Effects of eltrombopag on platelet count and platelet activation in Wiskott-Aldrich syndrome/X-linked thrombocytopenia

Abstract

Because Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) patients have microthrombocytopenia, hemorrhage is a major problem. We asked whether eltrombopag, a thrombopoietic agent, would increase platelet counts, improve platelet activation, and/or reduce bleeding in WAS/XLT patients. In 9 WAS/XLT patients and 8 age-matched healthy controls, platelet activation was assessed by whole blood flow cytometry. Agonist-induced platelet surface activated glycoprotein (GP) IIb-IIIa and P-selectin in WAS/XLT patients were proportional to platelet size and therefore decreased compared with controls. In contrast, annexin V binding showed no differences between WAS/XLT and controls. Eltrombopag treatment resulted in an increased platelet count in 5 out of 8 patients. Among responders to eltrombopag, immature platelet fraction in 3 WAS/XLT patients was significantly less increased compared with 7 pediatric chronic immune thrombocytopenia (ITP) patients. Platelet activation did not improve in 3 WAS/XLT patients whose platelet count improved on eltrombopag.

In conclusion: (1) the reduced platelet activation observed in WAS/XLT is primarily due to the microthrombocytopenia; and (2) although the eltrombopag-induced increase in platelet production in WAS/XLT is less than in ITP, eltrombopag has beneficial effects on platelet count but not platelet activation in the majority of WAS/XLT patients. This trial was registered at www.clinicaltrials.gov as #NCT00909363.

Figure 1

Platelet surface expression of activation markers in controls and WAS/XLT patients. The following markers were measured with and without agonist stimulation: (A) percentage of platelets positive for activated GPIIb-IIIa (measured by PAC1 binding), (B) mean fluorescence for activated GPIIb-IIIa (PAC1), (C) percentage of platelets positive for P-selectin, (D) mean fluorescence for P-selectin, (E), mean fluorescence for CD41, and (F) percentage of cells positive for PS (measured by annexin V binding). Low ADP, 0.5 μM; high ADP, 20 μM; low TRAP, 1.5 μM; high TRAP, 20 μM; low convulxin, 1 ng/mL; and high convulxin, 5 ng/mL. Results are expressed as mean ± SEM with n = 8 (n = 7 for panel F) for controls and n = 9 (n = 6 for panel F) for WAS/XLT patients, and analyzed with 2-way ANOVA with Bonferroni posttest. *Significant difference (P < .05).

Figure 2

Platelet surface-activated GPIIb-IIIa and P-selectin normalized to platelet surface CD41 fluorescence in controls and WAS/XLT patients. The following markers were measured with and without agonist stimulation: (A) FSC of platelets, (B) ratio of mean fluorescence for activated GPIIb-IIIa (PAC1) and CD41 mean fluorescence, and (C) ratio of mean fluorescence for P-selectin and CD41 mean fluorescence. Low ADP, 0.5 μM; high ADP, 20 μM; low TRAP, 1.5 μM; and high TRAP, 20 μM. Results are expressed as mean ± SEM with n = 8 for controls and n = 9 for WAS/XLT patients, and analyzed with 2-way ANOVA with Bonferroni posttest. *Significant difference (P < .05).

Figure 3

Platelet markers in WAS/XLT patients at day 0 and >30 days of eltrombopag treatment. Platelet count was measured in a Bayer-ADVIA autoanalyzer (A). The following makers were measured with and without agonist stimulation: (B) percentage of platelets positive for activated GPIIb-IIIa (measured by PAC1 binding), (C) mean fluorescence for activated GPIIb-IIIa (PAC1), (D) percentage of platelets positive for P-selectin, (E) mean fluorescence for P-selectin, and (F) percentage of cells positive for PS (measured by annexin V binding). Low ADP, 0.5 μM; high ADP, 20 μM; low TRAP, 1.5 μM; high TRAP, 20 μM; low convulxin, 1 ng/mL; and high convulxin, 5 ng/mL. Results are expressed as mean ± SEM with n = 3 for WAS/XLT patients and analyzed with Student t test (A) or 2-way ANOVA with Bonferroni posttest (B-F). *Significant difference (P < .05).

Figure 4

Platelet counts of WAS/XLT patients (n = 8) during treatment with eltrombopag. Eltrombopag doses varied by patient and ranged from 9 mg to 75 mg daily. Patients were considered responders if at least one platelet count increased to ≥50 × 10⁹/L, was double the baseline count, and bleeding was reduced. The temporary rise in platelet count at week 6 to >160 × 10⁹/L in subject 10 (a nonresponder) was the result of a platelet transfusion (A). Five patients continued treatment past 52 weeks (B).

Figure 5

Changes in AIPF in WAS/XLT patients using eltrombopag compared with age-matched chronic ITP patients responding to eltrombopag. Post-eltrombopag data were unavailable for 2 WAS/XLT patients. Two ITP patients had almost superimposable data so that there are 7 patients’ data but only 6 evident lines.