Abstract

Background: Patients with platelet counts less than 20 or 30 x 109/L have an increased risk of bleeding. Nevertheless, some patients with immune thrombocytopenia (ITP) have fewer bleeding symptoms than expected. In a previous communication (ASH 2014) we reported that these patients presented high microparticles (MP)-associated procoagulant activity to compensate bleeding risk and that cellular origin of these MPs were platelets and red cells. However, other mechanisms might be involved.

Objective: The aim of this work was to analyse the involvement of other factors to compensate bleeding risk in thrombocytopenic ITP patients. Moreover, the feasibility of using the coagulation global assays thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT) to test haemostasis in these patients was evaluated.

Methods: Fifty patients with chronic ITP with platelet count less than 50 x 109/L and twenty-five healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). 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) and aliquots were stored at -70ºC until analysis.

To assess the kinetics of clot formation, 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 Calibrated Automated Thrombogram (CAT) test at a final concentration of 1 pM tissue factor and 4 mM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). 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.

Fibrinolytic proteins and E-selectin was tested in PPP using commercialized kits.

Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant.

Results: PRP from ITP patients showed a prolonged CT (control: 550+ 95 sec, ITP: 890+165 sec, p<0.01), diminished alpha angle (control: 62.8+4.3, ITP: 53.5+7.5, p<0.05), and increased MCF (control: 46.7+3.1mm, ITP: 52.4+6.1 mm, p<0.05) and LI60 (control: 90.6+3.0% , ITP: 95.5+3.4, p<0.05) when compared with controls.

In order 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 between patients and healthy controls except for PAI-1 which level was increased in ITP patients (control: 14.7 ng/ml+11.7 ng/ml, ITP: 30.4+17.5, p<0.05). Since plasma PAI-1 might be increased as consequence of endothelial damage, plasma concentration of E-selectin, marker of endothelial injury, was determined. E-selectin was increased in samples from ITP patients (control: 10.5 ng/ml+3.9 ng/ml, ITP: 31.6+14.0, p<0.05). Moreover, MCF and LI60 ROTEM parameters correlated to E-selectin plasma concentration (Spearman r values 0.6643, p<0.001 for MCF; 0.6053, p<0.001 for LI60).

Thrombin generation in PPP was also measured and a shorter time to peak (control: 9.3+1.2 sec, ITP: 8.3+1.7 sec,p<0.05) and increased ETP (control: 1223.8+257.7 nMxmin, ITP: 1696.4+524 nMxmin,p<0.05) and peak (control: 225.7+82.8.1 nM, ITP: 330.4+106.1 nM,p<0.05) were observed in ITP patients.

Conclusions: We demonstrated that ITP patients presented a hypercoagulable profile that might be related, at least in part, to a reduced fibrinolysis mainly caused by an increase in PAI-1 level that seemed to be related to endothelial damage. Moreover ROTEM and CAT appeared to be useful tools for evaluating coagulant profile in ITP patients.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.