Alex Gatt

Thrombin generation testing in routine clinical practice: are we there yet?

Thrombin is the central enzyme in the coagulation cascade. Estimation of an individual’s potential to generate thrombin may correlate more closely with a hyper‐ or hypo‐coagulable phenotype, compared to traditional coagulation tests. The possible correlation and recent technical advances in thrombin generation measurement has caused a significant interest in the method and the development of commercial assays. Several variations of the assay exist depending on the defect to be investigated. Fluorogenic thrombin generation assays have acceptable intra‐laboratory variation but a higher inter‐laboratory variation. Variation in preanalytical variables makes comparisons between studies difficult. Thrombin generation is highly variable between individuals and there are suggestions that this may allow individualized treatment based on global haemostatic response in patients with bleeding disorders or on anticoagulant therapy. In patients with thrombotic disorders it may be possible to identify those at higher risk of recurrent thrombosis. For both scenarios, however, data from large prospective studies are lacking or inconclusive and a good relationship between thrombin generation and phenotype remains to be established. Further standardization of the assay is needed before large multicentre studies can be conducted and until then thrombin generation in routine clinical practice is not yet a reality.

Enhanced thrombin generation in patients with cirrhosis-induced coagulopathy

Summary.  Background: Prothrombin time (PT) and the international normalized ratio (INR) are still routinely measured in patients with liver cirrhosis to ‘assess’ their bleeding risk despite the lack of correlation with the two. Thrombin generation (TG) assays are global assays of coagulation that are showing promise in assessing bleeding and thrombosis risks. Aim: To study the relationship between the INR and TG profiles in cirrhosis‐induced coagulopathy. Methods: Seventy‐three patients with cirrhosis were studied. All TG parameters were compared with those from a normal control group. Contact activation was prevented using corn trypsin inhibitor. TG was also assayed in the presence of Protac®. The endogenous thrombin potential (ETP) ratio was derived by dividing the ETP with Protac® by the ETP without Protac®. Results: The INR (mean 1.7) did not correlate with the ETP and the velocity of TG (P > 0.05). There was no difference between the lag time and ETP of the two groups (P > 0.05). The velocity of TG was increased in cirrhosis (67.95 ± 34.8 vs. 45.05 ± 25.9 nM min−1; P = 0.016) especially in patients with INRs between 1.21 and 2.0. Both the ETP with Protac® and the ETP ratio were increased in cirrhosis (mean 1074 ± 461.4 vs. 818 ± 357.9 nM min, P = 0.004 and 0.80 ± 0.21 vs. 0.44 ± 0.15, P ≤ 0.0001, respectively). Conclusion: Despite a raised INR, TG parameters are consistent with a hypercoagulable profile in cirrhosis‐related coagulopathy. This confirms that the PT or INR should not be used to assess bleeding risk in these patients, and other parameters, such as TG, need to be explored as clinical markers of coagulopathy.

Patients with liver cirrhosis suffer from primary haemostatic defects? Fact or fiction?

Patients with cirrhosis can have abnormalities in laboratory tests reflecting changes in primary haemostasis, including bleeding time, platelet function tests, markers of platelet activation, and platelet count. Such changes have been considered particularly relevant in the bleeding complications that occur in cirrhosis. However, several studies have shown that routine diagnostic tests, such as platelet count, bleeding time, PFA-100, thromboelastography are not clinically useful to stratify bleeding risk in patients with cirrhosis. Moreover, treatments used to increase platelet count or to modulate platelet function could potentially do harm. Consequently the optimal management of bleeding complications is still a matter of discussion. Moreover, in the last two decades there has been an increased recognition that not only bleeding but also thrombosis complicates the clinical course of cirrhosis. Thus, we performed a literature search looking at publications studying both qualitative and quantitative aspects of platelet function to verify which primary haemostasis defects occur in cirrhosis. In addition, we evaluated the contribution of qualitative and quantitative aspects of platelet function to the clinical outcome in cirrhosis and their therapeutic management according to the data available in the literature. From the detailed analysis of the literature, it appears clear that primary haemostasis may not be defective in cirrhosis, and a low platelet count should not necessarily be considered as an automatic index of an increased risk of bleeding. Conversely, caution should be observed in patients with severe thrombocytopenia where its correction is advised if bleeding occurs and before invasive diagnostic and therapeutic procedures.

Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters.

