Evidence of altered haemostasis in an ovine model of venovenous extracorporeal membrane oxygenation support

This study was approved by the animal research and ethics committee of the Queensland University of Technology and the University of Queensland (approval numbers 1100000053 and 1000000025) and adhered to the Australian Code for the Care and Use of Animals for Scientific Purposes, Eighth Edition, 2013 (the code), of the National Health and Medical Research Council. A total of 27 South African meat merino (SAMM)/Border Leicester Cross ewes weighing between 39 and 58 kg (mean 49.5 kg) were randomly divided into four groups. Animals received either sham injury (n = 12) or smoke-induced acute lung injury (S-ALI) (n = 15) as detailed previously [9]. Sham injury animals were divided into healthy controls (ventilation only for 24 h) (n = 4) and ECMO controls (ECMO only for 24 h) (n = 8). S-ALI animals were divided into controls (S-ALI only, no ECMO for 24 h) (n = 7) and the test group of S-ALI + ECMO (n = 8) for 24 h. ECMO was instituted as per our previously validated model [10]. Briefly, animals placed on ECMO had a 21- to 23-French femoral venous cannula (Medtronic, Minneapolis, MN, USA) inserted into the internal jugular vein and positioned in the distal inferior vena cava (access cannula). A 19- to 21-French venous cannula (Medtronic) was placed at the superior vena cava-right atrium junction (return cannula). Intra-cardiac echocardiography was used to confirm the cannula position. A Jostra ROTAFLOW centrifugal pump (Maquet Cardiopulmonary, Rastatt, Germany) was used, and target flow rates of 3–4 L/minute were maintained. Porcine mucous heparin (1000 U; Pfizer Australia, West Ryde, Australia) was added to the circuit, and a heparin infusion was commenced at 4 U/kg/h to obtain a target ACT between 220 and 300 seconds. Sheep were continuously monitored for haemodynamics, fluid and electrolyte balances, and temperature. Pressure-controlled mandatory ventilation based on previous ovine models was used [11, 12]. An arterial partial pressure of carbon dioxide of approximately 30 mmHg and arterial partial pressure of oxygen >80 mmHg were managed via ECMO gas sweep. Mean arterial pressure was augmented by protocolised use of pressors, inotropes and balanced fluid resuscitation to maintain mean arterial pressure >65 mmHg. Bolus fluids and increased maintenance fluids (up to 500 ml/h) were administered when a persistent decrement in ECMO blood flows was observed and other potential causes for the same were excluded.

Whole blood platelet aggregometry was measured by change in impedance and expressed in aggregation units using the Multiplate® 5.0 platelet function analyser. Hirudin-anticoagulated blood was tested using adenosine diphosphate (ADP; 6.4 μmol/L) and collagen (3.2 μg/ml) agonists [13, 14].

Whole blood clot formation profiles were recorded by ROTEM® (Haemoview Diagnostics, Brisbane, Australia) with the EXTEM (thromboplastin-initiated coagulation), INTEM (contact factor-initiated coagulation), FIBTEM (thromboplastin-initiated coagulation with the platelet inhibitor cytochalasin D) and HEPTEM (contact factor-initiated coagulation with heparinase) activating reagents in accordance with the manufacturer’s instructions. Parameters evaluated included clotting time (CT), clot formation time (CFT) and maximum clot firmness (MCF).

To determine the effect of the experimental group on each outcome, a mixed effects linear regression model was fitted with random effects for sheep and time (within sheep). Potential confounders included the laboratory where routine and specialised coagulation testing was performed, weight of the animal at baseline, fluid balance and the ratio of INTEM-CT to HEPTEM-CT (used as a proxy measure to adjust for potential confounding due to heparin dosage). Eligible confounders with univariable P values <0.2 were entered into a multivariable model, and a stepwise backwards elimination process was applied until all variables remained significant at the 0.05 level. Statistical analyses were performed using the STATA^TM 13 statistical software package (StataCorp, College Station, TX, USA).

Sheep body temperature remained constant throughout the experiment (Fig. 1a). Significant decreases in pH (Fig. 1b) were seen in the S-ALI control (P < 0.001) and S-ALI + ECMO groups (P < 0.05) compared with healthy controls, although this was still within the normal range for ovine pH (7.32–7.5). S-ALI + ECMO groups also had decreased ionised calcium levels (P < 0.001) (Fig. 1c) and a significantly decreased circuit flow rate (P < 0.05) (Fig. 1d) compared with healthy controls. The mean and SD of all haematological and plasma-based coagulation parameters at selected time points are detailed in Table 1. Baseline measurements for FBE as well as routine and specialised coagulation tests did not differ significantly across the experimental groups. Results from each group were compared with those of the healthy control group. WCC (P = 0.004), RCC (P = 0.01) and Hb (P < 0.001) increased in the S-ALI controls. In contrast, WCC (P = 0.01), RCC (P < 0.001) and Hb (P < 0.001) levels were lower in the ECMO controls. S-ALI + ECMO animals also had lower WCC (P < 0.001). Platelet count remained unchanged across all groups. A prolonged PT was observed in both S-ALI controls (P < 0.001) and S-ALI + ECMO (P < 0.001) groups after 12 h of ECMO. aPTT, TCT and d-dimer did not differ significantly in any groups compared with healthy controls. Fibrinogen, FVIII and vWAg levels were lower in ECMO controls (P < 0.001). Fibrinogen, AT, FXII and protein C levels were lower in S-ALI controls (P < 0.001). With the combination of S-ALI + ECMO, there were reductions in coagulation factors FVIII and FXII as well as fibrinogen and vWAg. The levels of naturally occurring anti-coagulants AT and protein C were also lower in this group (P < 0.001). Predicted marginal means with 95% CIs over experimental group for measures of interest are shown in Table 2 along with overall Wald P values derived from mixed effects regression models.

Relative to healthy controls, collagen-induced platelet function was significantly higher in S-ALI controls (P = 0.001), ECMO controls (P < 0.001) and S-ALI + ECMO (P < 0.001) experimental groups (Fig. 2a). There was no significant difference in ADP platelet aggregation across any of the experimental groups (Fig. 2b).

S-ALI controls (P < 0.001) and the S-ALI + ECMO group (P = 0.03) had a significantly lower INTEM-MCF (Fig. 3a) than healthy controls. Both S-ALI controls (P = 0.007) and S-ALI + ECMO (P < 0.001) animals had a significantly lower HEPTEM-MCF (Fig. 3b). There was no significant difference in INTEM or HEPTEM-CFT across any of the experimental groups (data not shown). EXTEM-CFT was extended in S-ALI controls (P = 0.011) and further protracted in S-ALI + ECMO (P < 0.001) experimental animals (Fig. 3c). In addition, the overall clot quality as measured by the EXTEM-MCF was also lower in S-ALI controls (P = 0.031) and further decreased in S-ALI + ECMO (P < 0.001) (Fig. 3d).

Fibrinogen levels in all three experimental groups (P < 0.001) were significantly lower than in healthy controls (Fig. 4a). FIBTEM-CFT was prolonged in both S-ALI controls (P = 0.016) and the S-ALI + ECMO group (P = 0.001) (Fig. 4b). FIBTEM-MCF was lower in S-ALI controls (P < 0.001) and ECMO controls (P = 0.001) and was further reduced in S-ALI + ECMO (P < 0.001) groups (Fig. 4c).