Open Access

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

  • Margaret R. Passmore1Email author,
  • Yoke L. Fung1, 2,
  • Gabriela Simonova1, 3,
  • Samuel R. Foley1,
  • Sara D. Diab1,
  • Kimble R. Dunster1,
  • Michelle M. Spanevello4, 5,
  • Charles I. McDonald1,
  • John-Paul Tung1, 3,
  • Natalie M. Pecheniuk6,
  • Karen Hay7,
  • Kiran Shekar1 and
  • John F. Fraser1
Critical Care201721:191

https://doi.org/10.1186/s13054-017-1788-9

Received: 28 February 2017

Accepted: 5 July 2017

Published: 29 July 2017

Abstract

Background

Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used in the management of cardiopulmonary failure that is refractory to conventional medical and surgical therapies. The major problems clinicians face are bleeding and clotting, which can occur simultaneously. To discern the impact of pulmonary injury and ECMO on the host’s haemostatic response, we developed an ovine model of smoke-induced acute lung injury (S-ALI) and ECMO. The aims of this study were to determine if the ECMO circuit itself altered haemostasis and if this was augmented in a host with pulmonary injury.

Methods

Twenty-seven South African meat merino/Border Leicester Cross ewes underwent instrumentation. Animals received either sham injury (n = 12) or S-ALI (n = 15). Control animal groups consisted of healthy controls (ventilation only for 24 h) (n = 4), ECMO controls (ECMO only for 24 h) (n = 8) and S-ALI controls (S-ALI but no ECMO for 24 h) (n = 7). The test group comprised S-ALI sheep placed on ECMO (S-ALI + ECMO for 24 h) (n = 8). Serial blood samples were taken for rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests. Animals were continuously monitored for haemodynamic, fluid and electrolyte balances and temperature. Pressure-controlled intermittent mandatory ventilation was used, and mean arterial pressure was augmented by protocolised use of pressors, inotropes and balanced fluid resuscitation to maintain mean arterial pressure >65 mmHg.

Results

Rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests demonstrated that S-ALI and ECMO independently induced changes to platelet function, delayed clot formation and reduced clot firmness. This effect was augmented with the combination of S-ALI and ECMO, with evidence of increased collagen-induced platelet aggregation as well as changes in factor VIII (FVIII), factor XII and fibrinogen levels.

Conclusions

The introduction of an ECMO circuit itself increases collagen-induced platelet aggregation, decreases FVIII and von Willebrand factor, and induces a transient decrease in fibrinogen levels and function in the first 24 h. These changes to haemostasis are amplified when a host with a pre-existing pulmonary injury is placed on ECMO. Because patients are often on ECMO for extended periods, longer-duration studies are required to characterise ECMO-induced haemostatic changes over the long term. The utility of point-of-care tests for guiding haemostatic management during ECMO also warrants further exploration.

Keywords

Extracorporeal membrane oxygenation Thromboelastometry Coagulation Haemostasis Fibrinogen Platelet aggregation

Background

Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used in the management of cardiopulmonary failure that is unresponsive to conventional medical and surgical therapies. Despite its benefits, survival rates for ECMO are only 84%, 67% and 66% when used for respiratory failure and 62%, 67% and 56% when used for cardiac failure in neonates, paediatric patients and adults, respectively [1]. Bleeding events remain a common and serious complication during ECMO, occurring in approximately 30% of patients [24]. Authors of some reports have proposed that contact between the patient’s blood and the ECMO circuit can lead to activation of the coagulation, fibrinolysis and complement pathways [5]. The shift to a pro-coagulant state appears to be mediated primarily by thrombin, whilst there is an excessive fibrinolytic tendency mediated by plasmin, with the result being consumption of clotting factors, impaired platelet function, thrombocytopenia, and fibrinolysis [5]. The goal of the clinician is to minimise bleeding and transfusion requirements whilst avoiding micro- or macro-thrombus formation in the circuit and within the patient’s cardiovascular system [6]. This tightrope is a dynamic process; hence, a more complete understanding of how haemostasis is altered may improve bleeding management strategies and thus to reduce morbidity and mortality.

