Increased platelet activation is recognized in patients with sickle cell disease (SCD), but its pathogenesis and clinical relevance remain uncertain. Pulmonary arterial hypertension (PAH), an important complication of SCD, is characterized by a proliferative pulmonary vasculopathy, in situ thrombosis, and vascular dysfunction related to scavenging of nitric oxide (NO) by hemoglobin released into blood plasma during intravascular hemolysis. We investigated links between platelet activation, PAH and NO scavenging in patients with SCD. Platelet activation marked by activated fibrinogen receptor correlated to the severity of PAH (r ‫؍‬ 0.58, P < .001) and to laboratory markers of intravascular hemolysis, such as reticulocyte count (r ‫؍‬ 0.44, P ‫؍‬ .02). In vitro exposure of platelets to pathologically relevant concentrations of cell-free hemoglobin promoted basal-and agonist-stimulated activation and blocked the inhibitory effects on platelet activation by an NO donor. In patients with SCD, administration of sildenafil, a phosphodiesterase-5 inhibitor that potentiates NO-dependent signaling, reduced platelet activation (P ‫؍‬ .01). These findings suggest a possible interaction between hemolysis, decreased NO bioavailability, and pathologic platelet activation that might contribute to thrombosis and pulmonary hypertension in SCD, and potentially other disorders of intravascular hemolysis. This supports a role for NO-based therapeutics for SCD vasculopathy. This trial was registered at www.clinicaltrials.gov as no. IntroductionA chronic state of hemostatic activation in sickle cell disease (SCD) has been well documented by several investigators. Some have highlighted the role of the sickle red cell 1,2 ; others have highlighted the contribution of abnormal tissue factor and secondary thrombin generation by a dysfunctional endothelium, 3 the depletion of endogenous anticoagulants, 4 or the activation of white blood cells. 5 It is likely that the increased expression of adhesion molecules, tissue factor, and thrombin generation, the result of chronic endothelial dysfunction or acute injury, are all important contributors to the coagulopathy of SCD. 6 Increased platelet activation is another known component of hemostatic activation in patients with SCD 1,7-9 Increased percentages of platelets are activated during steady state in patients with SCD, and this accelerates during vaso-occlusive crisis (VOC). [7][8][9] The exact inciting mechanism and clinical consequences of this process remain unknown, but platelet activation hypothetically might play a role in the development of chronic vascular complications, such as pulmonary arterial hypertension, by secreting mitogenic and vasoactive substances that promote intimal hyperplasia. In patients without SCD, platelet-derived growth factor plays a fundamental role in the pathogenesis of plexogenic pulmonary hypertension, and pathologic platelet activation likely contributes to the in situ thrombosis in pulmonary hypertension, which has recently been reviewed. 10 Pulmonary a...

Background-In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy. Methods and Results-Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54 000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by Ͼ10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified Ϸ100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. Conclusions-The present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

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