Microparticle-Associated Thrombogenic Mechanism Might Compensate Bleeding Tendency in Patients with Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders with clonal bone marrow anomalies characterized by ineffective hematopoiesis, morphologic and functional hematopoietic cells abnormalities, and an increased risk of transformation to acute myeloid leukemia. Previous work from our lab has shown that MDS patients had an impairment in platelet activation, more externalization of phosphatidylserine (PS) indicating an increased platelet apoptosis and, in some cases, thrombocytopenia. However, these patients do not bleed at the frequency that would be expected taking into account these facts.

The aim of this work was to identify the existence of some compensatory mechanisms that could be counteracting the expected bleeding tendency in these patients.

Since apoptosis is accompanied by membrane blebbing and production of PS-exposing procoagulant microparticles (MP) shed from the plasma membrane, we assessed the thrombogenic capacity of MP and evaluate their correlation with exposure of PS. We also studied the ability of plasma to generate thrombin after stimulus.

Seventy-five patients with MDS, 36% female, mean age 77 years (range: 41 to 93 years) and sixty-eight healthy controls, 29% female, mean age 52 years (range 20 to 76 years) were included. Whole blood was collected in citrated tubes and centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma (PPP). PPP was subjected to 2 additional centrifugations at room temperature (first: 15 min at 1,500 g and second: 2 min at 13,000 g) following SSC-ISTH recommendations to obtain platelet free plasma (PFP). MP procoagulant activity was determined in PFP with the ZYMUPHEN MP-Activity kit (HYPHEN BioMed, Neuville sur Oise, France).

Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) as described by Hemker at final concentration of 1pM tissue factor and 4 microM phospholipids. Four CAT parameters were recorded: endogenous thrombin potential (ETP, total amount of thrombin generated over time), lag time (time to the beginning of the explosive burst of thrombin generation), time to peak (TTP, time to reach the maximum thrombin concentration) and peak height (maximum thrombin concentration obtained). Apoptosis was determined by assessing the level of PS externalization by the binding of FITC-Annexin V to platelets by flow cytometry. Thrombocytopenia was defined as platelet count less than 50,000 platelets/microliter.

Platelet count in MDS patients varied from 4,000 to 478,000 platelets/microliter. We observed an increased MP-associated procoagulant activity in MDS patients with thrombocytopenia compared to controls (p<0.05). This would be related to the increased apoptosis observed in platelets from MDS patients with thrombocytopenia (Spearman test r = 0.655, p <0.01). CAT evaluation of plasma procoagulant activity did not show differences between patients and controls in any of the measured parameters.

Our results showed an increased procoagulant activity associated to plasma MPs in MDS with thrombocytopenia that might contribute to the minor risk of bleeding observed in these patients. However, further studies are necessary to assess the clinical significance of this finding.

No relevant conflicts of interest to declare.