Unfractionated heparin is the systemic anti-coagulant most widely used during ECMO and is monitored primarily by activated clotting time (ACT). Viscoelastic tests using rotational thromboelastometry (ROTEM®) are gaining popularity because they assess whole blood coagulation and thus provide information on the dynamics of clot development, stabilisation and dissolution. A number of small studies suggest that ROTEM®-guided coagulation management could reduce bleeding episodes in ECMO patients [7, 8]. Whole blood platelet aggregometry using the cobas Multiplate® (Roche Diagnostics, Rotkreuz, Switzerland) is also increasing in use with a recent study demonstrating decreased platelet aggregation in ECMO patients [8].

Patients who require ECMO are critically ill, thus making it difficult to discern the relative contributions of a patient’s underlying pathology from the insult of the ECMO circuit per se. We hypothesised that the ECMO circuit itself alters haemostasis and that this becomes more pronounced in the presence of a pre-existing lung injury. To discern the ECMO effect from that of the underlying illness, we used an established ovine model of lung injury and placed the animal on ECMO support. Haemostasis was monitored using conventional coagulation assays, thromboelastometry and platelet aggregometry to determine how each physiological insult alters coagulation in this scenario.

Methods

Extracorporeal membrane oxygenation ovine model

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.

Blood sampling

Arterial blood samples and blood gas measurements (temperature, pH, ionised calcium) were collected at baseline; 15 minutes post-S-ALI (or sham injury) (S0), before ECMO commencement (S2); and 1, 2, 6, 12, 18 and 24 h post-ECMO. Blood samples were also taken for ROTEM® analysis, full blood examination (FBE) and Multiplate® whole blood platelet aggregometric testing. Samples for routine coagulation tests were centrifuged twice (15 minutes, 4 °C, 3000 × g) to obtain platelet-poor plasma and were subsequently stored at −80 °C until further analysis.

Full blood examination and routine coagulation tests

An FBE using the veterinary mode of the AcT diff™ haematology analyser (Beckman Coulter Australia Pty Ltd, Lane Cove West, Australia) was performed to assess the white cell count (WCC), red cell count (RCC), haemoglobin (Hb), haematocrit (Hct) and platelet count. Prothrombin time (PT), activated partial thromboplastin time (aPTT), factor VIII (FVIII) and FXII, anti-thrombin (AT), fibrinogen using the Clauss method, protein C, von Willebrand factor antigen (vWAg), thrombin clotting time (TCT) and d-dimer were performed on the ACL TOP® analyser (Instrumentation Laboratory/Werfen Australia, Artarmon, Australia) or the Stago STA-R Evolution analyser (Diagnostica Stago, Doncaster, Australia) following the manufacturer’s instructions. ACT was measured in fresh whole blood using kaolin tubes with the HEMOCHRON® 401 coagulation analyser (Soma Technology, Bloomfield, CT, USA).

Whole blood platelet function (Multiplate®)

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].

ROTEM®

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).

Statistical analysis

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 STATATM 13 statistical software package (StataCorp, College Station, TX, USA).

Results

Physiological, haematological and routine coagulation parameters

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.
Fig. 1

The effect of ECMO on physiological parameters. Temperature remained constant a, while S-ALI controls and S-ALI + ECMO animals had a decreased pH b. Ionised calcium levels c and the circuit flow rate d were decreased in S-ALI + ECMO compared with healthy controls. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 versus healthy controls. n = 4 healthy controls, n = 8 ECMO controls, n = 7 S-ALI controls, n = 8 S-ALI + ECMO. ECMO Extracorporeal membrane oxygenation, S-ALI Smoke-induced acute lung injury, B Baseline

Table 1

Routine and specialised haemostatic parameters by experimental group at selected time points

 

Ventilation only

S-ALI control

ECMO control

S-ALI + ECMO

B

24 h

B

24 h

B

24 h

B

24 h

Full blood examination

 WCC, ×109/L

9.8 (3.1)

10.2 (2.2)

7.8 (2.1)

21.8 (8.3)a

7.0 (2.2)

7.9 (1.7)a

6.6 (1.2)

9.1 (3.8)a

 RCC, ×1012/L

8.7 (0.68)

6.2 (0.6)

8.5 (0.78)

8.9 (1.36)a

8.0 (1.2)

4.8 (0.54)a

8.3 (0.87)

7.3 (1.2)

 Hb, g/L

97.8 (9.1)

70.3 (6.1)

96.1 (9.4)

99.1 (12)a

89.3 (12)

54.3 (5.4)a

93.4 (11)

83.5 (12)

 Hct

0.28 (0.02)

0.20 (0.02)

0.28 (0.03)

0.29 (0.04)a

0.26 (0.04)

