Abstract
Patients with Essential Thrombocythemia (ET) have abnormal platelet counts and function leading to increased thrombo-hemorrhagic events, which are the principal causes of morbidity and mortality in these patients. ET results from somatic mutations altering genes involved in intracellular signaling pathways: mutations in the JAK2 kinase gene are found in 50-60% of patients, mutations in the calreticulin (CALR) represents 30-40% of patients and mutations in MPL, the thrombooietin receptor, represents 5-10% of patients. JAK2 mutations lead to a constitutive activation of intracellular signaling pathway of JAK-STAT resulting in hyperactivated platelets. CALR has been shown to associate with MPL, the thrombopoietin receptor, and consequently activates its downstream signaling via JAK-STAT signaling. Interestingly, CALR -mutant patients have a better prognosis than JAK2 -mutant patients with less thrombotic events regardless of the platelet count. Despite the high frequency of CALR and JAK2 mutations, the mechanisms by which these mutations promote thrombosis are unknown. In this study, we characterized the platelet glycoprotein profile and investigated the molecular mechanisms leading to platelet activation of ET patients according to JAK2 or CALR mutations.
Venous blood samples from 14 patients with ET (8 CALR, 6 JAK2) and 9 healthy controls were used to 1) quantify platelet surface glycoproteins by flow cytometry. 2) Assessment of platelet activation status by fibrinogen binding and P-selectin exposure and 3) analysis of platelet lysates for western blot analysis.
Quantification of surface glycoproteins showed that 1) CALR- mutant platelets had significantly increased GP1ba surface expression when compared to controls and JAK2 -mutant platelets, which in contrast showed a slight decrease. 2) Both CALR- and JAK2 -mutant platelets had increased GPVI surface expression, but only JAK2-mutant levels reached statistical significance, 3) CALR -mutant platelets had significantly lower levels of Integrin b3 (CD61) when compared to controls and JAK2 -mutants showed no difference.
We next assessed the activation status of platelets from ET patients compared to controls at rest and we observed that JAK2 -mutant platelets had significantly higher fibrinogen bound and P-selectin exposure, while CALR -mutant platelets increased values failed to reach significance. We then evaluated the activating potential of ET platelets upon activation with TRAP-6 (T6) and Convulxin (CVX). Comparative analysis of fibrinogen binding to platelets showed no differences between groups regardless of the agonist used. However, JAK2 -mutant platelets showed significantly higher P-selectin exposure upon activation only with CVX. These results are consistent with the increase in GPVI surface expression observed in JAK2- mutant platelets and support the observations that CALR -mutant patients are have a lower thrombotic tendency than JAK2 -mutants. Western blot analysis of lysates from controls, JAK2 - and CALR -mutant platelets showed the appearance of high molecular phospho-Tyrosine proteins upon activation with CVX. According to their specific molecular weight we identified that activation with CVX but not T6, activates PLCg2. We also observed phosphorylation of JAK2 kinase upon activation, but not at rest, likely due to aspirin treatment of most patients.
Taken together, our data demonstrates that the hyperactive status of platelets from ET patients carrying JAK2 or CALR mutations, is dependent on the collagen receptor GPVI and its downstream signaling cascade. These studies suggest that ET patients can benefit from GPVI targeted therapy to reduce thrombotic risk.
Stone: Actinium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Merck: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sumitomo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Ono: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees. Steensma: Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; H3 Biosciences: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onconova: Consultancy; Takeda: Consultancy; Incyte: Equity Ownership. DeAngelo: Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Incyte: Consultancy, Honoraria; Celgene: Research Funding; Glycomimetics: Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; BMS: Consultancy; Immunogen: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; ARIAD: Consultancy, Research Funding; Shire: Honoraria; Blueprint Medicines: Honoraria, Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
