for population pharmacodynamic analysis of the drug effect and for describing the variability in baseline APTT. RESULTS: The population mean baseline APTT was 29 s, but large variations between individuals (s.d. 3.6 s) were observed. The variability between studies (1.3 s) and centres (1.8 s) were of less importance, though statistically significant. APTT increased in a nonlinear manner with increasing inogatran concentration and the relationship was well described by a combined linear and Emax model. A significant part of the overall variability could be ascribed to the APTT reagent and equipment used at the different study centres. These method-dependent differences were compensated for by including the lower limit of the normal reference range as a covariate, affecting both baseline and Emax, in the model. For the typical healthy subject and patient, the method-corrected population mean parameters were: APTTbaseline 35 and 31 s, slope 8.0 and 5.8 s x l x micromol(-1), Emax 36 and 34 s, and EC50 0.54 and 0.72 micromol x l(-1), respectively. The model predicted plasma concentration needed to double the APTT from the baseline value was 1.25 and 1.45 micromol x l(-1) in the healthy volunteer and patient, respectively. CONCLUSIONS: The nonlinear relationship between APTT and inogatran concentration in plasma was well described by a combined linear and Emax model. Pooling of data was made possible by incorporating a centre-specific characteristic of the assay method in the model. Patients had lower baseline APTT and appeared to have less pronounced effect of inogatran than young healthy subjects. (+info)