Microparticle-Associated Thrombin Generation and Procoagulant Activity Is Increased In Patients with Essential Thrombocythemia
Authors: Leuzzi Annamaria , Marchetti Marina , Panova-Noeva Marina , Tartari Carmen Julia , Russo Laura , Rambaldi Alessandro , Finazzi Guido , Barbui Tiziano , Woodhams Barry John , Falanga Anna 
Published: October 2019

Abstract
Abstract 1985
Essential Thrombocythemia (ET) is a chronic myeloproliferative neoplasm characterized by an increased thrombotic risk. Numerous quantitative and qualitative abnormalities of platelets and leukocytes, arising from the clonal proliferation of hematopoietic progenitor cells, have been described as responsible of the thrombophilic state in this disease. Recently, a high number of plasma microparticles (MP) of different cellular origin has been described in ET patients. MP are a key component of the hemostatic response and have been found increased in diseases at high thrombotic risk. To explore the contribution of plasma MP to the hypercoagulable state of patients with ET, in this study we aimed to characterize the MP functional procoagulant features. We used two different methods: the calibrated automated thrombogram (CAT), to determine the MP-associated thrombin generation (TG), and the P-PPL/1 assay (Stago R&D) to measure the MP-associated procoagulant activity (PCA). Both assays were performed in platelet free plasma (P-FP) obtained from 69 ET patients (24M/45F; 33 carriers of JAK2V617F mutation) and 67 control subjects (32M/35F). In a subgroup of 23 ET patients and 23 controls MP-associated TG and PCA were also determined in MP-free plasma (MP-FP). PFP was obtained by two serial centrifugations (4,000 rpm for 15 min, then 11,000 rpm for 10 min) and MP-FP by re-centrifuging P-FP at 14,000 rpm for 30 min. MP were isolated from the pellet. For the TG assay, 80 ul of P-FP or MP-FP and 20 ul of buffer were mixed, and TG started by adding CaCl2. The results were expressed as lag-time, peak, area under the curve (ETP), and time-to-peak (ttPeak). For P-PPL/1 assay, 100 ul of P-FP or MP-FP were mixed with 50 ul of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds.
The results show that P-FP from ET patients generated significantly higher quantity of thrombin compared to controls, as demonstrated by the shorter lag-time (22.1±8.7 vs 29.5±14.7 min; p=0.004) and time to peak (26.6±8.4 vs 33.3±13.2 min; p=0.004) and the significantly greater peak (52.9±24.9 vs 38.24±20.9 nM; p=0.002) and ETP (765.6±206.4 vs 537.5±295 nM*min; p=0.001). Similarly, the MP-associated PCA was significantly increased in ET patients (79±11 sec) compared to controls (89±11 sec; p<0.05). This increase was due to the presence of MP, as no TG and little PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples to the original values of P-FP for both assays. TG was significantly (p<0.05) increased in the JAK2V617F mutation carriers (lag-time: 19.5±7.4 min; peak: 59.8±26.9 nM; ttpeak: 23.9± 4.9 min) compared to wild-type subjects (lag-time: 24.5±9.1 min, peak: 45.4±20.6 nM, ttpeak: 29.3±10.3 min), while no significant differences were found for PCA. Significant correlations were found between the PCA by PPL-assay and the different parameters of TG assay [lag-time (R2= 0.414), peak (R2= -0.542), ETP (R2= -0.514)].
In conclusion, our results show that MP-associated TG capacity, as well as PCA, are increased in plasma from ET patients. The highest MP-associated TG was found in JAK-2 mutation carriers, who also are at higher risk for thrombosis compared to wild-type subjects. Our data provide evidence for a contribution of MP to the thrombophilic state of these patients and suggest to test MP associated TG and PCA in prospective studies to predict thrombosis in ET patients.

Disclosures:
No relevant conflicts of interest to declare.