Platelets from Sickle Cell Disease Individuals Demonstrate Increased Adhesive Properties That Are Reversed by Hydroxyurea Therapy in Association with Alterations in Intraplatelet cAMP and αIIbα3 Integrin Activation

Abstract

Sickle cell disease (SCD) is a chronic inflammatory disease that results in hemolytic anemia and vaso-occlusive events, the principal cause of morbidity in SCD patients. Whilst roles for red cells and leukocytes in the vaso-occlusive process in SCD are well established, the contribution of platelets to this process remains unclear. Increased platelet activation has been described in SCD patients, contributing to the hemostatic activation observed in the disease. Since the adhesion of platelets to the vascular endothelium and extracellular matrix (ECM) could potentially contribute to vaso-occulusion, this study compared the adhesive properties of platelets (PLTs) from healthy control subjects (CON), steady-state SCD patients (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day). Washed PLTs were ressuspended in Krebs solution (1.2×108 PLT/ml) and their adhesion to fibrinogen (FB, 50 μg/ml) coated 96-well plates evaluated utilizing static adhesion assays (15 min, 37°C). SCD PLTs demonstrated a significantly greater adhesion to FB than CON PLTs (26.0±4.0%; 15.8±2.0%, respectively, n≥13; p<0.05, Mann-Whitney test). In contrast, SCDHU PLT adhesion was significantly lower than that of SCD PLTs, being similar to that of CON PLTs (11.7±1.5%, n=13; p<0.01 comp. SCD). A thrombin stimulus (50 mU/ml) significantly increased CON, SCD and SCDHU adhesion to FB (26.2±2.1%; 33.8±3.7%; 23.5±3.7%, respectively, n≥13; p<0.001 comp. to basal, paired t test). Levels of the second messenger, cyclic adenosine monophosphate (cAMP), the reduction of which potentiates platelet activation, were found to be significantly decreased in the PLTs of SCD individuals compared to those of CON and SCDHU (5.4±0.6; 8.0±0.7; 7.1±0.4 pmol/108 PLT, respectively, n=7; P<0.05 for SCD comp. to CON and SCDHU). Interestingly, thrombin significantly reduced cAMP levels in CON and SCDHU (6.0±0.9; 5.4±0.5 pmol/108 PLTs, respectively, n=7, P<0.05 comp. to basal), but not SCD PLTs (4.7±0.6 pmol/108 PLT, n=7; P>0.05), providing further evidence that PLTs circulate in an activated state in SCD individuals. Flow cytometry studies demonstrated no alterations in the expressions of CD42b (GPIb molecule), CD62P (P-selectin), CD49b (α2 integrin subunit) and CD41a (αIIb integrin subunit) on the surface of SCD PLTs compared to CON and SCDHU (P<0.05, data not shown). However, binding of the PAC-1 antibody, which specifically recognizes activated αIIbβ3 integrin, was significantly higher on SCD PLTs than on CON and SCDHU PLTs (40.7±8.9%; 17.1±6.5%; 20.3±5.4%, respect., n≥8, p<0.05). The αIIbβ3 integrin, in its activated affinity, can bind to numerous soluble ligands that in turn may bridge the interaction between PLTs, endothelial cells and other cell types. As such, PLTs from SCD individuals in steady state circulate in an activated state and present an increased ability to adhere to the blood component, FB, compared to control PLTs. Increased adhesive properties were found associated with decreased intraplatelet cAMP and high affinity αIIbβ3 integrin expression. Interestingly, HU therapy was associated with a reversal in increased PLT adhesion coupled with augmented intraplatelet cAMP and reduced αIIbβ3 activation. Thus, SCD platelets may have an increased capacity for adhesion to the vascular wall and ECM components, where the presence of adhered and activated platelets may contribute to the vaso-occlusive process by facilitating leukocyte accumulation at the endothelium and the release of platelet factors that may stimulate further inflammation at the site.