Treatment of Primary Immune Thrombocytopenia with Thrombopoietin Receptor Agonists: Effect On Platelet Function and Plasma Thrombin Generation

Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopenia caused by autoantibodies against platelet proteins that lead to platelets destruction and suboptimal platelet production. Platelet count and bleeding phenotype vary widely between ITP patients. In spite of the low platelet number, some thrombocytopenic ITP patients seldom bleed which might indicate the presence of additional mechanisms that contribute to the haemostasis in such patients.

Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of patients with ITP and can noticeably increase platelet count in most of the ITP patients, even in those with severe refractory thrombocytopenia. The aim of this work was to study platelet function and thrombin generation in thrombocytopenic ITP patients before treatment with TPO-RA and once they had recovered the normal platelet count as consequence of the treatment.

Fourteen ITP patients with thrombocytopenia (TP-ITP, platelet count less than 100,000 platelets/microliter) were studied before starting TPO-RA treatment (4 patients with romiplostim and 10 patients with eltrombopag) and after reaching a platelet count higher than 100,000 platelets/microliter (NP-ITP). Thirty-three healthy subjects were included as the control group.

Platelet activation was determined by flow cytometry through binding of FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 microM TRAP activated platelets. Immature platelet fraction was determined labeling platelets with thiazole orange. Expression of fibrinogen receptor was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry through FITC-annexin V binding to phosphatidylserine (PS).

Thrombin generation was measured in platelet-free plasma by the method of Hemker (Calibrated Automated Thrombography, CAT). Activation was performed with a final concentration of 4 microM of phospholipids and 1 pM of tissue factor.

Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Values of p≤0.05 were considered statistically significant.

Platelets from TP- and NP-ITP patients showed a basal expression of activated fibrinogen receptor similar to controls. Platelets from TP-ITP patients presented a reduced ability for being activated by TRAP (binding of PAC-1 expressed as % of positive platelets: 89±19 % in controls and 56±22 % in TP-ITP, p<0.01). TPO-RA treatment did not improve platelet activation despite increasing platelet count (binding of PAC-1: 47±19 % in NP-ITP, p<0.01). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from either TP- or NP-ITP patients, expressed more PS than controls under basal conditions. Percentage of platelets that bound FITC-annexin V were: 47±12 % in controls, 63±13 % in TP-ITP and 60±15 % in NP-ITP, p<0.001). Percentage of immature platelets in TP-ITP patients were higher than in controls (0.8±0.9 % in controls and 6.6 ±4.0 % in TP-ITP, p<0.05) and was reduced by TPO-RA treatment (in NP-ITP patients: 2.2 ±1.7 %).

CAT experiments showed that TP-ITP patients had an increased endogenous thrombin potential (ETP, p<0.001) and reached higher maximum levels of thrombin (peak height, p <0.001) than controls. This procoagulant characteristic of plasma from TP-ITP patients was unchanged after treatment with TPO-RA and the recovery of a normal platelet count.

Our results showed that treatment of ITP patients with TPO-RA is effective for increasing platelet production but did not ameliorate platelet function or procoagulant conditions of the disease which may indicate that treatment with TPO-RA do not interfere with the mechanisms involved in impairment of platelet function and generation of a procoagulant state. This increased thrombin generation of plasma from ITP patients has to be taken under consideration when evaluating thrombotic risk of therapeutic treatments, and we would like to point out the need of performing more studies to elucidate causes of the increased thrombogenic potential observed in ITP patients.

No relevant conflicts of interest to declare.