Features of Microparticle-Associated Procoagulant Activity in Patients with Thrombocytopenias of Immune and Central Origin

Introduction: The risk of bleeding in patients with thrombocytopenia is increased with platelet counts less than 20 or 30 x 10⁹/L. Nevertheless, some patients with thrombocytopenia of peripheral and central origin (immune thrombocytopenia [ITP] and myelodysplastic syndromes [MDS] respectively) have fewer bleeding symptoms than expected. This fact suggests there may be compensatory mechanisms for the thrombocytopenia, such as the presence of microparticles (MPs).

Objective: The aim of this study was to evaluate and characterize the microparticle-associated procoagulant activity in ITP and MDS patients with thrombocytopenia.

Methods: Thirty-five patients with chronic ITP and twenty-six patients with MDS with a platelet count less than 50 x10⁹/L and twenty-five healthy controls were included. Blood cell counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 1,500 g for 15 min at 23°C. Platelet-poor plasma obtained was additionally centrifuged twice at 23°C (15 min at 1,500 g, and 2 min at 13,000 g, [PFP]) and aliquots were stored at -70ºC until analysis.

Phosphatidylserine-MP (Ph-MP) and tissue factor-MP (TF-MP)-dependent procoagulant activities were determined with the ZYMUPHEN kits (Hyphen BioMed, Neuville sur Oise, France) following the manufacturer’s instructions.

The identification of MP’s cell origin was determined by flow cytometry labeling MPs with Annexin-V-fluorescein and the following specific monoclonal antibodies (mAb) conjugated with phycoerythrin: anti CD41 mAb for platelets, anti CD14 mAb for monocytes, anti CD144 mAb for endothelial cells, anti CD235 mAb for red cells and anti CD45 mAb for leukocytes.

APRIL plasma levels were determined by ELISA (DuoSet ELISA, R&D Systems, Minneapolis,

MN, USA).

Results: Ph-MP associated procoagulant capacity in MDS and ITP patients was higher than in controls (p<0.01) whereas MP-TF associated procoagulant activity was only increased in MDS patients and practically negligible in ITP and control groups (p<0.01).

MPs analysis by flow cytometry of seventeen controls, twenty ITP and six MDS patients showed that ITP patients had an increased percentage of MPs from platelets and red cells, whereas MDS had an increased percentage of monocytes derived MPs. Proportion of MPs derived from other cell types were similar to the control group.

Increased percentage of monocyte-derived MPs in MDS patients is in accordance with the higher MP-TF-associated capacity observed in this group since monocytes are tissue factor rich cells. Nevertheless, no differences were found in monocyte count with control and ITP groups and monocyte count did not correlate with MPs associated procoagulant activity and the percentage of monocyte-derived MPs.

In the ITP group neither MP-Ph-associated procoagulant activity nor the percentage of red cell-derived MPs correlated with red cell count. On the contrary, MP-Ph-associated procoagulant activity and the percentage of platelet-derived MPs inversely correlated with platelet count.

ITP is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction. A proliferation-inducing ligand (APRIL) is a factor that promotes B-cell maturation and survival. All patients with ITP and thrombocytopaenia showed higher APRIL plasma levels than MDS and control groups (p<.01), which inversely correlated with platelet count and correlated to MP-Ph-associated procoagulant activity. These observations support the proposed pathogenic role of APRIL in the development of this disease and that the increase in platelet-derived MPs was due to peripheral platelet destruction.

Conclusion: Peripheral and central thrombocytopenias might present a MP-associated procoagulant activity to compensate bleeding risk present in these patients. Cellular origin of these MPs differed according to thrombocytopaenia etiology.