Thrombin generation in patients with acquired haemophilia and clinical bleeding risk

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Acquired haemophilia is a rare bleeding disorder characterized by autoantibodies directed against coagulation factor VIII and is frequently seen in elderly people. The clinical presentation of these patients is very diverse with life-threatening bleeds and occasionally a mild bleeding tendency. The authors of a British registry report did not find factor VIII activity (FVIII:C) or inhibitor titre at presentation to be prognostic factors (Collins et al, 2007). Factor VIII deficiency is believed to be a disease of thrombin generation. Classical clotting tests are based on the appearance of the fibrin clot, which appears when only 3–5% of total thrombin is formed (Brummel et al, 2002). These tests may not entirely reflect thrombin generation. Thrombin generation tests (TGT) have been shown to be useful in the assessment of bleeding tendency in congenital factors deficiencies (Al Dieri et al, 2002) and in haemophilia A (Dargaud et al, 2005), and these two reports identified some threshold values for endogenous thrombin potential (ETP) that could exclude a severe bleeding phenotype (ETP >20% of normal for congenital deficiencies and ETP >50% of normal for haemophiliacs). We used a calibrated automated thrombography (CAT) to evaluate four major parameters of thrombin generation [ETP, lag time (LT), peak height (PH) and time-to-peak (TTP)] to evaluate their power to characterize the bleeding phenotype of acquired haemophilia patients at diagnosis. Patients with acquired haemophilia were selected retrospectively from three French university hospitals. A total of 16 patients with acquired haemophilia A, were identified between 2002 and 2009; they had a complete clinical history and classical laboratory parameters at diagnosis. These patients had platelet-poor plasma (PPP) stored at −80°C usable for thrombin generation. The patients were divided into two groups severe and non-severe bleeders, as defined by an International Committee (Schulman et al, 2005). Median time between diagnosis and severe bleeding event was 1 d (from 0 to 82 d). A control population of plasma from healthy volunteers (n = 25) between 18 to 65 years of age was used. PPP was obtained after centrifuging twice at 2500 g for 15 min at 15°C, and supernatant was stored at −80°C until CAT analysis, when thrombin generation was measured. Experiments were conducted in triplicate for patient plasma samples and calibrator. Eighty microlitre of PPP was dispensed into each well of round-bottomed 96 well-microtitre plates. A mixture (20 μl) containing tissue factor and phospholipids was added to obtain a final concentration of 5 pmol/l and 4 μmol/l respectively (PPP Reagent; Stago, Paris, France). The starting reagent (20 μl) containing fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) and CaCl2 was dispensed by the Fluoroscan Ascent® fluorometer (Labsystem France SA, Les Ulis, France). Calibrator wells contained a thrombin solution of known activity c. 600 nmol/l (Thrombin calibrator; Stago). Five thrombin generation parameters: ETP, LT, PH, TTP and the thrombin generation velocity [PH/(TTP-LT)] were determined using Thrombinoscope™, version 3·1·0·55; Synapse BV, Maastricht, the Netherlands) (Hemker et al, 2003). Results between patients groups and controls were compared by the Wilcoxon’s test or t-Student test when appropriate. A P value of <0·05 was considered statistically significant.

Nine patients were assigned as non-severe bleeders and seven as severe bleeders. We then determined thrombin generation for these 16 patients. The patients showed no differences in associated clinical features or biological classical tests (FVIII:C, activated partial thromboplastin time [aPTT] and inhibitor titre). All parameters were altered in the patients compared to controls except for ETP in non-severe bleeders (Fig 1). ETP and peak height were decreased and LT or TTP increased. ETP and peak height were significantly lower in severe bleeders compared to non-severe. However, there was no significant difference in LT or TTP between the two groups. The thrombin generation velocity was significantly decreased in severe bleeders compared to non-severe. Thrombin generation parameters in severe bleeders were all <50% for ETP, <30% for PH or <17% for thrombin generation velocity when expressed against normal values. Thrombin generation could be measured in complete remission in seven patients, all of whom showed normalized thrombin generation parameters (Huth-Kuhne et al, 2009). There was a global linear correlation between FVIII and ETP (r2 = 0·28, P = 0·01), FVIII and PH (r2 = 0·59, P < 0·0001) but no correlation between FVIII and LT (r2 = 0·03, P = 0·47) and a hyperbolic relationship between FVIII and ETP or PH (Fig 2A,B). Our results confirm a correlation between FVIII and TGT results in acquired haemophilia. This correlation does not hold for low FVIII:C, so FVIII:C is not sufficient to determine these patients’ bleeding phenotype (Fig 2C,D). It has been demonstrated in previous studies that FVIII:C and anti-VIII levels are not prognostic indicators (Collins et al, 2007, Delgado et al, 2003). We have also demonstrated that the aPTT is not a prognostic indicator. Because classical coagulation test are based on the time to appearance of fibrin clot, they do not entirely reflect thrombin generation. Based on similar works in congenital haemophilia (Dargaud et al, 2005; van Veen et al, 2009) we conducted a retrospective study to assess the discriminatory power of TGT on bleeding phenotype in acquired haemophilia A patients. We found that ETP and PH were decreased in the severe bleeding phenotype compared to controls or non-severe bleeders. The results show that a threshold could be proposed to exclude severe bleeding phenotype based on TGT results. If ETP is >50%, PH is >30% or Thrombin velocity is >17% of normal values, a severe form can reasonably be excluded. This possibility of classifying patients according to their future bleeding phenotype at the time of diagnosis is of considerable interest for practitioners who treat these patients. Immunosuppression-related mortality is one of the leading causes of death in this disease (Levesque et al, 2009) and these results offer the possibility of considering new therapeutic strategies. Corticosteroids alone or a second-line immunosuppressant introduced later than usually recommended in non-severe bleeders would permit lighter immune suppression. These results and future prospects need to be confirmed in a prospective study to confirm the possibility that the bleeding phenotype may be defined by TGT.

Figure 1.

 Discrimination between severe bleeders, non-severe bleeders and controls at a tissue factor concentration of 5 pmol/l. Thrombin generation test parameters shown are endogenous thrombin potential (ETP; A), peak height (B), lag time (C) and time to peak (TTP; D). †Not significant,*P < 0·05, **P < 0·01, ***P < 0·005.

Figure 2.

 Endogenous thrombin potential (ETP; A) and peak height (B) as a function of FVIII:C for the whole population (diagnosis and complete remission). ETP (C) and peak height (D) for FVIII:C at diagnosis (no correlation was found at low level).

Conflict of interest

None declared.

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