Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients
Authors: Álvarez Román María Teresa , Justo Sanz Raul , Monzon Manzano Elena , Martín Salces Monica , Fernandez Bello Ihosvany , Rivas Muñoz Sergio , Rivas Pollmar María Isabel , Canales Miguel , Jiménez Yuste Victor , Butta Nora 
Published: October 2019

Abstract
Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have.
Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation.
Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) 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).
To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer.
Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP.
Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions.
To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses.
Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p<0.01; ADP-induced PAC1 binding: 63±14 % in controls and 50±23 % in ITP, p<0.05). Diminished responses to activation were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients.
Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p<0.05].
Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p <0.02; MF for TRAP-induced FVa binding: 44.1±11.4 in controls, 81.4±38 in ITP, p<0.001; MF for basal FXa binding: 45.7±18.4 in controls, 58.1±24 in ITP, p <0.005; MF for TRAP-induced FXa binding: 46.1±16.4 in controls, 72.0±24 in ITP, p<0.05). The lower the platelet count the higher increase in PS exposure (Spearman r =-0,518, p <0.001) and the union of FVa (Spearman r = -0.8571, p <0.001) and FXa (Spearman r = -0.7455, p<0.05).
Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP.
This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457

Disclosures
No relevant conflicts of interest to declare.