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Haemostatic changes in septic patients are complex, with both procoagulant and anticoagulant changes. Thirty-eight patients with severe sepsis and 32 controls were investigated by coagulation screens, individual factor assays, calibrated automated thrombography (CAT), whole blood low-dose-tissue factor activated (LD-TFA) Rotem and LD-TFA waveform analysis. Thirty-six of 38 patients had an abnormal coagulation screen. The mean levels of factors II, V (P < 0·05), VII, X, XI and XII, antithrombin and protein C (P < 0·01) was decreased in sepsis compared with controls. The mean factor VIII and fibrinogen level (P < 0·001) was increased. CAT in platelet rich and poor plasma showed a prolonged lag time (P < 0·02), decreased peak thrombin (P < 0·02) and delayed time to peak thrombin (P < 0·001) in sepsis patients, however, the endogenous thrombin potential was equivalent in sepsis and controls. In LD-TFA Rotem, septic patients had delayed clot times (P = 0·04) but an increased maximum velocity of clot formation (P < 0·01) and area under the clot elasticity curve (P < 0·01). LD-TFA waveform analysis showed a delayed onset time but an increased rate of clot formation (P < 0·005). In conclusion, global tests of haemostasis suggest that in this patient group, activation of haemostasis is delayed but once initiated thrombin generation and clot formation are normal or enhanced.
Sepsis is associated with complex changes in haemostasis. In severe cases, disseminated intravascular coagulation (DIC) may lead to consumption of platelets and coagulation factors resulting in clinical bleeding, whilst in other situations a compensated consumptive coagulopathy may be present, with fibrinogen levels and platelet number preserved or raised (Dempfle, 2004). Altered levels of both procoagulant factors and anticoagulant proteins have been described in patients with sepsis syndrome (Hesselvik et al, 1989; Mavrommatis et al, 2000; Dempfle, 2004; Dhainaut et al, 2005). Some changes, such as decreased levels of coagulation factors, predispose the patient to bleeding whilst others, such as raised factor VIII and fibrinogen and decreased levels of protein C and antithrombin, induce a prothrombotic state.
Critically ill patients with sepsis syndrome often develop multi-organ organ failure. This complication has a complex pathophysiology but is thought to be partly due to microvascular thrombosis (Dixon, 2004). This hypothesis is indirectly supported by studies showing that patients with sepsis have increased markers of activation of haemostasis, such as d-dimer, prothrombin fragment 1 + 2 and thrombin antithrombin complexes (TAT) (Gando et al, 1998; Mavrommatis et al, 2000; Amaral et al, 2004). Improved assessment of the balance between anticoagulant and prothrombotic changes may aid understanding of haemostatic changes associated with sepsis syndrome, give insight into the pathogenesis of multi-organ failure and ultimately aid the clinical management of patients.
Current concepts of haemostasis support the view that haemostasis is activated through low concentrations of tissue factor (Mann et al, 2003; Roberts et al, 2004) and this has been confirmed in in vivo models of sepsis (Taylor et al, 1991). It is also thought that the rate of clot formation, and in particular the rate of thrombin generation, is crucial for formation of a stable fibrin clot (Roberts et al, 2004). Routine coagulation screens are often prolonged in critically ill patients with sepsis syndrome (Dempfle, 2004). However, there is debate about the utility of these tests in assessing haemostasis (Mannucci, 2006; Reverter, 2006) and coagulation screens appear not to accurately reflect a patient's risk of bleeding (Segal & Dzik, 2005). This may be because the endpoint of these assays is early in the haemostatic process or because they are initiated by high concentrations of tissue factor or contact activators that do not reflect the physiological situation. Despite these shortcomings, prolonged coagulation screens are often treated with infusion of fresh frozen plasma (FFP), even though individual coagulation factor levels are often not significantly reduced (Chowdhury et al, 2004).
There has been recent interest in the role of global tests of haemostasis, such as thrombin generation assays, in the investigation of both acquired and congenital haemostatic defects (Al Dieri et al, 2002; Hemker et al, 2003; Luddington & Baglin, 2004). A thromboelastographic method, activated by low concentration tissue factor and measured using the Rotem® machine, has also been described (Sorensen et al, 2003). Updated software enabled the standard clot elasticity trace to be differentiated to give a velocity of clot strengthening, and the area under this curve gives a measure of the total amount of increasing clot strength (Sorensen et al, 2003). A third test is described here, which activates haemostasis through low concentrations of tissue factor in the presence of thrombomodulin and measures rate of fibrin clot formation using the first derivative of the MDA waveform.
Assays that are activated by low concentrations of tissue factor are susceptible to contact activation (Luddington & Baglin, 2004). Although activation of contact factors may occur in sepsis, depletion of these factors will not affect in vivo haemostasis and assays that are contact-activated may be misleading in the setting of sepsis. In the assays presented here, we therefore inhibited contact activation with corn trypsin inhibitor (CTI). We report the results of routine coagulation tests and three global haemostatic assays in critical ill patients with severe sepsis syndrome.
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The data reported here demonstrated that critically ill patients with sepsis syndrome have abnormal routine coagulation screens and delayed initiation of haemostasis, as measured by three low concentration tissue factor-activated global haemostatic assays. These assays further showed that, once haemostasis was activated, the propagation and clot formation phase was either normal or enhanced.
The three global haemostatic assays showed a similar pattern of results. The CAT assays demonstrated that, whilst initiation of thrombin generation was delayed and peak thrombin reduced, the total amount of thrombin, as measured by the ETP, was unchanged in the patients with sepsis compared with controls. Similarly, whilst the time to clot in the whole blood, low concentration tissue factor Rotem assay and the MDA waveform assay were also delayed, the rate of increasing fibrin clot elasticity and rate of clot formation was enhanced.