0.16 (0.01)a

0.28 (0.03)

0.24 (0.04)

 PLT, ×109/L

341 (173)

190 (110)

355 (186)

203 (111)

471 (95)

260 (80)

364 (140)

199 (60)

Routine coagulation tests

 PT, seconds

14.5 (1)

17.3 (1)

15.9 (1.1)

34.4 (5.4)a

12.9 (0.6)

17.4 (2.1)

14.3 (0.9)

35 (6.7)a

 aPTT, seconds

33 (6)

139 (76)

31 (6)

165 (42)

26 (4.6)

122 (64)

30 (6)

158 (57)

 Fibrinogen, g/L

2.5 (0.54)

3.5 (0.13)

2.1 (0.62)

2.5 (0.34)a

2.9 (0.47)

2.6 (0.16)a

3.0 (0.97)

1.5 (0.5)a

d-dimer, ng/ml

215 (85)

198 (70)

146 (44)

139 (82)

435 (192)

343 (217)

406 (213)

213 (136)

 TCT, seconds

13 (0)

21 (7)

13 (0)

81 (84)

12 (1)

156 (81)

13 (1)

44 (69)

Specialised coagulation tests

 FVIII, %

1531 (413)

1390 (414)

1347 (417)

1544 (238)

891(235)

408 (51)a

904 (166)

531 (242)a

 FXII, %

103 (17.7)

75 (10)

96 (30.9)

29 (11.5)a

151 (37.7)

67 (12.4)

107 (20.2)

16 (6.7)a

 Protein C, %

21.5 (2.5)

21.5 (1.3)

21 (7.4)

12.1 (3.2)

55 (13.4)

50 (11.2)

46 (11.4)

14 (4.5)a

 vWAg, %

136 (22.3)

130 (25.8)

96 (31.9)

140 (18.2)

94 (47.6)

88 (10.9)a

129 (30)

96 (25.3)a

 AT, %

85.8 (7.3)

58.3 (6.6)

84.1 (3.9)

27.1 (3.2)a

93.7 (8.8)

62.7 (6)

92.3 (1.9)

22 (4.3)a

Abbreviations: aPTT Activated partial thromboplastin time, AT Anti-thrombin, B baseline, ECMO Extracorporeal membrane oxygenation, FVIII Factor VIII, FXII Factor XII, Hb Haemoglobin, Hct Haematocrit, PLT Platelets, PT Prothrombin time, RCC Red cell count, S-ALI Smoke-induced acute lung injury, TCT Thrombin clotting time, vWAg von Willebrand factor antigen, WCC White cell count

Data are presented as mean (±SD)

a P < 0.05 over time vs ventilation only

Table 2

Predicted marginal mean values and 95% CIs for haemostatic parameters, by experimental group and overall Wald P values derived from mixed effects regression models

 

Ventilation only

S-ALI control

ECMO control

S-ALI + ECMO

Wald P value

WCC

8.4 (6.8–9.9)

5.5 (4.4–6.7)

6.0 (4.8–7.2)

4.9 (3.8–6)

0.004

RCC

6.9 (6.3–7.5)

7.3 (6.9–7.8)

5.0 (4.5–5.5)

6.2 (5.7–6.6)

<0.001

Hb

75 (70–81)

87 (83–91)

55 (50–60)

73 (69–77)

<0.001

PLT

195 (121–269)

248 (188–309)

238 (185–290)

207 (155–260)

0.609

PT

16 (13.4–18.6)

20 (17.7–22.2)

18.9 (16.7–21)

23.7 (22.1–25.3)

0.007

aPTT

113 (89–137)

117 (96–139)

100 (80–119)

124 (106–141)

0.164

TCT

68 (36–99)

96 (69–122)

159 (133–184)

104 (82–127)

0.063

d-dimer

211 (98–324)

157 (71–242)

332 (232–432)

276 (195–358)

0.852

Fib(C)

2.7 (2.4–3)

2.0 (1.7–2.3)

2.2 (1.9–2.4)

1.4 (1.2–1.6)

<0.001

FVIII

1161 (1016–1307)

1082 (956–1208)

529 (410–647)

505 (415–595)

<0.001

FXII

70 (59–81)

22 (13–31)

63 (55–72)

13 (6–20)

<0.001

vWAg

135 (120–151)

130 (118–142)

92 (79–106)

103 (92–114)

<0.001

Protein C

23 (17–29)

