Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms
Authors: Berens Christina , Rühl Heiko , Müller Jens , Oldenburg Johannes , Brossart Peter , Wolf Dominik , Pötzsch Bernd 
Published: October 2019

Abstract
Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex.
Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.
Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively.
Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a 'hyperreactivity' of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients.

Disclosures
Berens: Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.