Thrombin Generation in Paroxysmal Nocturnal Haemoglobinuria

INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. It is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic events (TEV). Venous thrombosis is the main cause of death. However its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We choose to demonstrate the hemostatic unbalance associated with PNH clone through a global coagulation assay. METHODS: We used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without thrombomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential, or ETP (ETP ratio) and of the upper limit of thrombin concentration, or peak (peak ratio) was done to identify the hypercoagulable state. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%) (n=23, PNH), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%) (n=25, AA), and controls (n=39, NN). Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood tests, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TEV through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TEV (PNH n=14, AA n=12 and NN n=33); the second step, done only in patients, included individuals using hormonal contraceptives, with positive TB tests, diagnosis of any asymptomatic infection or TEV associated to temporary risk factors that occurred in a period longer than one year since inclusion in the study (PNH n=23, AA n=25). The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. The study was approved by the local Ethics Committee, and performed in accordance with the Helsinki Declaration. RESULTS: The presence of the PNH clone >= 10% was associated with TM inefficiency in reducing the ETP [ETP ratio PNH 0,68 (0,32-0,93), AA 0,49 (0,20-0,81) and controls 0,48 (0,19-0,89) p <0,05, PNH x AA p=0,032, PNH x NN p=0,004)] and in reducing the peak [peak ratio PNH 0,84 (0,46-1,03), AA 0,65 (0,28-0,96) and controls 0,60 (0,25-0,99) p <0,05, PNH x AA p= 0,041, PNH x NN p= 0,001)]. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP ratio and the activity of the von Willebrand factor (FvW:RCo) (Spearman rank correlation = 0,59, p<0,05), whereas a negative correlation was observed between ETP ratio and the levels of protein C (PC) (Pearson correlation coefficient = -0.59, p<0,05). The PNH group presented the shortest time to peak (min) [PNH 5,9 (5,3-7,3), AA 6,8 (5,0-8,8), NN 6,1 (4,7-8,7) p<0,05, PNH x AA p=0,037). CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The positive and negative correlations found respectively between ETP ratio, FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Inflammation secondary to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor, leading to lower PC activation. Our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statins) may explain the aggressive nature of thrombophilia in PNH and the development of TEV even in those taking oral anticoagulants.