In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Abstract

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. (Blood. 2012;119(17): 4066-4072) IntroductionImmune thrombocytopenia (ITP) is an autoimmune condition characterized by antibody-mediated accelerated platelet destruction and suboptimal platelet production, which leads to various degrees of thrombocytopenia and bleeding symptoms. 1,2 Thrombopoietin receptor (TPO-R) agonists have recently been developed for the treatment of patients with ITP, and can dramatically increase platelet counts and reduce bleeding symptoms in the majority of patients, including some with severe refractory thrombocytopenia. [3][4][5] Eltrombopag, is an oral, small-molecule TPO-R agonist that interacts with the transmembrane domain of the receptor in a noncompetitive manner with endogenous TPO, thereby activating intracellular signal transduction pathways and leading to enhanced megakaryocyte differentiation, proliferation, and platelet production. 6 Patients with ITP often have few bleeding symptoms despite very low platelet counts, suggesting that ITP platelets are highly functional. [7][8][9] The TPO-R is expressed on circulating platelets, 10 and although TPO stimulation in vitro does not directly activate platelets, it potentiates platelet reactivity to several agonists, including adenosine diphosphate (ADP), thrombin, and collagen. 11-13 Administration of eltrombopag to healthy volunteers does not increase platelet aggregation or platelet surface expression of P-selectin or activated GPIIb/IIIa. 14 However, the effect of eltrombopag on platelet function in vivo in thrombocytopenic patients is unknown and requires study because of the potential for thrombosis with higher (...

Safety and efficacy of eltrombopag for treatment of chronic immune thrombocytopenia: results of the long-term, open-label EXTEND study

“…Even though in some cases it may be difficult to ascertain the accurate onset of a thrombotic event, and thus an accurate corresponding platelet count, 88% of the 16 patients who experienced TEEs did so with platelets lower than their maximum study platelet counts and half experienced TEEs at platelets below the normal range ( Figure 4). This is consistent with published reports failing to document platelet activation with eltrombopag 38 and do not demonstrate that high platelet counts per se are the main determinant of these events. Biomarkers of thrombophilia are being investigated to better understand the etiology of TEEs in eltrombopag-treated ITP patients.…”

Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP

“…Whole blood flow cytometric analysis of platelet surface P-selectin and activated glycoprotein (GP)IIb-IIIa was performed essentially as previously described 10,11 using sodium citrate-anticoagulated blood. Briefly, aliquots of whole blood (10 mL) were incubated (15 minutes at room temperature) with fluorescently labeled monoclonal antibodies (10 mL) and either 0.5 mM adenosine 59-diphosphate (ADP) (Bio/Data, Horsham, PA), 20 mM ADP, 1.5 mM protease activated receptor 1 (PAR1) thrombin receptor-activating peptide (TRAP) SFLLRNamide (Bachem, Torrance, CA), 20 mM TRAP, or N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES)-Tyrode's buffer (10 mM HEPES, 137 mM sodium chloride, 2.8 mM potassium chloride, 1 mM magnesium chloride, 12 mM sodium hydrogen carbonate, 0.4 mM sodium phosphate dibasic, 5.5 mM glucose, and 0.35% w/v bovine serum albumin, pH 7.4) (5 mL).…”

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

“…Platelet surface-activated GPIIb-IIIa, P-selectin, and annexin V binding were measured relative to the isotype control as percentage positive cells and mean fluorescence intensity (MFI) for activated GPIIb-IIIa, P-selectin, and CD41, as previously described. 20 Established voltages for forward scatter (FSC) and side scatter (SSC) were used to present platelets in the middle of the dot plot (FSC-SSC). Cells with high FSC-SSC were gated out.…”