The fluorogenic calibrated automated thrombin-generation assay is influenced by contact pathway activation in platelet-rich and platelet-poor plasma. This influence lessens with increasing tissue factor (TF) concentrations and is inhibited by corn trypsin inhibitor (CTI). CTI is expensive and at what TF concentration its influence becomes irrelevant is unclear. Spiking of factor VIII (FVIII)-depleted plasma with FVIII, in samples without CTI, shows a plateau of thrombin generation at low normal FVIII levels. Given the association with thrombosis at high levels, a continuing increase in thrombin generation would be expected. We studied the effect of CTI on this relation by spiking experiments up to 4.8 IU/ml at 1 pmol/l TF and compared the influence of CTI at 1 and 5 pmol/l in platelet-poor plasma. CTI significantly influences thrombin generation in platelet-poor plasma at 1 pmol/l TF (difference of means for endogenous thrombin potential of 232.5 nmol/l per min, P < 0.0001) and peak of 48 nmol/l (P < 0.0001)) but not at 5 pmol/l. Spiking experiments without CTI confirm the hyperbolic relation between FVIII coagulant activity (FVIII:C) and endogenous thrombin potential with a plateau at 0.70–1.40 IU/ml. With CTI, a near-linear response up to 1.0 IU/ml was found with a plateau at 2.4–4.8 IU/ml. For peak thrombin, no plateau was reached with CTI. The present study confirms and extends previous data on CTI and the relationship between FVIII:C and thrombin generation. CTI is not necessary at 5 pmol/l TF, and thrombin generation remains dependent on FVIII:C up to 4.8 IU/ml at 1 pmol/l with CTI. Higher levels than previously thought may be needed to normalize thrombin generation.

Evaluation of coagulation abnormalities in acute liver failure

BACKGROUND & AIMS In acute liver failure (ALF), prothrombin time (PT) and its derivative prothrombin time ratio (PTR) are elevated, and are considered predictors of increased bleeding risk. We aimed at determining whether increased PT/PTR reflects the haemostatic potential and bleeding risk in ALF patients. METHODS Twenty consecutive ALF patients were recruited. Samples were analysed on admission for standard laboratory clotting tests (e.g. PT), thromboelastography (TEG), individual pro and anticoagulant factors and thrombin generation (TG) kinetics with and without Protac, a snake venom protein C activator, and microparticle assay. TG was also measured in 20 age and sex matched healthy volunteers. RESULTS PT was significantly raised (50.7s ± 7.2, p=0.0001) but did not correlate with TEG parameters. TEG tracings were consistent with a hypocoagulable state in 20%, normal in 45%, and hypercoagulable in 35% of the patients. There was a concomitant and proportional reduction in plasma levels of both procoagulants and natural anticoagulant proteins, in conjunction with a significant elevation in plasma levels of factors-VIII (FVIII) and Von Willebrand factor, and microparticles, culminating in an overall efficient, albeit reduced, thrombin generation capacity in comparison with healthy individuals. A heparin-like effect (HLE) was also noted in most patients. No significant clinical bleeding complications occurred and no blood transfusions were required. CONCLUSIONS In ALF, despite grossly deranged PT in all patients, estimation of bleeding risk suggests that the coagulation disturbance in ALF patients is complex and heterogeneous for which an individualised approach is required.

Thrombin generation assays are superior to traditional tests in assessing anticoagulation reversal in vitro

Even though new anticoagulants are being devised with the notion that they do not require regular monitoring, when bleeding occurs, it is important to have an antidote and a reliable test to confirm whether the anticoagulant effects are persisting. We examined the effects of five heparinoids, unfractionated heparin (UFH), tinzaparin, enoxaparin, danaparoid and fondaparinux on the traditional APTT and anti-Xa assays as well as on the calibrated automated thrombogram (CAT). We also studied the ability of protamine sulphate (PS), NovoSeven, FEIBA and FFP to reverse maximum anticoagulation induced by the different heparinoids. The CAT was the only test to detect the coagulopathy of all the anticoagulants. PS produced complete reversal of UFH, and this could be monitored with all three tests. Tinzaparin can also be completely neutralised in vitro with high doses of PS, but the maximum enoxaparin reversal achieved with PS is only approximately 60%. Fondaparinux does not significantly affect the APTT and PS has no significant effect on its reversal. Only NovoSeven was able to correct the fondaparinux induced CAT abnormalities whilst having no effect on the anti-Xa level. None of the reversal agents was very effective in danaparoid spiked plasma but NovoSeven, at high dose, increased the ETP by 40% and reduced the anti-Xa level from 0.93 to 0.78 IU/ml. We conclude that the CAT is superior to the traditional coagulation tests in that it not only detects the coagulopathy of all the heparinoids but can be also be used to monitor their reversal.

Inhibitor development in haemophilia according to concentrate. Four-year results from the European HAemophilia Safety Surveillance (EUHASS) project.