15 (10–20)

51 (47–56)

14 (9–18)

<0.001

INTEM-CFT

323 (154–492)

332 (179–486)

211 (115–306)

235 (121–348)

0.468

INTEM-MCF

67 (55–79)

52 (41–63)

64 (57–71)

58 (52–64)

<0.001

HEPTEM-CFT

92 (35–149)

89 (46–132)

136 (85–187)

105 (65–146)

0.5416

HEPTEM-MCF

69 (65–74)

67 (63–71)

70 (65–74)

60 (57–63)

0.001

EXTEM-CFT

108 (87–130)

144 (127–160)

105 (90–121)

179 (164–194)

<0.001

EXTEM-MCF

75 (71–78)

71 (68–74)

71 (68–74)

63 (61–65)

<0.001

FIBTEM-MCF

29 (26–33)

23 (20–26)

23 (20–26)

16 (14–19)

<0.001

Abbreviations: aPTT Activated partial thromboplastin time, AT Anti-thrombin, CFT Clot formation time, ECMO Extracorporeal membrane oxygenation, Fib(C) Clauss fibrinogen, FVIII Factor VIII, FXII Factor XII, Hb Haemoglobin, MCF Maximum clot firmness, PLT Platelets, PT Prothrombin time, RCC Red cell count, S-ALI Smoke-induced acute lung injury, TCT Thrombin clotting time, vWAg von Willebrand factor antigen, WCC White cell count

Data are presented as predicted mean (95% CI)

Platelet function (Multiplate®)

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).
Fig. 2

Platelet aggregometric function for each group of animals. a Collagen-induced AUC was significantly higher in S-ALI controls, ECMO controls and S-ALI + ECMO groups, while b ADP was not significantly different. Data are presented as mean ± SEM. ***P < 0.001 versus healthy controls. n = 4 healthy controls, n = 8 ECMO control, n = 7 S-ALI controls, n = 8 S-ALI + ECMO. ADP Adenosine diphosphate; B Baseline, ECMO Extracorporeal membrane oxygenation, S-ALI Smoke-induced acute lung injury

Thromboelastometry

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).
Fig. 3

The effect of ECMO on INTEM, HEPTEM and EXTEM parameters. INTEM and HEPTEM-MCF a and b in the S-ALI controls and S-ALI + ECMO groups was significantly lower than in healthy controls. c EXTEM-CFT was prolonged in both S-ALI controls and S-ALI + ECMO. d EXTEM-MCF was subsequently lower in both S-ALI controls and S-ALI + ECMO. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 versus healthy controls. n = 4 healthy controls, n = 8 ECMO controls, n = 7 S-ALI controls, n = 8 S-ALI + ECMO. B Baseline, CFT Clot formation time, ECMO Extracorporeal membrane oxygenation, MCF Maximum clot firmness, S-ALI Smoke-induced acute lung injury

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).
Fig. 4

The effect of ECMO on FIBTEM parameters. a Clauss fibrinogen was significantly decreased in all three groups compared with healthy controls. b FIBTEM-CFT was prolonged in both S-ALI controls and S-ALI + ECMO groups, while c FIBTEM-MCF was lower in S-ALI controls, ECMO controls and S-ALI + ECMO. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus healthy controls. n = 4 healthy controls, n = 8 ECMO controls, n = 7 S-ALI controls, n = 8 S-ALI + ECMO. B Baseline, CFT Clot formation time, ECMO Extracorporeal membrane oxygenation, FibC Clauss fibrinogen, MCF Maximum clot firmness, S-ALI Smoke-induced acute lung injury

Discussion

In clinical practice, it is the critically ill who are placed on ECMO. This ovine smoke injury and ECMO model has revealed that the introduction of an ECMO circuit alone leads to an increase in collagen-induced platelet aggregation and a reduction in the levels of coagulation factors FVIII, FXII and fibrinogen. When a host with S-ALI is placed on ECMO, these haemostatic alterations are augmented.

Studies on the impact of ECMO on platelet number document variable results. A number of case series and reviews suggest the ECMO circuit may lead to thrombocytopenia through platelet consumption [1517]; however, two recent studies demonstrated that ECMO is not associated with decreased platelet numbers [18, 19]. Consistent with these latter reports, we observed no significant decrease in platelet numbers in our ovine ECMO model.