The findings of this study may be explained by the measured levels of individual coagulation factors in the light of currently accepted models of haemostasis. Coagulation is thought to be activated through the tissue factor/factor VII pathway, which generates a small amount of thrombin, insufficient to clot fibrinogen, but required for the activation of factors V and VIII and expression of negatively charged phospholipids on platelets. The rapid burst of thrombin generation required to form a stable clot is then driven through the tenase complex of factor IXa and factor VIIIa and the prothrombinase complex of factor Xa and factor Va localised on the negatively charged phospholipid surface of activated platelets (Mann et al, 2003; Roberts et al, 2004). Activation of haemostasis in sepsis has been shown to be through the tissue factor pathway (Taylor et al, 1991; Levi et al, 2003).
In the sepsis group of patients, it is likely that the decreased levels of factors VII, X and II led to a delay in the generation of sufficient initial thrombin to activate factors V and VIII and to stimulate expression of platelet phospholipids. The major components of the intrinsic pathway (factors IXa and VIIIa), however, were preserved or increased in sepsis patients and hence, once the cofactors had been activated, thrombin and fibrin generation proceeded normally or was enhanced. This mechanism also explains the prolonged PT and aPTT tests, which are sensitive to the initiation rather than the propagation phase of haemostasis.
Measurement of individual coagulation factors and anticoagulants in this study demonstrated a pattern similar to those previously reported (Hesselvik et al, 1989). The procoagulant factors II, V, VII, X and XI as well as the anticoagulant factors antithrombin and protein C, were decreased compared with normal. The level of these factors correlated with serum albumin, suggesting that hepatic dysfunction as well as consumption played a role. Some coagulation factors, such as factor VIII and fibrinogen, were increased whilst factor IX was stable. The raised fibrinogen (but not factor VIII) correlated with CRP confirming that the increase was the result of an acute phase reaction. It is unclear what effect the combined changes in individual coagulation factors and anticoagulant have on global haemostasis, especially as some patients were also thrombocytopenic. As a result, it was not clear from the measurement of individual factors whether patients were at increased risk of bleeding, thrombosis or both. It is possible that some parameters of the global haemostatic tests may give information about the balance between pro- and anti-coagulant changes. The low concentration tissue factor-activated MDA waveform assay described here, for example, measures the rate of fibrin clot formation in PPP in the presence of thrombomodulin. This means the assay is sensitive to the protein C concentration in the sample in addition to the procoagulant clotting factors. The CAT and Rotem assays are not sensitive to the reduced levels of antithrombin and protein C and would underestimate the procoagulant state in patients.
In routine clinical practice, clinicians rely on the PT and aPTT to assess a patient's haemostatic status. Prolongation of these assays is interpreted as evidence of an anticoagulated state and FFP is often infused, particularly at the time of invasive procedures. This contrasts with previous findings of raised markers of activation of haemostasis, such as prothrombin fragment 1 + 2 and TAT, in sepsis syndrome (Gando et al, 1998; Mavrommatis et al, 2000; Amaral et al, 2004). We have previously shown that abnormal coagulation screening tests are not a reliable guide to coagulation factors levels in critically ill patients (Chowdhury et al, 2004) and there continues to be a debate regarding the utility of coagulation screens to predict bleeding during invasive procedures in the context of hepatic dysfunction (Segal & Dzik, 2005; Mannucci, 2006; Reverter, 2006). This issue was further highlighted by our finding that factor XII was the most commonly reduced individual coagulation factor in the cohort of septic patients as previously reported (Hesselvik et al, 1989). Reduced factor XII is an important cause of a prolonged aPTT, but it is not required for haemostasis and so the prolongation of the aPTT related to a low factor XII is misleading and likely to be one of the major reasons why the aPTT is of limited utility in assessing clinical bleeding risk in septic patients.
The prolonged coagulation screening tests and the delayed initiation of haemostasis demonstrated by the three global tests probably reflect similar processes. If this is the case then the initiation time in the global assays may also have limited utility in assessing an individual patient's risk of bleeding and need for blood product support. It is possible that other parameters measured on the global assays that assess the propagation phase of haemostasis may be more useful. Although in our study the septic patients, as a group, had some results suggestive of a prothrombotic state, individuals had parameters of global haemostasis below the normal range, which may indicate an increased risk of bleeding during invasive procedures.
The present study aimed to establish the effect of sepsis on global haemostatic assays. It did not aim to link these results to either bleeding or thrombotic complications in these patients and patients who were bleeding were not recruited into the study. The role of these assays in the management of patients remains to be defined. Further studies are underway to link clinical relevant bleeding endpoints to global assays of haemostasis in this patient population.
Baseline levels and dynamic changes in haemostatic factors have been associated with outcome in critically ill patients with sepsis and used as evidence to support a pathophysiological link between coagulation abnormalities and multi-organ failure (Dhainaut et al, 2005). Biphasic clot waveform has also been shown to predict clinical outcome in sepsis (Toh et al, 2003). There were too few patients included in this study to investigate whether any parameters measured were predictive of prognosis or outcome.
We conclude that global assays activated by low concentrations of tissue factor show that critically ill patients with sepsis have delayed activation of haemostasis but, once initiated, thrombin and clot formation are normal or enhanced. The results may help to explain why routine coagulation screen tests poorly predict bleeding in sepsis. Whether global assays will be useful in clinical practice will only be established once they are linked to relevant end points of bleeding, end organ failure and clinical outcomes.