Inhibitor development represents the most serious side effect of haemophilia treatment. Any difference in risk of inhibitor formation depending on the product used might be of clinical relevance. It was this studys objective to assess inhibitor development according to clotting factor concentrate in severe haemophilia A and B. The European Haemophilia Safety Surveillance (EUHASS) was set up as a study monitoring adverse events overall and according to concentrate. Since October 2008, inhibitors were reported at least quarterly. Number of treated patients was reported annually, specifying the number of patients completing 50 exposure days (Previously Untreated Patients, PUPs) without inhibitor development. Cumulative incidence, incidence rates and 95 % confidence intervals (CI) were calculated. Data from October 1, 2008 to December 31, 2012 were analysed for 68 centres that validated their data. Inhibitors developed in 108/417 (26 %; CI 22-30 %) PUPs with severe haemophilia A and 5/72 (7 %; CI 2-16%) PUPs with severe haemophilia B. For Previously Treated Patients (PTPs), 26 inhibitors developed in 17,667 treatment years [0.15/100 treatment years; CI 0.10-0.22) for severe haemophilia A and 1/2836 (0.04/100; (CI 0.00-0.20) for severe haemophilia B. Differences between plasma-derived and recombinant concentrates, or among the different recombinant FVIII concentrates were investigated. In conclusion, while confirming the expected rates of inhibitors in PUPs and PTPs, no class or brand related differences were observed.

Coagulation and fibrosis in chronic liver disease

In the hepatic tissue repair mechanism, hepatic stellate cells (HSCs) are recruited at the site of injury and their changes reflect paracrine stimulation by all neighbouring cell types, including sinusoidal endothelial cells, Kupffer cells, hepatocytes, platelets and leucocytes. Thrombin converts circulating fibrinogen to fibrin, promotes platelet aggregation, is a potent activator of endothelial cells, acts as a chemoattractant for inflammatory cells and is a mitogen and chemoattractant for fibroblasts and vascular smooth muscle cells. Most of the cellular effects elicited by thrombin are mediated via a family of widely expressed G-protein-coupled receptors termed protease activated receptors (PARs). All known members of the PAR family stimulate cell proliferation/activation in a rat HSC line. Thrombin receptors are constitutively expressed in the liver, and their expression increases in parallel with the severity and/or the duration of liver disease. In human studies, thrombotic risk factors were found to be independently associated with the extent of fibrosis; severity of hepatitis C virus (HCV)-associated liver disease appears to be less in patients with haemoihilia when compared with those with HCV alone. Several studies, based mostly on rat models, demonstrate that anticoagulants or antiplatelet agents prevent hepatic necrosis and fibrosis by acting on HSCs. These drugs could be therapeutic agents in patients with chronic liver disease and specific studies should be initiated.

Long-term outcomes of previously untreated myeloma patients: responses to induction chemotherapy and high-dose melphalan incorporated within a risk stratification model can help to direct the use of novel treatments

Induction chemotherapy followed by high‐dose melphalan (HDM) is the standard treatment for fitter patients with myeloma. The place of bortezomib and the thalidomide analogues within this treatment paradigm is yet to be established. We sought to identify patients who may benefit from the introduction of novel agents during their initial management. An intention‐to‐treat analysis was performed on 383 patients with newly diagnosed myeloma eligible for HDM to determine whether the extent of response to induction therapy and HDM correlated with long‐term survival. Early response [complete response (CR) and partial response (PR)] to induction therapy was predictive of overall survival (OS) [median OS, 7·47 years for responders (CR and PR) versus 4·89 years for non‐responders; P = 0·035]. The attainment of CR at 3 months post‐HDM correlated with a prolonged progression‐free survival (PFS) (median PFS, 7·4 years in CR group versus 5·3 years in non‐CR group; P = 0·023). This data suggests that, at every stage of treatment, the aim should be to achieve CR. Patients with suboptimal responses could be offered alternative therapy. We propose a multiparametric risk‐adapted model that includes response to induction chemotherapy and HDM, for identifying patients who may benefit from novel approaches to treatment.

Wide variation in thrombin generation in patients with atrial fibrillation and therapeutic International Normalized Ratio is not due to inflammation.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a 5–20% annual risk of stroke. Warfarin reduces this risk by at least 60%. Despite adequate anticoagulation within the target International Normalized Ratio (INR) range of 2·0–3·0, some patients still experience thrombotic and bleeding events. It is now possible to assess the intensity of anticoagulation with automated thrombin generation (TG) tests, such as the calibrated automated thrombogram (CAT). These tests were compared and an inverse relationship was found between the INR and CAT in 143 elderly AF patients. There was equally good correlation between the concentration of factors II, VII, IX and X and the INR and TG parameters. The peak thrombin was most strongly associated with the concentration of prothrombin fragment 1 + 2 in plasma. There was wide variability in TG parameters in patients with identical INR values, sometimes up to a fourfold difference. This TG variability in individuals with the same INR is not due to inflammation, at least when the latter is measured as the concentration of factor VIII coagulant activity, von Willebrand factor antigen, high sensitivity C‐reactive protein and fibrinogen. It was concluded that, although the TG and INR were closely correlated there was wide variability in peak thrombin and endogenous thrombin potential in patients within the INR therapeutic range, the cause of which remains unclear.