Vassiliki Galea

The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles

Sickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

Tissue factor over-expression by human pancreatic cancer cells BXPC3 is related to higher prothrombotic potential as compared to breast cancer cells MCF7

Cancer histology influences the risk of venous thromboembolism and tissue factor (TF) is the key molecule in cancer-induced hypercoagulability. We investigated the relation between TF expression by pancreatic and breast cancer cells (BXPC3 and MCF7 respectively) and their capacity to trigger in vitro thrombin generation in normal human plasma. Flow cytometry and Western blot analysis for TF expression were performed using murine IgG1 monoclonal antibody against human TF. Real-time PCR for TFmRNA was also performed. Activity of TF expressed by cancer cells was measured with a specific chromogenic assay. Thrombin generation in PPP was assessed using calibrated automated thrombogram. Cancer cells were added to platelet poor plasma from healthy volunteers. In separate experiments cells were incubated with the anti-TF antibody at concentration that completely neutralized the activity of recombinant human TF on thrombin generation. BXPC3 cells expressed significantly higher amounts of functional TF as compared to MCF7 cells. Incubation of BXPC3 and MCF7 cells with PPP resulted in acceleration of the initiation phase of thrombin generation. BXPC3 cells manifested higher procoagulant potential than MCF7 cells. The incubation of BXPC3 or MCF7 cells with the anti-TF monoclonal antibody which resulted in reversal of their effect on thrombin generation. The present study establishes a link between the amount of TF expressed by cancer cells with their procoagulant activity. Both studied types of cancer cells trigger thrombin generation but they have different procoagulant potential. The procoagulant activity of BXPC3 and MCF7 cells is related to the amount of TF expressed. Kinetic parameters of thrombogram are the most relevant for the detection of the TF-dependent procoagulant activity of cancer cells. TF expression is one of the mechanisms by which cancer cells manifest their procoagulant potential but it is not the unique one. The present experimental model will allow the characterization the procoagulant fingerprint of cell lines from the same or different histological types of cancer.

Impact of breast cancer stage, time from diagnosis and chemotherapy on plasma and cellular biomarkers of hypercoagulability

BackgroundIn breast cancer patients routine thromboprophylaxis is not recommended but individualized risk assessment is encouraged. The incorporation of hypercoagulability biomarkers could increase the sensitivity of risk assessment models (RAM) to identify patients at VTE risk. To this aim we investigated the impact of cancer-related characteristics on hypercoagulability biomarkers.MethodsThrombin generation (TG) assessed with the Thrombogramme-Thrombinoscope®, levels of platelet derived microparticles (Pd-MP) assessed with flow cytometry, procoagulant phospholid dependent clotting time (PPL-ct) measured with a clotting assay and D-Dimers (were assessed in a cohort of 62 women with breast cancer and in 30 age matched healthy women.ResultsPatients showed significantly higher TG, Pd-MP, D-Dimers levels and shortened PPL-ct compared to the controls. The PPL-ct was inversely correlated with the levels of Pd-MP, which were increased in 97% of patients. TG and D-Dimers were increased in 76% and 59% of patients respectively. In any stage of the disease TG was significantly increased as compared to the controls. There was no significant difference of TG in patients with local, regional of metastatic stage. There was no significant difference in Pd-MP or Pd-MP/PS+ between the subgroups of patients with local or regional stage of cancer. Patients with metastatic disease had significantly higher levels of Pd-MP and Pd-MP/PS+ compared to those with regional stage. The D-Dimers increased in patients with metastatic stage. In patients on chemotherapy with less than 6 months since diagnosis TG was significantly higher compared to those on chemotherapy who diagnosed in interval > 6 months. Patients with metastatic disease had significantly higher levels of Pd-MP and D-Dimers compared to those with non-metastatic disease.ConclusionIn breast cancer patients the stage, the time elapsed since the diagnosis and the administration of chemotherapy are determinants of cellular and plasma hypercoagulability. The levels and the procoagulant activity of Pd-MP are interconnected with the biological activity and the overall burden of cancer. TG reflects the procoagulant properties of both breast cancer and chemotherapy in the initial period of cancer diagnosis. Thus the weighted incorporation of the biomarkers of cellular and plasma hypercoagulabilty in RAM for VTE might improve their predictive value.