While platelet numbers were unaffected by ECMO, it is possible that haemostasis in our ovine ECMO model may have been impacted by perturbations in platelet function. To investigate this possibility, we conducted whole blood platelet function assays. We and others have previously demonstrated the utility of human platelet function assays in the ovine setting [13, 14]. The platelet ADP response in SAMM/Border Leicester Cross sheep is comparable to that of humans [14], and in our study, there was no change with ADP-induced platelet aggregation in any of the sheep groups compared with healthy controls. In contrast, researchers in several studies have reported an impaired platelet response with ADP in adults [8, 20] as well as children on ECMO [21]. Patients are often on ECMO for extended time periods, and because our study involved only a 24-h observation period, it is conceivable that ADP-dependent platelet dysfunction may develop with prolonged exposure. While the collagen response in SAMM/Border Leicester Cross sheep is lower than that of humans [14], we observed significant increases in collagen-triggered platelet function with S-ALI and ECMO individually and sequentially. The shear stress from the ECMO circuit is thought to damage endothelial cells, thus exposing collagen and inducing platelet aggregation [22]. In addition, shear stress has been shown to induce amplification of platelet micro-particle production and shedding [23], as well as to prime platelets for faster activation [24].

SAMM/Border Leicester Cross sheep have similar levels of von Willebrand factor (vWF) to humans, but they have significantly higher levels of FVIII [14]. Results from this study demonstrate that while S-ALI does not affect circulating FVIII or vWF, the introduction of the ECMO circuit in both ECMO controls and S-ALI + ECMO resulted in a significant reduction in vWF and FVIII levels. Because our analysis was corrected for fluid administration, this effect cannot be attributed to haemodilution alone. Rather, exposure of blood to the increased shear stress of the ECMO circuit may be causative. Increased shear stress, as seen in ventricular assist devices (VADs), total artificial hearts and aortic valve stenosis, has been reported to cause alterations in the configuration of vWF and reduced levels of high-molecular-weight multimers (HMWM) of vWF [25]. Patients on long-term VADs have been reported to develop acquired von Willebrand syndrome, which has been linked to the loss of HMWM [26, 27]. In a study of eight ECMO patients, researchers reported relatively stable FVIII levels but a decreased ristocetin cofactor to vWF ratio, indicating the possible loss of HMWM of vWF [17, 28]. While no change to FVIII levels has been recorded in human studies, we speculate that the higher levels of FVIII in sheep enabled us to detect this change more easily.

Because ROTEM is routinely used to provide rapid results for managing bleeding in our intensive care unit [29], we also applied it to assess whole blood coagulation in this model. The shortened INTEM CTs in both the ECMO groups are consistent with reduced FVIII and vWF. As expected with heparin administration, prolongation of the aPTT did not change significantly across groups. With respect to measures of the extrinsic pathway, both S-ALI controls and S-ALI + ECMO samples demonstrated an increase in PT to more than double the baseline time after 24 h of ECMO support. These results suggest that whole blood coagulation assays (thromboelastometry) may be more informative and sensitive than plasma-based assays (aPTT and PT) when monitoring ECMO patients [30].

Our results showed that both S-ALI and ECMO individually compromised fibrinogen function, and the combined insult of S-ALI + ECMO produced a cumulative effect. Interestingly, serial monitoring revealed a transient early decrease in fibrinogen level and function following the initiation of ECMO, which begins normalising after 12 h. Fibrinogen is one of the first plasma proteins to be adsorbed onto the ECMO circuit and has been shown to be associated with platelet consumption [31]. Whether fibrinogen levels return to baseline levels and function after 24 h remains to be investigated.

d-Dimer levels did not change significantly in any of the groups, but protein C and AT levels decreased with time in both S-ALI controls and S-ALI + ECMO. Studies in patients with burn injuries and inhalational trauma showed increased thrombin-AT complexes and fibrin degradation products with decreased levels of protein C and AT consistent with pulmonary coagulopathy [32]. A series of ovine models of smoke inhalation injury have shown that aerosolised anticoagulants such as heparin, recombinant human AT and recombinant activated protein C reduce airway obstruction and improve oxygenation [3335]. Because this was a 24-h model, we were unable to determine if the compromise to the extrinsic pathway continued, plateaued or recovered with time.

