Haemostasis in Immune Trombocytopenia Patients Responders to Agonists of Thrombopoietin Receptor
Authors: Butta Nora , Fernandez Bello Ihosvany , Álvarez Román María Teresa , Martín Salces Monica , Justo Sanz Raul , Monzon Manzano Elena , Rivas Pollmar María Isabel , Rivas Muñoz Sergio , Canales Miguel , Jiménez Yuste Victor 
Published: November 2019

Introduction: The goal of treatment of patients with immune thrombocytopaenia (ITP) is to raise platelet count to a level that will minimize or stop bleeding. One of the therapeutic strategies employed is to augment platelet production with agonists of the thrombopoietin receptor (TPOR-A). Clinical trials for TPOR-A comparing their effects with placebo showed a reduction in proportion of patients reporting bleeding (Tarantino et al, Blood Coag Fibrinolysis, 2013, 24:284-296). This effect might rely on the increase in platelet count, but other factors known to compensate bleeding risk in thrombocytopenic ITP patients might be regulated by TPOR-A.

Objective: We aimed to compare haemostasis in ITP patients untreated with any pharmacologic agent (UT-ITPp) and treated with TPOR-A (TPOA-Rp) using the coagulation global assays thromboelastrometry (ROTEM®) and Calibrated Automated Thrombogram (CAT).

Methods: Thirty chronic UT-ITPp [platelet count: (86±60)x109 platelets/L], twenty six responders to TPOR-A [16 with Romiplostim® and 10 with Revolade®, platelet count: (132±46)x109 platelets/L] and fifty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). Aliquots were stored at -80ºC until analysis.

Non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded.

Plasma thrombin generation was measured in PPP using the CAT test at a final concentration of 1 pM tissue factor and 4 microM phospholipids. We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak.

Platelet activation was determined by flow cytometry through PAC1-FITC binding after stimulation with 100 micromol/L thrombin receptor-activating peptide 6 (TRAP). Fibrinolytic proteins were tested in PPP using commercialized kits.

Comparisons of quantitative variables were made with SPSS.22 software. Values of p≤0.05 were considered statistically significant.

Results: Platelets from all patients with ITP had a defect in their ability to be activated, as shown by the lower PAC1 binding (p<0.001).

No differences were observed among groups for thrombin generation except for an increase in ETP in both groups of ITP patients [control: 1240±320 nMxmin, UT-ITPp: 1464±443 nMxmin (p<0.05), TPOA-Rp: 1533±390 nMxmin (p<0.05)].

In ROTEM experiments, PRP from UT-ITPp and TPOA-Rp showed a prolonged CT [control: 550± 95 sec, UT-ITPp: 1090±75 sec (p<0.05), TPOA-Rp: 859±209 sec (p<0.05)]. Only UT-ITPp presented a diminished alpha angle [control: 60.3±7.5, UT-ITPp: 49.3±7.3 (p<0.05), TPOA-Rp: 55.8±5.58]. On the other hand, TPOA-Rp had an increased MCF (control: 46.5±2.1 mm, UT-ITPp: 50.2±5.6 mm, TPOA-Rp: 57.9±4.4 mm, p<0.05) and LI60 (control: 91.8±3.1 %, UT-ITPp: 94.1±3.1 %, TPOA-Rp: 97.6±1.1 %, p<0.05).

To evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed among the two groups of patients and healthy controls except for PAI-1 which level was increased in TPOA-Rp (control: 12.9±12.3 ng/ml, UT-ITPp: 17.3±12.5 ng/ml, TPOA-Rp: 46.8±20.8, ng/ml p<0.05).

Conclusions: As we previously described, TPOR-A treatment increased platelet count but did not ameliorate their function (Álvarez Román et al, Thromb Haemost. 2014, 112:65-72). Nevertheless, ITP patients responders to TPOR-A showed a haemostasis unbalanced on a hypercoagulable profile due, at least in part, to a hipofibrinolytic pattern mainly caused by an increase in PAI-1 plasma level. This fact, together with the increment in platelet count caused by TPOR-A, might help to protect these patients from bleeding.

This work was supported by a grant from the FIS-FEDER PI15/01457

Disclosures

No relevant conflicts of interest to declare.