Fluid resuscitation with 0.9% saline alters haemostasis in an ovine model of endotoxemic shock.

INTRODUCTION: Fluid resuscitation is a cornerstone of severe sepsis management, however there are many uncertainties surrounding the type and volume of fluid that is administered. The entire spectrum of coagulopathies can be seen in sepsis, from asymptomatic aberrations to fulminant disseminated intravascular coagulation (DIC). The aim of this study was to determine if fluid resuscitation with saline contributes to the haemostatic derangements in an ovine model of endotoxemic shock. MATERIALS AND METHODS: Twenty-one adult female sheep were randomly divided into no endotoxemia (n = 5) or endotoxemia groups (n = 16) with an escalating dose of lipopolysaccharide (LPS) up to 4 µg/kg/h administered to achieve a mean arterial pressure below 60 mmHg. Endotoxemia sheep received either no bolus fluid resuscitation (n = 8) or a 0.9% saline bolus (40 mL/kg over 60 min) (n = 8). No endotoxemia, saline only animals (n = 5) underwent fluid resuscitation with a 0.9% bolus of saline as detailed above. Hemodynamic support with vasopressors was initiated if needed, to maintain a mean arterial pressure (MAP) of 60-65 mm Hg in all the groups. RESULTS: Rotational thromboelastometry (ROTEM®) and conventional coagulation biomarker tests demonstrated sepsis induced derangements to secondary haemostasis. This effect was exacerbated by saline fluid resuscitation, with low pH (p = 0.036), delayed clot initiation and formation together with deficiencies in naturally occurring anti-coagulants antithrombin (p = 0.027) and Protein C (p = 0.001). CONCLUSIONS: Endotoxemia impairs secondary haemostasis and induces changes in the intrinsic, extrinsic and anti-coagulant pathways. These changes to haemostasis are exacerbated following resuscitation with 0.9% saline, a commonly used crystalloid in clinical settings.

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

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.

A comprehensive study of ovine haemostasis to assess suitability to model human coagulation.

INTRODUCTION: Similarities in size, anatomy and physiology have supported the use of sheep to model a wide range of human diseases, including coagulopathy. However, coagulation studies involving sheep are limited by the absence of high quality data defining normal ovine coagulation and fibrinolysis. MATERIALS AND METHODS: Full blood examination, routine and specialised coagulation tests, rotational thromboelastometry and whole blood platelet aggregometry was performed on 50 healthy Samm & Border Leicester Cross ewes and compared to corresponding human ranges. Intraspecies breed and gender variability was investigated by comparison to a smaller population of 13 healthy Merino wethers. RESULTS: Ovine coagulation was similar to human according to routine coagulation methods (PT, aPTT, TCT, Fib(C)) and some specialised coagulation tests (vWF, AT, Plasmin Inh). Despite these similarities, ovine secondary haemostasis demonstrated substantial differences to that of human. Rapid initiation of the contact activation pathway, high levels of FVIII, low Protein C, greater overall clot firmness and a reduced capacity for clot lysis was documented in sheep. In addition, ADP and collagen agonists precipitated a reduced primary haemostatic response in sheep relative to human. Intraspecies differences in whole blood platelet aggregometry between the cohorts of sheep indicate the need for breed-specific normal ranges. CONCLUSIONS: The application of a board spectrum of coagulation assays has enabled elucidation of the similarities as well as differences between ovine and human coagulation. The new knowledge generated from this study will guide the design of future translational coagulation studies in ovine models.

Molecular mechanisms influencing NK cell development: implications for NK cell malignancies.

Natural Killer (NK) cells are important effector cells in both the innate and adaptive immune responses. Although they were identified almost 40 years ago, our understanding of how and where NK cells develop is rudimentary. In particular, we have only a limited understanding of the signaling pathways that need to be activated to cause NK cell commitment and maturation. Knowledge of this process is important as disruptions can lead to the development of highly aggressive NK cell malignancies. In this review, we discuss the known molecular mechanisms that trigger NK cell commitment, prompt them to mature and finally allow them to become functional killers. Known disruptions in this developmental process, and how they may contribute to malignancy, are also addressed.