Introduction Compound heterozygosity for β-thalassemia/hemoglobin E (β-thal/HbE) is a prevalent hemoglobinopathy in Southeast Asia, with an estimated 3,250 new cases per year in Thailand. The incidence of thrombosis is increased in these patients, and is accentuated following splenectomy. Proposed thrombophilic mechanisms include increased phosphatidylserine exposure on abnormal red cells and on activated platelets, endothelial dysfunction, and reduced levels of natural regulators of coagulation including proteins C and S, and antithrombin. Thrombomodulin (TM) is a 75-kD transmembrane protein and thrombin receptor that functions as a cofactor in the activation of protein C and thrombin-activatable fibrinolysis inhibitor. The soluble form of thrombomodulin (sTM) has been used as a probe in plasma thrombin generation (TG) assay protocols to evaluate resistance to the TM/activated protein C pathway in hypercoagulable states including sickle cell anemia. However, no previous study has evaluated sTM-modified plasma TG in β-thal/HbE.

Objectives

  • Compare TG (+/- added sTM) in β-thal/HbE patients (with or without splenectomy) and healthy controls.

  • Evaluate the contribution of splenectomy to circulating extracellular vesicle (EV) numbers and prothrombotic activity in β-thal/HbE and healthy controls.

Methods The study population included 54 patients with β-thal/HbE (of whom 35 were post-splenectomy) and 26 healthy controls. All subjects were of Thai origin, and the 3 groups were equally balanced by age and gender. TG was initiated in platelet poor plasma (PPP) with 1pM tissue factor and 10.4 µM phospholipid. Where noted, 15 nM sTM was added prior to the addition of tissue factor. TG parameters included slope, peak thrombin and area under the curve (AUC). The ‘AUC ratio’ (AUC + sTM/AUC - sTM) was calculated where indicated. The dilute Russell's Viper Venom Time (RVVT) assay, without added exogenous phospholipid, was used to evaluate prothrombinase activity associated with EVs in PPP. EVs in PPP were enumerated in PPP by nanoparticle tracking analysis.

Results Surprisingly, all TG parameters in β-thal/HbE patients were significantly reduced compared to healthy controls (p<0.0005). Within the β-thal/HbE cohort, TG parameters were less decreased in splenectomized patients compared to non-splenectomized patients (p<0.05). Simultaneous evaluation of the activated protein C pathway by the addition of sTM reduced net TG in all 3 groups, as expected. However, as evidenced by the AUC ratio, TM resistance was more profound in non-splenectomized than splenectomized β-thal/HbE patients. Neither transfusion dependence nor gender were significantly associated with the sTM-modified TG parameters in β-thal/HbE patients. In the dilute RVVT, the splenectomized group had a significantly shorter clotting time in PPP than both the non-splenectomized and control groups (p<0.0001 and p=0.0013 respectively), while the latter two groups did not differ from each other(p=0.65). Both categories of β-thal/HbE patients demonstrated increased numbers of circulating EVs compared to controls. However, increased prothrombinase activity in splenectomized subjects did not correlate with the numbers of EVs.

Conclusion Splenectomized β-thal/HbE patients manifest reduced TG potential but less resistance to soluble thrombomodulin compared to non-splenectomized patients. Additionally, shortening of the Russell Viper Venom Time suggests the presence of more circulating procoagulant phosphatidylserine-expressing EVs in splenectomized patients, although numerically, the absolute numbers of EVs was identical to non-splenectomized patients. In aggregate, these data suggest that prothrombinase activity in cell-free plasma is increased in β-thal/HbE, particularly following splenectomy. Future studies to evaluate thrombin generation in whole blood would be of interest, given the likely roles of red cells and platelets in this disorder.

Key points

  • β-thal/HbE patients demonstrate less plasma thrombin generation (TG) potential than controls, albeit to a lesser degree in splenectomized individuals

  • However, resistance to thrombomodulin is less profound in the splenectomized compared to the non-splenectomized cohort. This may reflect more efficient activated protein C anticoagulant activity on circulating extracellular vesicles (EVs)

  • EV-associated prothrombinase activity is increased in the splenectomized group which may reflect greater availability of phosphatidylserine.

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2025
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