Veena Chantarangkul

Evidence of normal thrombin generation in cirrhosis despite abnormal conventional coagulation tests

The role played by coagulation defects in the occurrence of bleeding in cirrhosis is still unclear. This is partly due to the lack of tests that truly reflect the balance of procoagulant and anticoagulant factors in vivo. Conventional coagulation tests such as prothrombin time and activated partial thromboplastin time are inadequate to explore the physiological mechanism regulating thrombin, because they do not allow full activation of the main anticoagulant factor, protein C, whose levels are considerably reduced in cirrhosis. We used a thrombin generation test to investigate the coagulation function in patients with cirrhosis. Thrombin generation measured without thrombomodulin was impaired, which is consistent with the reduced levels of procoagulant factors typically found in cirrhosis. However, when the test was modified by adding thrombomodulin (i.e., the protein C activator operating in vivo), patients generated as much thrombin as controls. Hence, the reduction of procoagulant factors in patients with cirrhosis is compensated by the reduction of anticoagulant factors, thus leaving the coagulation balance unaltered. These findings help clarify the pathophysiology of hemostasis in cirrhosis, suggesting that bleeding is mainly due to the presence of hemodynamic alterations and that conventional coagulation tests are unlikely to reflect the coagulation status of these patients. In conclusion, generation of thrombin is normal in cirrhosis. For a clinical validation of these findings, a prospective clinical trial is warranted where the results of thrombin generation in the presence of thrombomodulin are related to the occurrence of bleeding. (HEPATOLOGY 2005;41:533–558.)

An Imbalance of Pro- vs Anti-Coagulation Factors in Plasma From Patients With Cirrhosis

BACKGROUND & AIMS Patients with cirrhosis have an increased tendency to develop thromboses despite the longer coagulation times of their plasma, compared with that of healthy individuals. We investigated whether plasma from cirrhotic patients has an imbalance of pro- vs anti-coagulation factors. METHODS We analyzed blood samples from 134 cirrhotic patients and 131 healthy subjects (controls) for levels of pro- and anti-coagulants and for thrombin generation in the presence or absence of thrombomodulin (the main physiologic activator of the protein C anticoagulant pathway). RESULTS The median ratio of thrombin generation (with/without thrombomodulin) was higher in patients (0.80; range, 0.51-1.06) than controls (0.66; range, 0.17-0.95), indicating that cirrhotic patients are resistant to the action of thrombomodulin. This resistance resulted in greater hypercoagulability of plasma from patients of Child-Pugh class C than of class A or B. The hypercoagulability of plasma from patients of Child-Pugh class C (0.86; range, 0.70-1.06) was slightly greater than that observed under the same conditions in patients with congenital protein C deficiency (0.76; range, 0.60-0.93). Levels of factor VIII, a potent pro-coagulant involved in thrombin generation, increased progressively with Child-Pugh score (from Child-Pugh class A to C). Levels of protein C, one of the most potent naturally occurring anti-coagulants, showed the opposite trend. CONCLUSIONS The hypercoagulability of plasma from patients with cirrhosis appears to result from increased levels of factor VIII and decreased levels of protein C-typical features of patients with cirrhosis. These findings might explain the risk for venous thromboembolism in patients with chronic liver disease.

The international normalized ratio calibrated for cirrhosis (INRliver) normalizes prothrombin time results for model for end‐stage liver disease calculation

The model for end‐stage‐liver‐disease (MELD) is a mathematical score used to prioritize patients for liver transplantation and includes results for creatinine, bilirubin, and prothrombin time (PT) expressed as international normalized ratio (INR). The rationale of using the MELD rests on the assumption that the score would be the same across the country if the methods used to measure the variables yield the same numerical results regardless of the testing laboratory. Evidence was provided that specific methodologies may influence the MELD, and the PT‐INR was identified as the most important. This study was designed to provide information on the between‐thromboplastin variability and to explore alternatives to obviate such variability. Fifty‐seven patients with cirrhosis were selected, and their PTs were measured with 7 thromboplastins. The thromboplastins were previously calibrated by testing plasmas from patients on vitamin K antagonists and healthy subjects to assign the international sensitivity index (ISIvka) needed to convert PT into INR. Each of the thromboplastins was also assigned an ISIliver by substituting in the calibration the plasmas from vitamin K antagonist patients with plasmas from patients with cirrhosis. INR and MELD values for individual patients were calculated by using the ISIvka or the ISIliver. The mean INRvka obtained with the 7 thromboplastins were significantly different (P < 0.001). Conversely, the mean INRliver were not. Similarly, the mean MELDvka were significantly different (P < 0.001), but those differences were abrogated for the MELDliver. Conclusion: The alternative thromboplastin calibration using plasmas from patients with cirrhosis instead of from vitamin K antagonist patients is feasible and may resolve the variability of the MELD to prioritize patients for transplantation. (HEPATOLOGY 2007.)

