Assessment of Changes of Membrane Properties and Platelets Function in Patients with Chronic Myeloid Leukaemia

Background. Thrombosis is frequent complication in patients with chronic myeloid leukemia (CML). Qualitative and quantitative modifications of platelet membrane receptors are involved in alteration of platelet function. Moreover the properties of cellular membrane may influence the expression and activity of receptors. The aim of our study was to determine whether changes of platelet membrane properties (lipid bilayer fluidity and resting transmembrane potential) may be correlated with the modified expression of platelet receptors and altered platelet function in CML.

Materials and methods. This retrospective study included 30 patients with CML as well as 10 healthy volunteers. Platelet function was analyzed by optical platelet aggregometry using as agonists ADP, collagen, ristocetin and epinephrine. Lipid bilayerfluidity (FA) of platelet membrane was evaluated by fluorescence anisotropy technique using as marker 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA DPH). The resting transmembrane potential was measured by fluorescence spectroscopy using 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (DiSC3-5). Depending on platelet response to different reagents used for platelet aggregation, we divided the CML patients into two groups: patients with normal response (amplitude > 60%) and patients with absent/low response. Platelet receptor expression was analyzed by flow cytometry for adhesion markers (CD 42a and CD 42b) and aggregation markers (CD61, CD41).

Results and discussion. It has been observed a statistically significant difference in FA for advanced-phase of CML patients when compared to controls (P=0.02). Patients with molecular remission presented a significantly lower FA (more fluid lipid bilayer) than patients without molecular remission (median value 0.1445 (95% CI 0.10-0.16) compared to median value 0.1694 (95% CI 0.13-0.29), P=0.05). The inhibitors of tyrosine kinase (TKI) turned into less fluid the lipid bilayer: the value of FA significantly increased between untreated status FA=0.064 vs. after 2 months of treatment FA=0.119. This analysis was performed only for a small group (4 patients) and has to be confirmed on a larger population of patients under treatment. The amplitude and slope of the platelet aggregation curves are significantly lower for CML patients than for healthy volunteers, for all reagents except ristocetin; the platelet response was more influenced in CML patients-accelerated and blastic phase. The CD61/CD41 expression (GP IIbIIIa) presented statistically significant lower values in CML patients (median value CD 61=76.63 (59.18-84.6); CD 41=55.3 (43.7-71.42)) versus controls (median value: CD61= 96.74 (71.36-98.16); CD 41=89.03 (55.67-92.92)).There was not diference in expression of CD42a/CD42b in the CML group compared with control groupPatients in advanced or blastic phase has a lower value of GP IIbIIIA expression (median value: CD 61=70.02 (41.36-83.35); CD 41= 45.45 (29.21-67.78)) than patients in chronic phase (median value: CD 61=84.63 (66.98-94.92); CD 41= 72.82 (59.78-86.85)). There was no statistical significant correlation between the lipid bilayer fluidity and platelet transmembrane resting potential. The average value of membrane potential of platelets from CML patients was -62±15 mV in accordance with values found in literature.

Conclusions. CML patients have a low expreser expression of platelet aggregation receptors and consequent lower parameters of amplitude and slope on aggregation curves, but an increased lipid bilayer fluidity. A normal response to ristocetin was observed, although the platelet receptors expressions were low. TKI decreased the lipid bilayer fluidity and confirmation of this result on larger population is undergoing. The influence of platelet transmembrane resting potential on platelet aggregation response needs to be analysed on a higher number of patients.