An inappropriate blood-to-anticoagulant ratio can cause an artifactual prolongation of the activated partial thromboplastin time (APTT) and prothrombin time (PT). In a drug safety study in dogs, w e observed a 4-to 5-second increase in the APTT from baseline coincident with increased hematocrit values (56% to 65%) secondary to drug-induced vomiting and diarrhea. The PT and platelet counts were unchanged, and there was no clinical evidence of bleeding associated with venipuncture. Although we were unable to sample the same dogs to investigate the possible effect of hemoconcentration on the prolonged APTT, the question was addressed by an in vitro study. The hematocrit value for citrated blood samples collected from t is well documented in human patients that a marked I increase in the hematocrit can artifactually prolong the APTT and PT because of an inappropriate blood-to-anticoagulant ratio.' When the hematocrit value is above 55%, there is a relative decrease in plasma volume. As a result, when the blood is centrifuged, the plasma fraction will contain an increased concentration of sodium citrate, causing excessive anticoagulation. The ideal concentration in the standard blood/anticoagulant (9: 1) mixture must be maintained between 0.109 mol/L (3.2%) and 0.129 mol/L (3.8%) of sodium citrate for accurate interpretation of the APTT and PT results.2In a drug toxicity study conducted in beagle dogs, we observed a 4-to 5-second prolongation for the APTT in dogs with high hematocrits (ie, 57% to 65%) ( Table I). The hemoconcentration was attributed to dehydration associated with drug-induced vomiting and diarrhea. The observation prompted an in vitro assessment of the effects of increased hematocrit on the APTT values in dogs.A 4.5-mL whole blood specimen anticoagulated with 0.5 mL of 0.129 mol/L (3.8%) buffered citrate solution (9: 1) was obtained from 3 healthy beagle dogs. A spun hematocrit (Drummond Microhematocrit, Broomall, PA) was determined for each specimen. The blood tubes were centrifuged at 2200 rpm for 15 minutes at 4°C and aliquots of plasma and red blood cell layer were retained to prepare blood specimens with high hematocnts.The 3 dogs were bled again as described above. Appropriately measured aliquots of this whole blood were dispensed into each of two 2.7 mL tubes each containing 0.3 mL of 0.129 mol/L buffered citrate. One tube was filled completely with whole blood, the other tube was filled with whole blood and an aliquot of hemoconcentrated citrated blood. The proper fill level was maintained in all tubes. A spun microhematocrit was determined on all tubes before centrifugation at 2200 rpm for 15 minutes at 4°C to harvest test plasma for determination of the PT and APTT by a photometric clot detection method using the Electra 700 (Medical Laboratory Automation Inc, Mt. Vernon, NY). Dade Actin Activated Cephaloplastin Reagent (liquid rabbit brain) and Dade Calcium Chloride, 0.02 mol/L (Baxter Diagnostics, Deerfield, IL) were used for APTT determinations and Dade Thromboplastin C (dried rabbit br...
SKF-99085, an acyl-CoA:cholesterol acyltransferase (ACAT) was evaluated in male and female Sprague-Dawley rats at oral doses of 0, 10, 100, or 400 mg/kg/day for 6 months as part of the preclinical safety assessment of this drug candidate. In male rats given 400 mg/kg/day SKF-99085, hemorrhage and death were observed in males during the first month of the study, prompting collection of blood samples at weeks 6, 17, and 24 to monitor coagulation parameters. A dose-related increase in activated partial thromboplastin time (APTT) and Thrombotest clotting time (TCT) was observed in all male drug-treated groups. Mean APTT values for male rats given 10, 100, or 400 mg/kg/day were increased maximally to 17.5, 20.8, and 34.7 s (control, 15.4-16.0 s), and mean TCT values were increased to 86, 100, and >300 s (control, 71-74 s), respectively. Mean prothrombin times (PT) for male rats given 400 mg/kg/day were increased to 16.5 s (control, 12.9-13.1 s). Activities of factors II, VII, IX, and X were decreased in males at dosages of 10, 100, or 400 mg/kg/day. Factor V and VIII activities were unaffected. In summary, the drug-related hemorrhagic disorder observed in male rats given high doses of the ACAT inhibitor SKF 99085 was attributed to a reduction in the activity of vitamin-K-dependent coagulation factors. In contrast to humans and some other species, the APTT and TCT were more sensitive than the PT in detecting this effect.
SummaryThe effect of human recombinant tissue-type plasminogen activator (rt-PA) on parameters of hemostasis and systemic plasminogen activation was studied in the dog and rat. Effects on screening coagulation times, fibrinogen concentration, fibrin/fibrinogen degradation products, and plasminogen and α2-anti- plasmin (α2-AP) activities in plasma were examined following single bolus injections of 0.5-5.0 mg/kg, single and repeated 3 hr infusions of 0.75-7.5 mg/kg and 24 hr infusions of 6.0 and 30.0 mg/kg administered intravenously to dogs. Rats receiving single or 14 daily injections of 5.0-30.0 mg/kg i.v. were similarly monitored. Systemic fibrinogenolysis (>50% decrease in fibrinogen, plasminogen or α2-AP values) was observed in dogs receiving ≥1.0 mg/kg as a bolus, ≥3.75 mg/kg (20.8 μg or 1.19 × 104 IU kg−1min−1) as a 3 hr infusion and >6 mg/kg (4.2 μg or 2.42 × 103IU kg−1min−1) as a 24 hr infusion; and in rats treated with bolus injections of 30 mg/kg rt-PA. Clinical and laboratory indications of impaired hemostasis and bleeding (anemia, prolonged coagulation times and post-mortem evidence of hemorrhage) were associated with these effects, which together were dose-dependent and influenced by the rate of infusion. The incidence of major hemorrhage was variable and limited to animals receiving prolonged (24 hr) or repeated infusions.
SKF-99085, an acyl-CoA:cholesterol acyltransferase (ACAT) was evaluated in male and female Sprague-Dawley rats at oral doses of 0, 10, 100, or 400 mg/kg/day for 6 months as part of the preclinical safety assessment of this drug candidate. In male rats given 400 mg/kg/day SKF-99085, hemorrhage and death were observed in males during the first month of the study, prompting collection of blood samples at weeks 6, 17, and 24 to monitor coagulation parameters. A dose-related increase in activated partial thromboplastin time (APTT) and Thrombotest clotting time (TCT) was observed in all male drug-treated groups. Mean APTT values for male rats given 10, 100, or 400 mg/kg/day were increased maximally to 17.5, 20.8, and 34.7 s (control, 15.4-16.0 s), and mean TCT values were increased to 86, 100, and >300 s (control, 71-74 s), respectively. Mean prothrombin times (PT) for male rats given 400 mg/kg/day were increased to 16.5 s (control, 12.9-13.1 s). Activities of factors II, VII, IX, and X were decreased in males at dosages of 10, 100, or 400 mg/kg/day. Factor V and VIII activities were unaffected. In summary, the drug-related hemorrhagic disorder observed in male rats given high doses of the ACAT inhibitor SKF 99085 was attributed to a reduction in the activity of vitamin-K-dependent coagulation factors. In contrast to humans and some other species, the APTT and TCT were more sensitive than the PT in detecting this effect.
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