Circulating Derived Tissue Factor Positive Microparticles and Thrombin Generation a Promising Tool for Identifying Cases at Increased Risk of Thrombotic Complications

Introduction: Sickle cell disease (SCD) is linked to hypercoagulability and risk of venous thromboembolism. It is characterised by high concentrations of erythrocyte-derived microparticles (EC-d-MP). In addition, in SCD endothelial cells and monocytes can be activated, which in turn are sources of tissue factor bearing MPs (TF-MP). Their impact on the thrombin generation process remains unclear.

Aim: We hypothesised that in SCD TF-MP and procoagulant phospholipids from endothelial cells and monocytes are implicated in blood hypercoagulability and potentially enhance thrombin generation .

Materials and methods: Consecutive patients with steady state SCD (n=68) and 31 age and sex-matched patients in painful crisis (n=31) and 30 healthy individuals (controls) were included. Flow cytometric analysis was performed with a precise number of standard beads (Megamix®) to determine MVs count, and specific conjugated antibodies were used to determine the cellular origin of the MVs. Microparticles derived from erythrocytes (RBd-MP), platelets (Pltd-MP), monocyte (Mod-MP), endothelial cell (ECd-MP) were identified using an anti-CD235a, CD61/CD41a, CD14, CB106 and CD41 monoclonal antibodies and FITC labelled annexin V. Total TF-MP was evaluated by CY-Quant MV-TF activity (BioCytex ,Marseille, France). Cellular origin of TF on MPs, samples were triple labelled with Cy5-labeled annexin V, a cell type–specific PE-labeled MoAb against either CD14 or CD144 and a FITC- MoAb against TF. Thrombin generation (TG) in platelet poor plasma was measured by CAT assay using PPP-reagent 5pM in presence and absence of thrombomodulin (TM) . Procoagulant phospholipid dependent activity was assessed by the Procoag-PPL assay, TM and TFPI were measured by Asserachrom TM and Asserachrom TFPI (Stago, Asnieres, France).

Results: The mean age of SCD patients in painful crisis and steady state were 25.1 ± 9.6 (range: 18–39 years) and 26.3 ± 8.1 years (range: 18–40 years), respectively. The mean age in the control group was 27.4 ± 7.2 years (range: 16–36 years). Total MPs were significantly elevated for both groups of patients with SCD in steady as compared to the controls (p<0.001). A significant difference was observed between the steady state patients and crisis patients (p<0.001). In both groups of SCD patients, RBd-MP, Pltd-MP expressing or not PS, ECd-MP and Mod-MP were significantly increased as compared to the control group (p<0.001). The RBd-MP, RBd-MP-PS+, Ed-MP and Mod-MP were significantly elevated in patients in crisis compared to those at steady state SCD (p<0.001, p<0.001, p<0.05, p<0.001 respectively). The TF-MPs were derived from endothelial cells and monocytes but not from red blood cells or platelets. Patients either in crisis or at steady state SCD showed significantly higher total TF-MPs T as compared to the control group (p<0.001). Patients in crisis had significantly higher TF-MP as compared to those at steady state SCD (p<0.001). Mod-MP-TF+ and Ed-MP-TF+ were significantly elevated in both patient groups as compared to the control group (p<0.001). Patients in crisis had significantly higher MP levels as compared to those at steady state SCD (p<0.001). TG was significantly increased in SCD patients as compared to the control. Among thrombogram parameters, the mean rate index (MRI) and Peak of thrombin were significantly higher in the SCD-patient group as compared to the control group (p<0.01). The MRI was significantly increased in patients with crisis as compared to those at steady state SCD (p<0.01). The lag-time and the ETP were not significantly different between SCD patients and the control group. The ETP +/- TM ratio increased significantly in both patients’ groups as compared to the controls (p<0.001), with a significant difference between steady state and crisis (p<0.01). The TM, and TFPI and the PPL-ct were significantly decreased in patients as compared to the controls (p<0.01). The PPL-ct was inversely correlated with the levels of Rb-MP/PS+ and Pd-MP/PS+.( p<0.02).The Rb-MP/PS+ were positively correlated with MRI (p<0.02). The concentration of the total MPs-TF+, Mod-MP-TF+ was positively correlated with MRI and Peak (p<0.01).

Conclusion: In SCD patients the TF bearing MPs derived from endothelial cells or monocytes could, contribute to thrombotic events and may be involved in painful crisis. Circulating MVs TF + may be considered as a potential biomarker for disease severity in SCD patients.