The G20210A mutation of the prothrombin gene in patients with previous first episodes of deep-vein thrombosis: prevalence and association with factor V G1691A, methylenetetrahydrofolate reductase C677T and plasma prothrombin levels.

A common G to A transition at nucleotide 20210 of the prothrombin gene is associated with an increased risk for deep-vein thrombosis (DVT) and high plasma levels of prothrombin. We calculated the prevalences of prothrombin G20210A, factor V G1691A (also associated with high risk for DVT) and homozygous methylenetetrahydrofolate reductase (MTHFR) C677T (associated with increased susceptibility to develop hyperhomocysteinemia) in 118 patients with a first episode of DVT and in 416 healthy controls. 15.9% of the patients and 2.3% of the controls had prothrombin G20210A (odds ratio [OR]: 8.7, 95% C.I.: 3.8-21.4); 21.1% of the patients and 3.2% of the controls had factor V G1691A (OR 7.8, 3.9-17.1); 20.5% of the patients and 21% of the controls had homozygous MTHFR C677T (OR: 1.0, 0.7-1.2). Exclusion of patients with other hereditary risk factors for DVT did not substantially modify the results. Mutant factor V and prothrombin coexisted in three patients but in no control. The concomitant presence of the MTHFR mutation did not increase the thrombotic risk associated with prothrombin G20210A. 63.2% of individuals with prothrombin G20210A had plasma levels of prothrombin in the upper quartile of distribution. After adjustment for age and sex, subjects with prothrombin levels in the upper quartile carried a slightly higher risk for thrombosis than those with lower prothrombin concentrations (OR: 1.9, 1.1-3.2). In conclusion, we found that prothrombin G20210A is relatively common in Italy and is associated with high prothrombin levels and an 8.7-fold increase in the risk for DVT. Such risk is independent of the coexistence of other known inherited risk factors for thrombosis and increases in patients with associated mutant factor V. Whether it is due to the associated increase in plasma prothrombin levels remains to be established.

High thrombin generation measured in the presence of thrombomodulin is associated with an increased risk of recurrent venous thromboembolism.

Summary.  Background: The assessment of the risk of recurrent venous thromboembolism (VTE) is important to determine the optimal duration of secondary prophylaxis. The risk can be estimated by measuring individual parameters reflecting hypercoagulability. Because of the large numbers of such putative parameters, the assessment in individual patients is complex. Application of global assays reflecting the pro‐/anti‐coagulant balance in vivo would be desirable. Objectives: To investigate the relationship between recurrent VTE and thrombin generation (TG). Patients‐methods: Two hundred and fifty‐four patients were followed‐up after a first episode of unprovoked, objectively documented VTE for a period of 2.7 years after discontinuation of treatment with vitamin K antagonists. TG was measured 1 month after discontinuation of treatment as endogenous thrombin potential (ETP), peak thrombin and lag‐time in the presence or absence of thrombomodulin. The study outcome was objectively documented symptomatic recurrent VTE. Results: Patients with ETP or peak (measured in the presence of thrombomodulin) of >960 nm*min or >193 nm had hazard ratios (HR) (95% CI) for recurrent VTE of 3.41 (1.34–8.68) or 4.57 (1.70–12.2) as compared with those with an ETP <563 nm*min or peak <115 nm. Patients with lag‐time <14.5 min had HR of 3.19 (1.29–7.89) as compared with those with lag‐time >20.8 min. HR for ETP, peak or lag‐time measured in the absence of thrombomodulin were smaller than those measured in the presence of thrombomodulin. Conclusions: The measurement of TG helps to identify patients at higher risk of VTE recurrence. The increased risk may be better appreciated if the test is performed in the presence of thrombomodulin.

Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile

Summary.  Background: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. Objectives, patients and methods: This case–control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Results: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet‐rich plasma than controls (P < 0.05). By multivariate logistic regression, only non‐null mutations were confirmed as an independent predictor of a mild clinical phenotype. Conclusions: These results indicate that non‐null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet‐rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.