As with any animal model, there were species-associated limitations. While SAMM/Border Leicester Cross sheep have aPTT, PT, TCT and fibrinogen levels similar to those in humans, they have significantly higher FVIII and half the protein C levels. In addition, coagulation measurements were performed using assays optimised for humans and were referenced against human calibration plasma with assigned values traceable to National Institute for Biological Standards and Control standards. The 24-h duration of the experiment was also a limitation because most patients remain on ECMO support for days to weeks. The complexity of the study and the high cost of the experiment prevented us from extending the duration of ECMO. Longer-duration studies are required to determine if the observed haemostatic changes are transient or stable or whether they will escalate.

The predominant challenge for a clinician caring for a patient on ECMO is making an informed assessment of the bleeding and clotting risks of each patient. Assessment of the patient’s haemostasis includes consideration of the pathophysiology, degree of organ failures and extent of tissue trauma during cannulation. Thus, a holistic approach to haemostatic management is needed to balance all these factors. This incremental ovine model enabled us to show that S-ALI + ECMO induced decreases in FVIII, FXII and vWAg and altered platelet response. While factor assays cannot be performed quickly, both ROTEM and whole blood platelet aggregometry provide rapid information on whole blood coagulation and thus may be used to guide the use of blood product support, factor replacement, anti-coagulation therapy and anti-fibrinolytics. Future research on the utility of ROTEM and whole blood platelet aggregometry of ECMO patients is required to determine their efficacy to support real-time haemostatic management in this cohort.

Conclusions

This animal model enabled us to demonstrate that the introduction of an ECMO circuit itself alters haemostasis, specifically by increasing collagen-induced platelet aggregation and reducing levels of fibrinogen, FVIII and vWF. In addition, the model demonstrated that these haemostatic perturbations are amplified in a host with pre-existing pulmonary injury. Thus, the introduction of ECMO support on its own alters haemostasis in the first 24 h. Longer-duration studies are required to characterise the haemostatic changes beyond this time. Additional efficacy studies are also required to assess the potential of point-of-care coagulation and platelet function tests for guiding haemostatic management during ECMO.

Abbreviations

ACT: 

Activated clotting time

ADP: 

Adenosine diphosphate

aPTT: 

Activated partial thromboplastin time

AT: 

Anti-thrombin

B: 

Baseline

CFT: 

Clot formation time

CT: 

Clotting time

ECMO: 

Extracorporeal membrane oxygenation

FBE: 

Full blood examination

Fib(C): 

Clauss fibrinogen

FVIII: 

Factor VIII

FXII: 

Factor XII

Hb: 

Haemoglobin

Hct: 

Haematocrit

HMWM: 

High-molecular-weight multimers

MCF: 

Maximum clot firmness

PLT: 

Platelets

PT: 

Prothrombin time

RCC: 

Red cell count

ROTEM: 

Rotational thromboelastometry

S-ALI: 

Smoke-induced acute lung injury

SAMM: 

South African meat merino

TCT: 

Thrombin clotting time

VAD: 

Ventricular assist device

vWAg: 

von Willebrand factor antigen

vWF: 

Von Willebrand factor

WCC: 

White cell count

Declarations

Acknowledgements

Not applicable.

Funding

This work was supported by funds from the National Health and Medical Research Council, The Prince Charles Hospital Foundation, and Pathology Queensland Study Education & Research Trust Fund. JFF received funding from the Office of Health and Medical Research, Queensland Health. The Australian government funds the Australian Red Cross Blood Service to provide blood, blood products and services to the Australian community. The funding bodies had no influence on the design of the study; the collection, analysis or interpretation of data; or the writing of the manuscript.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Authors’ contributions

MRP, YLF, GS, MMS, JPT, KH and NMP performed analysis and interpretation. JFF, YLF and KS conceived of and designed the study. SDD, KRD, MRP, GS, SRF, CIM, JFF, KS and YLF performed sheep studies. KH performed statistical analysis. All authors reviewed and approved the final version of the manuscript.

