Hydroxyurea Therapy Is Associated with Decreased Platelet Aggregation Responses and Activation in Sickle Cell Disease.

Abstract 2565

Poster Board II-542

Sickle cell disease (SCD) is characterized by a hypercoagulable state that may contribute to the initiation and propagation of vaso-occlusion. Platelets (PLTs) circulate in an activated state in SCD individuals and both increased and unaltered platelet aggregation responses to adenosine phosphate (ADP) stimulation have been reported in SCD adults. PLTs play an important role in vascular inflammation, where platelet adhesion to the inflamed vascular wall and thrombus formation can, in turn, trigger platelet release of inflammatory substances that can further activate the endothelium and leukocytes. This study compared the aggregation responses of PLTs from healthy control individuals (CON), steady-state SCD and SCD patients on hydroxyurea therapy (SCDHU; 20–30mg/kg/day) following stimulation with three soluble agonists. Venous blood from CON, SCD and SCDHU subjects was collected in sodium citrate. Platelet rich plasma (PRP; 250 000 PLTs/ml) was separated and preincubated for 3 min (37°C) with 5μM ADP, 1 μg/ml human collagen type I (Col) or 500 mU/ml thrombin (TB). Aggregation was then determined (5 min, 37°C, constant stirring at 1000 rpm) using a Chrono-log® 700 aggregometer and platelet-poor plasma as a reference. PAC-1 and anti-CD49b antibody binding were determined by flow cytometry. Following ADP stimulation, CON and SCD PLTs presented statistically similar aggregations (77.7±4.8%, n=14; 70.8±8.9%, n=5, respectively, p>0.05, ANOVA, Bonferroni); in contrast SCDHU PLT platelet aggregation was significantly lower (60.4±8.2%, n=7; p<0.05) than CON PLT aggregation. In response to TB, CON and SCD PLTs, again, presented statistically similar aggregations (86.5±4.4%, n=15; 85.3±7.5%, n=4; p>0.05), whilst SCDHU PLT aggregation was significantly lower than CON PLT aggregation (70.0±6.6%, n=7). ADP and TB stimulate PLT aggregation largely by activation of the α_IIbβ₃ integrin. Accordingly, α_IIbβ₃-integrin activation, as determined by PAC-1 antibody binding was found to be significantly lower on SCDHU PLTs than on SCD PLTs (11.1±3.7%; 28.6±6.4%; 12.4±4.2% PAC-1 binding for CON, SCD, SCDHU PLT, respect., n≥13, p<0.05 SCD comp. CON/SCDHU); furthermore α_IIbβ₃ activation negatively correlated with levels of fetal hemoglobin (HbF, %) in SCD/SCDHU patients (Spearman r= −0.756, p<0.01, n=24). Interestingly, Col stimulation of SCD PLT (98.6±9.9%, n=5) resulted in a significantly higher aggregation response than that of CON PLT (67.0±5.8%, n=15, p<0.01) and SCDHU PLT aggregation in response to Col was significantly lower than that of SCD PLT (63.8±6.6%, n=6, p<0.01). Expression of the collagen receptor, integrin α₂β₁, subunit (CD49b) was not altered on SCD or SCDHU PLTs, compared to CON (70.5±4.2%; 70.1±4.6%; 68.5+5.2%, respect., n≥13, p>0.05). Hydroxyurea (HU) therapy was found to be associated with a significant decrease in the aggregation properties of platelets from SCD individuals. Decreased aggregation was coupled with a lower presentation of the α_IIbβ₃-integrin in its activated conformation, which may occur as a consequence of elevated levels of HbF in these individuals. The mechanism by which HU may decrease PLT aggregation in response to Col is not clear, although activation of the α₂β₁ integrin and participation of the GPVI collagen receptor should be studied further. Results provide insights into the mechanisms involved in platelet aggregation and activation in SCD and suggest that HU therapy may benefit SCD patients by reducing the aggregation and inflammatory potential of PLTs in the microcirculation.