Dilutional coagulopathy and venous stasis in rabbits

Female New Zealand White rabbits 3–4 months old and weighing 2.2–3.2 kg (Manfred Bauer, Neuenstein-Lohe, Germany) received care in compliance with the European Convention on Animal Care, and the study was approved by the local governmental authorities. The animals were housed individually in wire-steel cages at 21–23°C and 50% relative humidity under a 12 h/12 h light-darkness cycle. The animals had free access to tap water and were fed rabbit pellets (Deukanin, Deutsche Tiernahrung Cremer GmbH & Co. KG, Düsseldorf, Germany) ad libitum.

Venous stasis was induced 10 minutes after the end of infusion; for analysis of longitudinal response to 4F-PCC, stasis was induced at varying intervals up to 168 hours post-infusion. Any observed thrombi were graded according to a scoring system from 0 to 3, and their wet weights were determined. Thrombus scores were defined as: 0 (no clot), 1 (one or a low number of small clots, too small to determine weights), 2 (not fully occluding clot, with measurable weight) or 3 (fully occluded clot). Histopathological investigations were also performed.

To assess thrombogenicity, we used a modification of the Wessler model of venous stasis-induced thrombosis in rabbits [28–30], as described previously [19] and illustrated in Fig 1. The primary endpoints of the study were thrombus score (based on number of clots, degree of occlusion and potential for determination of measurable thrombus weight) and thrombus wet weight after venous thrombosis. Secondary endpoints included assessment of histopathology.

(A) 4F-PCC in rabbits with dilutional coagulopathy, using modified Wessler model of venous stasis-induced thrombosis; (B) 4F-PCC in rabbits with edoxaban-induced coagulopathy using modified Wessler model of venous stasis-induced thrombosis; (C) 4F-PCC in rats, using a ferric chloride-induced model of arterial thrombosis. Haemodilution was used only in the experiments indicated. 4F-PCC, 4-factor prothrombin complex concentrate; h, hours; min, minutes; t, time.

For analysis of longitudinal response to 4F-PCC, venous stasis was induced at 10 minutes, 45 minutes, 6 hours, 24 hours, 72 hours and 168 hours post-infusion. Procoagulant effects were determined by exposing the contralateral jugular vein and isolating a segment of approximately 2 cm, causing a complete stasis in the isolated segment. Thirty minutes after stasis induction the vein segment was excised and dissected in sodium citrate solution.

Female New Zealand White rabbits were subjected to two cycles of haemodilution, separated by a 45-minute interval; animals received 15 mL/kg salvaged erythrocytes part way through this interval (Fig 1A). Haemodilution was only used in some experiments; where haemodilution was used, it was performed in advance of any treatment administration. Treatments included 4F-PCC, aPCC, rFVIIa, TXA and FCH. Animals were randomly allocated to treatment groups (n = 5–18 per group). Treatments were administered via IV infusion, with doses selected to range from therapeutic to supratherapeutic. TXA (15 mg/kg) was administered 5 minutes prior to 4F-PCC infusion as an IV bolus, as was FCH (100 mg/kg); FCH administration continued in parallel with 4F-PCC administration.

Haemodilution was conducted as described in [1]. Animals were subjected to haemodilution in phases by withdrawal of 30 mL/kg blood and infusion of 30 mL/kg hydroxyethyl starch (HES; Infucoll^® 6% solution; Schwarz Pharma) pre-warmed to 37°C. This procedure was repeated after 45 minutes. After 30 minutes, during the interval between the two cycles of blood withdrawal and HES infusion, the animals received 15 mL/kg salvaged erythrocytes, prepared from withdrawn whole rabbit blood by centrifugation for 10 min at 800×g, washing in normal saline and resuspension in Ringer’s lactate.

4F-PCC (Beriplex^®/Kcentra^®, CSL Behring GmbH, Marburg, Germany) was reconstituted in water for injection (B. Braun Melsungen AG, Melsungen, Germany) to a final FIX potency of 28 IU/mL, as per label instructions [2], and administered via IV infusion at doses of 50, 100, 200, 300, 400 and 500 IU/kg. 3F-PCC (Bebulin^® VH, Baxter Healthcare Corporation, Westlake Village, USA) [3] was reconstituted in sterile water for injection and administered at 300 IU/kg. Activated PCC (Factor Eight Bypassing Activity [FEIBA] Baxter AG, Vienna, Austria) [4] was reconstituted in water for injection and administered at doses of 5, 10, 50, 75 and 100 IU/kg. rFVIIa (NovoSeven^®, Novo Nordisk A/S, Bagsværd, Denmark) [5] was reconstituted in histidine diluent and administered at doses of 10, 50, 90, 180 and 300 μg/kg.

TXA (Carinopharm GmbH, Elze, Germany; provided as solution [100 mg/mL]) was administered at a dose level of 15 mg/kg, 5 minutes prior to 4F-PCC infusion as an IV bolus; this was considered a therapeutic dose. FCH (RiaSTAP^®, CSL Behring GmbH, Marburg, Germany) was reconstituted in water for injection and a dose of 100 mg/kg was administered as an IV bolus beginning 5 minutes prior to 4F-PCC infusion and continuing in parallel with 4F-PCC administration [6]. Equivalent volumes of isotonic saline [0.9%, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany] were used in the negative control groups. In rabbits with dilutional coagulopathy, there were 29 groups in total with n = 5–18 per group.

Following necropsy, the lung, kidney, liver, heart and brain were harvested and fixed in 10% Neutral Buffered Formalin for histopathological analysis. Histopathological investigations (Covance Laboratories, Harrogate, UK) of lung, kidney, liver, heart and brain tissue were conducted for all treatment groups for positive signals of thrombus formation following venous stasis. Tissue was processed and embedded in paraffin wax blocks, and sections with a nominal thickness of 5 μm were stained with haematoxylin and eosin prior to examination. Histopathology was assessed in accordance with the agreed definitive Histopathology Technical Method.