Escalating doses of intravenous APAC demonstrate antithrombotic effect in pigs

Figures

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  Fig. 1. Design and execution of the study. Time points for administration of APAC or UFH (black arrows) and timing of blood sample collections (black points) for subsequent analysis of pharmacodynamic variables are shown. At timepoints indicating simultaneous administration of APAC or UFH and blood collection, blood was collected before the dosing. Escalating doses of APAC were assessed in Pig 1 (n = 1) to select the initial dose levels (a), in Pig 2–6 (n = 5) for comparative repeated dosing (b), and in Pig 7 and 8 (n = 2) to estimate the effects at a supra-high dose (c). Schedule for the co-administration of aspirin and APAC (n = 3) is shown in Panel d. UFH was administered in Pig 12 and 13 (n = 2) (e). Protamine sulfate (140 IU/kg) was studied as a reversal agent for APAC or UFH at the end of the experiments

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  Fig. 2. Assessment of effective doses of intravenous APAC by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in plasma. APTT (a), TT (b) and PT (c) were followed in plasma after administering escalating doses of APAC (0.15, 0.25, 0.5 and 0.75 mg/kg) (Pig 1; n = 1). The time points of APAC dosing were 0, 75, 150, and 225 min. Blood collection times and respective results are presented by solid symbols. Results represent relative to the baseline value (APTT, 68 s; TT, 28 s and PT, 7.7 s). Protamine sulfate (140 IU/kg, open triangle) was administered as an antidote to all animals at 15 min after the highest APAC dose (at 240 min). Maximal APTT and TT detection times were 200 s and 100 s, respectively. Red symbols indicate the reach of the maximal detection time (upper limit of detection)

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  Fig. 3. Effect of escalating doses of intravenous APAC on activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in plasma. The results represent fold-changes from the baseline on APTT (a), TT (b) and PT (c) followed in plasma after administering escalating doses of APAC at 0.25, 0.5 and 0.75 mg/kg (Pig 2–6; n = 5). The time points of APAC dosing were at 0, 75, and 150 min. Blood collection times are presented by solid symbols. Results are presented in relation to baseline values (ranges for APTT 35–156 s, for TT 27–41 s and for PT 8–11 s). Protamine sulfate (140 IU/kg, open triangle) was administered as an antidote to all animals at 15 min after the highest APAC dose (at 165 min). Maximal APTT and TT detection times were 200 s and 100 s, respectively. Red symbols indicate the reach of the maximal detection time. Please refer to Supplemental Figure S2 for individual data. *p < 0.05, **p < 0.01, ***p < 0.001, relative to the baseline value

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  Fig. 4. Effect of escalating doses of intravenous APAC on intrinsic pathway-triggered blood coagulation InTEM. Administration of escalating doses of APAC was followed with rotational thromboelastometry (ROTEM; InTEM) at 0.25, 0.5 and 0.75 mg/kg (Pig 2–6; n = 5). Variables reflecting the rate of initial fibrin formation (coagulation time, CT) (a), the interaction of platelets and fibrin to form a stable clot (clot formation time, CFT) (b), and maximum clot firmness (MCF) (c) are shown. The time points of APAC dosing were at 0, 75, and 150 min. Blood collection times are presented by solid symbols. Results are presented in relation to baseline values (ranges for CT 90–145 s, for CFT 26–47 s and for MCF 71–82 s). Protamine sulfate (140 IU/kg, open triangle) was administered to all animals at 15 min after the highest APAC dose (at 165 min), and it uniformly reversed the effect of APAC. *p < 0.05, **p < 0.01, ***p < 0.001, relative to the baseline value

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  Fig. 5. Effect of escalating doses of intravenous APAC or UFH on activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. APTT and TT were followed in plasma after the escalating dosing of APAC (0.5, 0.75 or 1.5 mg/kg) in Pigs 7–8 (n = 2) (a and c). The dosing time points for APAC or UFH were 0, 75, and 150 min. The effect of UFH was tested at 0.5, 0.75 and 1.5 mg/kg similarly in Pigs 12–13 (n = 2) (b and d). Blood collection time points are presented by solid symbols. Results are shown in relation to baseline values (ranges for APTT 83–105 s, and for TT 22–32 s). Protamine sulfate (140 IU/kg, open triangle) was administered to all animals 15 min after the highest APAC or UFH dose (165 min). The maximal detection time for APTT was 200s, and for TT 100 s. The red symbols indicate the reach of maximal detection time

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  Fig. 6. Effects of escalating intravenous APAC or UFH doses prolonged activated clotting time (ACT) in whole blood. ACT after administration of escalating doses of APAC at (0.5, 0.75 or 1.5 mg/kg) (panel a, Pig 7–8; n = 2) or UFH at 0.5, 0.75 and 1.5 mg/kg (panel b, Pigs 12–13; n = 2). Prolongation of ACT as compared to baseline (%) vs. the dose of anticoagulant is shown on panel c (dose-response curves, APAC: red, UFH: green). ED50: effective dose needed for 50% of the effect

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  Fig. 7. Antiplatelet effects of APAC alone and co-administered with aspirin. Collagen-induced platelet aggregation (CIPA) at increasing administered doses of APAC (0.25, 0.50 and 0.75 mg/kg) (Panel a, Pig 4–6; n = 3). Co-administration of aspirin (500 mg) and APAC (1.5 mg/kg) were carried-out in three animals and followed by CIPA (Panel b, Pigs 9–11; n = 3). Results are expressed as the extent of maximal aggregation (∆transmission %). The dose-response effect of APAC on CIPA (red circles and solid lines) as compared to 500 mg aspirin alone (blue triangle and dotted line) (c). *p < 0.05, relative to the baseline value