Platelet Apoptosis and Agonist Mediated Activation In Myelodysplastic Syndromes

Abstract 1866

Myelodysplastic syndromes (MDS) comprise distinct disorders characterized by dysplastic and ineffective hematopoiesis that seems to be related to an increased apoptosis of bone marrow cells (Nimer, Blood 111: 4841, 2008). Clinical manifestations in MDS range from a diverse degree of anemias, leuko- or thrombocytopenias to severe transfusion-dependent peripheral pancytopenias. Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS. Many of the features of apoptosis such as membrane fragmentation, microvesiculation and phosphatidylserine (PS) exposure are observed during platelet activation to a procoagulant state, raising the possibility that apoptosis may regulate platelet function.

The aim of this work was to determine whether a correlation exists between apoptosis and activation processes in platelets from MDS patients.

Twenty six patients diagnosed MDS and classified according to WHO-2008 were included: 6 with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), 6 with refractory RA with excess blasts-1 (RAEB-1) and 7 with cytopenia with multilineage dysplasia (RCMD) associated with isolated 5q deletion. Twenty six healthy donors were included as control group.

Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane PS exposed under basal conditions and after stimulation with a PAR-1 receptor agonist (TRAP, SFLLRN, thrombin receptor-activating peptide 6). Levels of pro- apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis of western blots performed with platelet lysates.

Platelet activation was determined through FITC-fibrinogen, FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 mM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (aIIb and b3 subunits) was determined by flow cytometry with specific mAbs.

Platelets from RA, RARS and RCMD patients expressed more PS than control ones under basal conditions (p<0.05) as well as after 100 mM TRAP stimulation (p<0.05). Moreover, platelets from these MDS patients expressed more Bax protein than control group (p<0.05).

On the other hand, PS exposure and Bax content in platelets from RAEB-1 patients were similar to controls, but they expressed a higher amount of Bcl-2 (p<0.05). No correlation was observed between PS exposure or Bax expression and platelet number.

Platelets from all MDS patients showed an impaired activation by TRAP, even when PS exposure was higher than in control group. This diminished response to TRAP was not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients.

Our results suggest that platelets from RA, RARS and RCMD patients are more apoptotic than control ones and that a correlation between platelet surface PS and activation does not seem to exist. Moreover, dissimilarity in expression pattern of apoptotic proteins among MDS types indicates differences in the intracellular mechanisms underlying these pathologies.