Lobectomy and postoperative thromboprophylaxis with enoxaparin improve blood hypercoagulability in patients with localized primary lung adenocarcinoma

BACKGROUND Patients with lung adenocarcinoma undergoing surgery are in high risk for VTE and receive routine post-operative thromboprophylaxis with LWMH. AIM We investigated markers of hypercoagulability in patients with primary localized adenocarcinoma and the modifications induced by lobectomy and postoperative administration of enoxaparin. MATERIALS AND METHODS Patients suffering from localised primary lung adenocarcinoma (n=15) scheduled for lobectomy were studied. The control group consisted of 15 healthy age and sex-matched individuals. Blood was collected before anaesthesia induction and after surgery, at several intervals until the 7th post-operative day. Samples were assessed for thrombin generation, phosphatidylserin expressing platelet derived microparticles expressing (Pd-MP/PS(+)), tissue factor activity (TFa), FVIIa and TFPI levels, procoagulant phospholipid dependent clotting time and anti-Xa activity. RESULTS At baseline, patients showed increased thrombin generation and Pd-MP/PS(+). After lobectomy thrombin generation significantly decreased. Administration of enoxaparin attenuated thrombin generation. In about 50% of samples collected post-operatively an increase of thrombin generation occurred despite the presence of the expected anti-Xa activity in plasma. At the 7th post-operative day, 3 out of 15 patients showed a significant increase of thrombin generation. CONCLUSION In patients with localized lung adenocarcinoma, hypercoagulability is characterized by high thrombin generation and increased concentration of Pd-MP/PS(+). Tumor mass resection is related with attenuation of thrombin generation, which is inhibited by postoperative thromboprophylaxis with enoxaparin. The response to enoxaparin is not predicted by the concentration of the anti-Xa activity in plasma. The assessment of thrombin generation during prophylaxis with enoxaparin allows to identify patients with high residual plasma hypercoagulability.

Effect of Low Molecular Weight Heparins and Fondaparinux Upon Thrombin Generation Triggered by Human Pancreatic Cancer Cells BXPC3

Low molecular weight heparins (LMWHs) and fondaparinux are widely used for prophylaxis and treatment of venous thromboembolic disease in cancer patients. However, the optimization of the antithrombotic treatment especially in patients with adenocarcinoma of the pancreas is a challenging issue. The understanding of the mechanism of action of the LMWHs and fondaparinux in cancer-induced hypercoagulability might help to optimize antithrombotic treatment. To this aim, we investigated the influence of BXPC3 pancreas adenocarcinoma cells on the antithrombotic activity of LMWHs and fondaparinux. Thrombin generation (TG) in normal platelet poor (PPP) and platelet rich plasma (PRP) spiked with clinically relevant concentrations of dalteparin, enoxaparin, nadroparin tinzaparin and fondaparinux was assessed with the Calibrated Automated Thrombogram assay. BXPC3 (5 cells/μl) were added to plasma. The mean rate index (MRI) of the propagation phase of TG and the endogenous thrombin potential (ETP) were analyzed. The IC50 of the studied compounds were determined and compared on the basis of anti-Xa and anti-IIa equivalent units. We demonstrate that the specific antithrombin (AT)-dependent anti-Xa activity of LMWHs and fondaparinux almost selectively inhibits the propagation phase of TG. The synergy between the anti-Xa and anti-IIa activities of LMWHs rather than the selective inhibition of FXa warrants abrogation of TG. The mean molecular weight and anti-Xa/anti-IIa ratio of the AT-dependent agents cannot predict the alteration of their capacity to inhibit TG. Tinzaparin was the most potent inhibitor of TG than the other LMWHs. Enoxaparin was more potent than nadroparin and dalteparin.