The Effects of Hydroxyurea on the Thrombin Generation and Microparticles in Sickle Cell Anemia Patients

The clinical course of sickle cell disease (SCD) is punctuated by episodic vascular occlusive events. The possibility that activation of the clotting system plays a contributory role in these complications is supported by abundant clinical data during both steady-state disease and pain crisis. Hydroxyurea therapy induces fetal haemoglobin, improves laboratory parameters and reduces acute clinical complications of SCD, but despite an abundance of evidence for coagulation and platelet activation, it remains incompletely defined whether these changes contribute to the reduced thrombin generation. This study is designed to evaluate coagulation profiles of patients with SCA in steady state and to determine whether hypercoagulable state is modified or not in patients on hydroxyurea therapy.

We studied erythrocyte derived microparticles (Ed-MP) and platelet derived microparticles (Pd-MP) expressing or not expressing phosphatidylserine (PS) in patients with steady state SCD and we evaluated their specific procoagulant activity and their impact on thrombin generation process. A total of 92 steady state SCD patients were included in the study, of which 19 were under treatment with hydroyurea. The control group consisted of 30 healthy age and sex matched controls. Microparticles in whole blood were assessed using flow cytometry. Ed-MP and Pd-MP were identified using an anti-CD235 and CD41 monoclonal antibodies and annexin V. Thrombin generation in platelet poor plasma (PPL) was measured by CAT assay using PPP-reagent 5pM (Thrombinoscope, The Netherlands). Procoagulant phospholipid dependent activity in plasma was assessed by the Procoag-PPL assay (Diagnostica Stago, France). Thrombomodulin (TM) levels were measured by enzyme-linked immunosorbent assay (Elisa) Asserachrom thrombomodulin (Diagnostica Stago, Asnieres, France).

Hydroxyurea treated patients had lower counts of leukocytes, reticulocytes and platelets and an increased mean hemoglobin concentration as compared to non treated patients. Leukocyte and reticulocytes counts of treated patients were higher than those of controls. Platelets counts did not differ between treated and untreated patients. Patients on treatment with hydroxyurea had significantly lower levels of Ed-MP/PS⁺ and Ed-MP compared to untreated patients. The concentration of Pd-MP/PS⁺ and Pd-MP were not significantly different between hydroxyurea treated and non treated patients. The Ed-MP/PS+ showed a significant inverse correlation with Hb F (p<0.05). Thrombogram parameters, lag-time, ttPeak, Peak and MRI were significantly different between hydroxyurea treated patients and non treated patients. In hydroxyurea treated patients in contrast to the untreated ones no correlation was found between Ed-MP/PS⁺ and Ed-MP and parameters of thrombin generation. Among hydroxyurea treated patients 68% showed MRI levels higher than the UNL. Stratification groups of treated patients according to the levels of microparticles with Ed-MP/PS⁺ or Pd-MP/PS⁺ concentration higher than the UNL showed non significant difference compared to entirely group of patients. The PPL concentration was significantly lower in the SCD-treated patient compared to untreated patients (p<0.05). In contrast to platelet-derived-microparticles, the numbers of erythrocyte-derived-microparticles differed between patients with and without hydroxyurea during steady state. In patients treated with hydoxyurea, platelets were correlated with Ed-MP, Pd-MP with and without PS+ (p<0.05), but any of the others parameters showed one association. Procoagulant phospholipids and thrombomodulin were increased in SCD with and without hydroxyurea compared with controls group (p<0.05).

Treatment with hydroxyurea result in decreases in plasma markers of thrombin generation, and may decrease coagulation activation by reducing PS expression on the surface of both RBCs and platelets in addition to being a NO donor hydro may also decrese haemostatic activation by its effect in decreasing the white blood cell count and particularly monocytes that express TF, furthermore the beneficial effects of hydroxyurea may be due to vasodilatationand decressed platelet and coagulation activation following NO production.

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