Ethics approval and consent to participate

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.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Critical Care Research Group, University of Queensland and the Prince Charles Hospital
(2)
School of Health and Sport Sciences, University of the Sunshine Coast
(3)
Research and Development, Australian Red Cross Blood Service
(4)
Cancer Care Services, Royal Brisbane and Women’s Hospital
(5)
University of Queensland
(6)
School of Biomedical Sciences, Faculty of Health, Queensland University of Technology
(7)
QIMR Berghofer Metro North Hospital and Health Service Statistics Unit

References

  1. Extracorporeal Life Support Organization (ELSO). ECLS registry report international summary. Ann Arbor, MI: ELSO; 2016.Google Scholar
  2. Aubron C, Cheng AC, Pilcher D, Leong T, Magrin G, Cooper DJ, et al. Factors associated with outcomes of patients on extracorporeal membrane oxygenation support: a 5-year cohort study. Crit Care. 2013;17(2):R73.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Brogan TV, Thiagarajan RR, Rycus PT, Bartlett RH, Bratton SL. Extracorporeal membrane oxygenation in adults with severe respiratory failure: a multi-center database. Intensive Care Med. 2009;35(12):2105–14.View ArticlePubMedGoogle Scholar
  4. Zangrillo A, Landoni G, Biondi-Zoccai G, Greco M, Greco T, Frati G, et al. A meta-analysis of complications and mortality of extracorporeal membrane oxygenation. Crit Care Resusc. 2013;15(3):172–8.PubMedGoogle Scholar
  5. Oliver WC. Anticoagulation and coagulation management for ECMO. Semin Cardiothorac Vasc Anesth. 2009;13(3):154–75.View ArticlePubMedGoogle Scholar
  6. Extracorporeal Life Support Organization (ELSO). ELSO anticoagulation guideline. Ann Arbor, MI: ELSO; 2014.Google Scholar
  7. Faraoni D, Levy JH. Algorithm-based management of bleeding in patients with extracorporeal membrane oxygenation. Crit Care. 2013;17(3):432.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Nair P, Hoechter DJ, Buscher H, Venkatesh K, Whittam S, Joseph J, et al. Prospective observational study of hemostatic alterations during adult extracorporeal membrane oxygenation (ECMO) using point-of-care thromboelastometry and platelet aggregometry. J Cardiothorac Vasc Anesth. 2015;29(2):288–96.View ArticlePubMedGoogle Scholar
  9. Riedel T, Fraser JF, Dunster K, Fitzgibbon J, Schibler A. Effect of smoke inhalation on viscoelastic properties and ventilation distribution in sheep. J Appl Physiol. 2006;101(3):763–70.View ArticlePubMedGoogle Scholar
  10. Shekar K, Fung YL, Diab S, Mullany DV, McDonald CI, Dunster KR, et al. Development of simulated and ovine models of extracorporeal life support to improve understanding of circuit-host interactions. Crit Care Resusc. 2012;14(2):105–11.PubMedGoogle Scholar
  11. Lange M, Traber DL. The authors reply: Preclinical evaluation of epinephrine nebulization to reduce airway hyperemia and improve oxygenation after smoke inhalation injury. Crit Care Med. 2011;40(1):356.View ArticleGoogle Scholar
  12. Vidal Melo MF, Harris RS, Layfield D, Musch G, Venegas JG. Changes in regional ventilation after autologous blood clot pulmonary embolism. Anesthesiology. 2002;97(3):671–81.View ArticlePubMedGoogle Scholar
  13. Baumgarten A, Wilhelmi M, Kalbantner K, Ganter M, Mischke R. Measurement of platelet aggregation in ovine blood using a new impedance aggregometer. Vet Clin Pathol. 2010;39(2):149–56.View ArticlePubMedGoogle Scholar
  14. Foley SR, Solano C, Simonova G, Spanevello MM, Bird RJ, Semple JW, et al. A comprehensive study of ovine haemostasis to assess suitability to model human coagulation. Thromb Res. 2014;134(2):468–73.View ArticlePubMedGoogle Scholar
  15. Cheung PY, Sawicki G, Salas E, Etches PC, Schulz R, Radomski MW. The mechanisms of platelet dysfunction during extracorporeal membrane oxygenation in critically ill neonates. Crit Care Med. 2000;28(7):2584–90.View ArticlePubMedGoogle Scholar
  16. Glick D, Dzierba AL, Abrams D, Muir J, Eisenberger A, Diuguid D, et al. Clinically suspected heparin-induced thrombocytopenia during extracorporeal membrane oxygenation. J Crit Care. 2015;30(6):1190–4.View ArticlePubMedGoogle Scholar
  17. Panigada M, Artoni A, Passamonti SM, Maino A, Mietto C, L’Acqua C, et al. Hemostasis changes during veno-venous extracorporeal membrane oxygenation for respiratory support in adults. Minerva Anestesiol. 2016;82(2):170–9.PubMedGoogle Scholar
