Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension, and nitric oxide scavenging by cell-free hemoglobin

Abstract

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. NCT00352430.

Figure 1

Platelet activation in control participants and in patients with SCD at steady state and during VOC. Each dot represents the percentage of activated platelets from an individual patient with SCD or control participant, as determined by flow cytometry detection of platelet cell-surface (A) activated fibrinogen receptors and (B) P-selectin. Significant P values are indicated as determined by unpaired t test. P-selectin values were analyzed by the Mann-Whitney test because these results were not normally distributed. Boxes indicate median and interquartile ranges, and whiskers indicate ranges.

Figure 2

Effects of agonist-induced activation on platelets from patients with SCD or healthy participants. (A) ADP reactivity of platelets from patients with SCD and PAH. The curves represent the mean percentage plus or minus SEM of activated platelets (activated GPIIb/IIIa) after their incubation with increasing concentrations of ADP. Patients with SCD and secondary pulmonary hypertension (SCD + PAH; ▲) reached higher degrees of activation with significantly lower concentrations of ADP compared with control participants (■) and patients with SCD without PAH (SCD − PAH; ●). (B) Reactivity of platelets from patients with SCD and PAH to activation by TRAP is compared with that of platelets from healthy control participants or patients with SCD without PAH.

Figure 3

Effects of cell-free hemoglobin and NO exposure on platelet activation. (A) The in vitro exposure of purified human cell-free hemoglobin to platelets from healthy donors increased the percentage of activated platelets as indicated by cell-surface expression of activated GPIIb/IIIa (Plt + Hb; ■). The NO donor MAHMA-NONOate (50 μM) inhibited baseline activation, but this inhibitory effect was overcome by the NO-scavenging activity of cell-free hemoglobin in a dose-dependent manner (Plt + Hb + NO; ▲). (B) Platelet activation was induced significantly by cell-free hemoglobin. Activation was inhibited by the NO donor, but this inhibition was eliminated by the addition of 50 μM cell-free hemoglobin. The markers and bars represent the mean percentages (± SEM) of activated platelets after 3 experiments of healthy donor samples (*P < .05 compared with baseline samples; †P < .05 compared with NO alone; paired t test). (C) The induction of platelet activation by ADP was nearly abolished by the NO donor, but this action was again inhibited by cell-free hemoglobin (50 μM).

Figure 4

Effects of sildenafil on platelet activation of patients with SCD and PAH. The lines connect the percentages of activated platelets of 12 patients before and after (off-on) a dose of sildenafil. The percentage of activated platelets decreased after sildenafil from 30.6% (± 4.4%) to 17.3% (± 3.6%) for activated GPIIb/IIIa (P = .01, paired t test), and from 6.3% (± 1.6%) to 3.2% (± 0.6%) for P-selectin (P = .08).