Investigation on platelet adhesion in sickle cell disease and the role of cyclic nucleotides in this adhesion

Author(s)

Renata Proença Ferreira

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2009-08-28

Examining board members

Nicola Amanda Conran Zorzetto; Joyce Maria Annichino Bizzacchi; Maria Stella Figueiredo

• Advisor: Nicola Amanda Conran Zorzetto; Fernando Ferreira Costa

Abstract

Sickle cell disease (SCD) is caused by a point mutation that results in the formation of Hemoglobin S (HbS). The polymerization of deoxygenated HbS causes deformation of red cells, which then adopt a sickle shape and become rigid and fragile. Hemolytic anemia and vaso-occlusive events are the main cause of morbidity in SCD; abnormal adhesion of white and red cells to the endothelium decreases the blood flow in the microcirculation and appears to be the main factor involved in vaso-occlusion. The objective of this study was to compare the adhesive properties of platelets from healthy individuals (AA platelets) with those of platelets from SCD patients (SS platelets) and from patients on HU therapy (SSHU platelets), as well as the adhesion molecules and signaling pathways that may be involved in this adhesion. The basal adhesion of SS platelets to fibrinogen (FB) was significantly higher than that of AA platelets; in contrast, SSHU platelets demonstrated a similar adhesion to that of AA platelets. Platelets from AA, SS and SSHU individuals, when stimulated with thrombin (TB), all presented significantly higher adhesions when compared with their basal adhesions. In contrast, there were no significant differences between the adhesions of AA, SS and SSHU platelets when collagen was used as a ligand. Flow cytometry was utilized to compare the expression and activation of the main adhesion molecules on AA, SS and SSHU platelets and identified a significantly higher expression of the ?IIb?3 integrin in its activated conformation on the surface of the SS platelets, in relation to the AA and SSHU. Expression of the P-selectin adhesion molecule (CD62P) was also found to be increased on the surface of SSHU platelets. Adhesion assays utilizing specific antibodies for the activated ?IIb?3 and P-selectin indicated a possible role for the ?IIb?3 integrin in the adhesion of platelets to FB. The cyclic nucleotide, cyclic adenosine monophosphate (cAMP) is an important inhibitor of platelet aggregation. Intraplatelet levels of cAMP were found to be significantly lower in SS platelets compared to AA and SSHU platelets. Co-incubation of platelets with TB significantly reduced levels of cAMP in the AA and SSHU platelets, but in SS platelets this decrease was not significant. Interestingly, levels of fetal hemoglobin (HbF) in SCD patients (both SS and SSHU) were found to correlate significantly with levels of platelet AMPc. With regard to intraplatelet levels of cyclic guanosine monophosphate (cGMP), there were no significant differences found between AA, SS and SSHU platelets, however following a TB stimulus AA platelets demonstrated a significant increase in intracellular cGMP. Phosphodiesterase 3A (PDE3A) is the main cyclic nucleotide hydrolyzing enzyme in platelets. Incubation of SS platelets, but not AA or SSHU platelets, with cilostazol (specific inhibitor of PDE3A) resulted in a significant reduction in their adhesion to FB, suggesting that PDE3A activity in SS platelets may be activated and that augmentation of cAMP is capable of reverting increased SS platelet adhesion. Additional experiments indicate that Protein kinase A (PKA), PKG and PKC dependent signaling pathways are not involved in the altered adhesion of SS platelets. The functional activity of SS platelets was found to be altered in platelet aggregation assays, however further experiments are required to draw conclusions from these data. Results of this study suggest that platelets from SCD individuals circulate in an activated state and that they present a greater ability to adhere to proteins that may be encountered on the vessel wall. This augmented adhesion is associated with decreased levels of intraplatelet cAMP and activation of the ?IIb?3 integrin. These alterations appear to be reversed in patients on HU therapy. Results suggest that platelets may have an important role in the vaso-occlusive process.When activated these cells are an important source of inflammatory mediators that may, in turn, result in an exacerbation of cellular activation at the vaso-occlusive site. (AU)