  18. Abrams D, Baldwin MR, Champion M, Agerstrand C, Eisenberger A, Bacchetta M, et al. Thrombocytopenia and extracorporeal membrane oxygenation in adults with acute respiratory failure: a cohort study. Intensive Care Med. 2016;42(5):844–52.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Dzierba AL, Roberts R, Muir J, Alhammad A, Schumaker G, Clark J, et al. Severe thrombocytopenia in adults with severe acute respiratory distress syndrome: impact of extracorporeal membrane oxygenation use. ASAIO J. 2016;62(6):710–4.View ArticlePubMedGoogle Scholar
  20. Mutlak H, Reyher C, Meybohm P, Papadopoulos N, Hanke AA, Zacharowski K, et al. Multiple electrode aggregometry for the assessment of acquired platelet dysfunctions during extracorporeal circulation. Thorac Cardiovasc Surg. 2015;63(1):21–7.View ArticlePubMedGoogle Scholar
  21. Saini A, Hartman ME, Gage BF, Said A, Gazit AZ, Eghtesady P, et al. Incidence of platelet dysfunction by thromboelastography-platelet mapping in children supported with ECMO: a pilot retrospective study. Front Pediatr. 2015;3:116.PubMedGoogle Scholar
  22. Poole AW, Watson SP. Regulation of cytosolic calcium by collagen in single human platelets. Br J Pharmacol. 1995;115(1):101–6.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Miyazaki Y, Nomura S, Miyake T, Kagawa H, Kitada C, Taniguchi H, et al. High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. Blood. 1996;88(9):3456–64.PubMedGoogle Scholar
  24. Sheriff J, Bluestein D, Girdhar G, Jesty J. High-shear stress sensitizes platelets to subsequent low-shear conditions. Ann Biomed Eng. 2010;38(4):1442–50.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood. 2006;108(6):1903–10.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Heilmann C, Geisen U, Beyersdorf F, Nakamura L, Benk C, Trummer G, et al. Acquired von Willebrand syndrome in patients with extracorporeal life support (ECLS). Intensive Care Med. 2012;38(1):62–8.View ArticlePubMedGoogle Scholar
  27. Nascimbene A, Neelamegham S, Frazier OH, Moake JL, Dong JF. Acquired von Willebrand syndrome associated with left ventricular assist device. Blood. 2016;127(25):3133–41.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Bembea MM, Schwartz JM, Shah N, Colantuoni E, Lehmann CU, Kickler T, et al. Anticoagulation monitoring during pediatric extracorporeal membrane oxygenation. ASAIO J. 2013;59(1):63–8.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Pearse BL, Smith I, Faulke D, Wall D, Fraser JF, Ryan EG, et al. Protocol guided bleeding management improves cardiac surgery patient outcomes. Vox Sang. 2015;109(3):267–79.View ArticlePubMedGoogle Scholar
  30. Prakash S, Wiersema UF, Bihari S, Roxby D. Discordance between ROTEM® clotting time and conventional tests during unfractionated heparin-based anticoagulation in intensive care patients on extracorporeal membrane oxygenation. Anaesth Intens Care. 2016;44(1):85–92.Google Scholar
  31. Lubnow M, Philipp A, Foltan M, Bull Enger T, Lunz D, Bein T, et al. Technical complications during veno-venous extracorporeal membrane oxygenation and their relevance predicting a system-exchange – retrospective analysis of 265 cases. PLoS One. 2014;9(12):e112316.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Hofstra JJ, Vlaar AP, Knape P, Mackie DP, Determann RM, Choi G, et al. Pulmonary activation of coagulation and inhibition of fibrinolysis after burn injuries and inhalation trauma. J Trauma. 2011;70(6):1389–97.View ArticlePubMedGoogle Scholar
  33. Enkhbaatar P, Cox RA, Traber LD, Westphal M, Aimalohi E, Morita N, et al. Aerosolized anticoagulants ameliorate acute lung injury in sheep after exposure to burn and smoke inhalation. Crit Care Med. 2007;35(12):2805–10.View ArticlePubMedGoogle Scholar
  34. Maybauer MO, Maybauer DM, Fraser JF, Traber LD, Westphal M, Enkhbaatar P, et al. Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis. Crit Care Med. 2006;34(9):2432–8.View ArticlePubMedGoogle Scholar
  35. Murakami K, McGuire R, Cox RA, Jodoin JM, Bjertnaes LJ, Katahira J, et al. Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep. Shock. 2002;18(3):236–41.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2017

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