The International Normalized Ratio calibrated for rivaroxaban has the potential to normalize prothrombin time results for rivaroxaban-treated patients: results of an in vitro study

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The coagulopathy of cirrhosis assessed by thromboelastometry and its correlation with conventional coagulation parameters

BACKGROUND Thromboelastometry allows continuous registration of the blood viscoelastic changes upon activation by cephaline or tissue-factor plus calcium-chloride. The technique is used as a near-patient-testing device to guide transfusion in cardiac surgery or liver transplantation and less to investigate hemostasis in acquired or congenital coagulopathies. AIMS (i) Review of the coagulopathy associated with cirrhosis and (ii) report on its investigation by thromboelastometry in comparison with conventional coagulation parameters. METHODS We investigated citrated blood samples from 51 adult cirrhotics for the following thromboelastometry parameters: coagulation-time (CT), clot-formation-time (CFT), maximum-clot-firmness (MCF). RESULTS Relatively few patients [14/51(27%)] were identified as abnormal by CT; in contrast, a greater proportion were identified by the CFT [41/51(80%)] or MCF [39/51(76%)]. CFT and MCF were correlated with the platelet-count, antithrombin and fibrinogen. Prothrombin time (PT) was correlated with CFT and MCF. None of the coagulation parameters were correlated with CT. The correlation of the Child-Pugh-score (taken as index of severity) versus MCF or PT was -0.457(p < 0.001) or 0.484(p < 0.001), suggesting MCF as a suitable prognostic index. CFT and MCF, but not CT obtained ROC curves that were useful to distinguish patients from healthy individuals. CONCLUSIONS Thromboelastometry, currently used to assist liver transplantation is also suitable for investigating stable cirrhosis. CFT and MCF are the most interesting parameters to be considered for future clinical studies needed to assess their value as measures of bleeding-risk and prognosis in this category of patients.

Detection of the Imbalance of Procoagulant Versus Anticoagulant Factors in Cirrhosis by a Simple Laboratory Method

Patients with cirrhosis possess an imbalance in procoagulant versus anticoagulant activity due to increased factor VIII and decreased protein C. This imbalance can be detected by thrombin‐generation assays performed in the presence/absence of thrombomodulin (predicate assay) that are not readily available in clinical laboratories. We sought to assess this hypercoagulability with a simpler thrombin‐generation assay performed in the presence/absence of Protac, a snake venom that activates protein C in a manner similar to thrombomodulin (new assay). We analyzed blood from 105 patients with cirrhosis and 105 healthy subjects (controls). Results for the predicate‐assay or the new‐assay were expressed as ratio (with:without thrombomodulin) or as Protac‐induced coagulation inhibition (PICI%). By definition, high ratios or low PICI% translate into hypercoagulability. The median(range) PICI% was lower in patients (74% [31%‐97%]) than controls (93% [72%‐99%]; P < 0.001), indicating that patients with cirrhosis are resistant to the action of Protac. This resistance resulted in greater plasma hypercoagulability in patients who were Child class C than those who were A or B. The hypercoagulability of Child C cirrhosis (63% [31%‐92%]) was similar to that observed for patients with factor V Leiden (69% [15%‐80%]; P = 0.59). The PICI% values were correlated with the levels of protein C (rho = 0.728, P < 0.001) or factor VIII (rho = −0.517, P < 0.001). Finally, the PICI% values were correlated with the predicate assay (rho = −0.580, P < 0.001). Conclusion: The hypercoagulability of plasma from patients with cirrhosis can be detected with the new assay, which compares favorably with the other markers of hypercoagulability (i.e., high factor VIII and low protein C) and with the predicate‐assay based on thrombin‐generation with/without thrombomodulin. Advantages of the new assay over the predicate assay are easy performance and standardized results. Prospective trials are needed to ascertain whether it is useful to predict thrombosis in patients with cirrhosis. HEPATOLOGY 2010

Acquired coagulation disorders: revisited using global coagulation/anticoagulation testing

Acquired coagulation defects are characterized by a decrease of both pro‐ and anti‐coagulants. Because of this, we hypothesise that global tests, such as the prothrombin and partial thromboplastin times (PT and APTT), might be unsuitable for their investigation. Indeed, these tests are not good predictors of bleeding in acquired coagulopathies as they are in the congenital ones. This article discusses the possible reasons for this, using cirrhosis and the neonatal period as epitomes of acquired coagulation defects. Both display normal thrombin generation in the presence of thrombomodulin, in spite of prolonged PT and APTT. We surmise that, because of their design, the PT and APTT are responsive to thrombin generated as a function of pro‐coagulants, but much less to thrombin inhibited by the anti‐coagulants, especially protein C, which is activated to a limited extent in the absence of thrombomodulin. In conclusion, the PT and APTT can tell us whether or not a patient is deficient in one or more pro‐coagulants, but not whether this deficiency is counterbalanced by a parallel deficiency of anti‐coagulants. Thrombin generation assays are more suitable than PT and APTT for use in acquired coagulation defects.