A Study of Haemostatic Parameters in Patients with Philadelphia-Negative Myeloproliferative Neoplasms. Correlation with, Clinical, Laboratory, Molecular, and Treatment Characteristics
Authors: Giannakopoulou Nefeli , Politou Marianna , Diamantopoulos Panagiotis Theodorou , Korakakis Dimitris , Efstathopoulou Maria , Kassi Theodora , Kontandreopoulou Christina-Nefeli , Zoi Katerina , Giannopoulos Andreas , Dimou Maria , Panayiotidis Panayiotis , Viniou Nora-Athina 
Published: October 2019

Abstract
Introduction
Patients with Philadelphia-negative myeloproliferative neoplasms (PN-MPN) namely polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF) are at a higher risk for arterial and venous thrombosis that constitute a major cause of morbidity and mortality. Global coagulation assays such as thromboelastography, may be more efficient to evaluate the patient's thrombotic risk. The aim of the present study was to examine the hemostatic profile of patients with PN-MPN and correlate it with clinical, laboratory, treatment, and molecular characteristics including mutational analysis of JAK2, MPL, CALR, and polymorphisms of poly(ADP ribose) polymerase (PARP1), since a correlation of specific mutations with PARP1 polymorphisms has been reported in the literature.
Materials and methods
The study included adult patients with a confirmed diagnosis of PN-MPN according to the revised 2016 WHO classification. A written informed consent was obtained from all patients. The presence of splenomegaly, vascular events, PN-MPN specific therapy, and anticoagulation treatment were recorded. All the patients were assessed with complete blood count, routine coagulation tests [PT, INR, aPTT and fibrinogen, D-Dimers analyzed with the automatic coagulation analyzer Sysmex (Siemens)], platelet function performed with PFA-100 (COL, EPI, ADP), and global hemostatic potential assessed with ROTEM® Tromboelastometry (EXTEM), recording clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), lysis index at 30 (LI30) and 60 (LI60) minutes, and α angle.
Mutation profiles of JAK2, MPL and CALR were defined using peripheral blood DNA. JAK2 and MPL mutations were detected using a standard PCR and CALR mutations using an HRMA-PCR assay. The rs1136410/PARP-1 (V762A) single nucleotide polymorphism (SNP), was detected with an RFLP method using the enzyme AciΙ (New England Biolabs, USA) and the digestion products were evaluated by polyacrylamide gel electrophoresis.
Statistical analysis was performed using IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA).
Results
Seventy-four patients were included in the study (22 PV, 47 ET, 5 MF) with a median age of 63 years (25-87) and 68 healthy controls for the SNP/PARP1 study. At the time of sample collection, 71 (95.9%) patients were under treatment [hydroxyurea (HU), 57 (77.0%); anagrelide, 19 (25.7%); ruxolitinib, 9 (12.2%); interferon alpha, 2 (2.7%); an alkylating agent, 4 (5.4%)]. In terms of anticoagulation, 47 (63.5%) patients were on aspirin, 4 (5.4%) on clopidogrel, 7 (9.5%) on a combination of the two, 3 (4.1%) on a vitamin K antagonist, and 2 (2.7%) on a Xa-inhibitor. Twenty-two (29.7%) patients had abnormally high D-Dimers (>0,5mg/l). Nineteen (25.7%) patients had developed thrombosis after diagnosis (8, ischemic stroke; 4, coronary artery disease (CAD); 3, deep vein thrombosis (DVT); 1, symptomatic carotid stenosis; 2, a combination of stroke and CAD; 1, a combination of CAD and DVT). Among 69 patients who were not receiving anticoagulation, 5 (7,2%) had an abnormal CT, 4 (5,7%) had abnormal CFT, 3 (4,3%) had an increased α angle, and 18 (26%) had an increased MCF value.
Women had shorter CFT, higher α angle, and higher MCF (p<0.05 for all parameters). Patients with ET had higher MCF compared to PV and MF. Patients with mutated JAK2, CALR, or MPL had higher WBC and shorter CFT (p<0.05).
Patients receiving anagrelide or alkylating agents, had statistically significant shorter CFTs, higher α angles, and higher MCFs compared to the ones receiving HU.
Among 54 patients taking aspirin COL-EPI was normal in 10. Among 11 patients taking clopidogrel COL-ADP was normal in 5, implying that the antiplatelet treatment may not be sufficient in certain cases. No correlations were found between PARP1 polymorphic status and any of the studied parameters, nor between patients and healthy controls.
Discussion
Global assays such as thromboelastography are more useful than conventional hemostatic laboratory tests in depicting the hypercoagulable state in MPN. They may be useful in combination with other parameters such as the mutational status in identifying patients with MPN at higher risk for thrombosis and guide clinicians for the type of treatment (both cytoreductive and anticoagulants). Tests of platelet function assessment may help the clinicians adjust the type and dose of antiplatelet therapy.

Disclosures
No relevant conflicts of interest to declare.