Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

Abstract

Key Points• Platelets from essential thrombocythemia patients have an intrinsic impairment in the P13kinase/Rap1/integrin ␣IIb␤3 signaling pathway.• This explains the clinical observation that in vitro platelet aggregation is impaired in patients with essential thrombocythemia. IntroductionThe myeloproliferative disorders (MPDs) are a group of clonal hemopoietic disorders characterized by increased proliferation of one or more of the myeloid, erythroid, or megakaryocytic cell lineages. The MPDs include polycythemia vera (PV; excess production of red cells) and essential thrombocythemia (ET; excess production of platelets) and other disorders. 1 The clinical course of untreated MPDs is dominated by arterial and venous thrombosis, which are major causes of mortality and morbidity in patients with early stage disease. [2][3][4][5] Abnormal bleeding occurs in 5% to 10% of patients with ET during the disease course, particularly gastrointestinal, urogenital, and intracranial. 5,6 Several independent groups demonstrated in 2005 that the defect in the majority of patients with MPDs is a somatic V617F gain-of-function mutation in the gene encoding Janus kinase 2 (JAK2) in hematopoietic progenitor cells. 3 JAK2 V617F is present in peripheral blood cells, including platelets, 7,8 in virtually all patients with PV and approximately 60% of patients with ET. 8,9 In ET, JAK2 V617F increases the risk of thrombosis by approximately 2-fold 10,11 and the risk of thrombosis is even higher in ET patients with Ͼ 50% JAK2 V617F allele load. 12 The JAK2V617F mutation is also present in some patients presenting with thrombosis who do not meet the diagnostic criteria for MPDs. [13][14][15] Bleeding risk in ET appears not to be associated with the presence of JAK2 V617F. Flow cytometry studies demonstrated that JAK2V617F platelets express higher levels of platelet activation markers, such as P-selectin, compared with platelets from patients without the mutation, 16,17 suggesting that increased platelet activation may contribute to the thrombotic phenotype. A recent study also observed that platelet-mediated thrombin production is increased in ET patients. 18 In contrast, numerous studies performed over the past 35 years clearly showed that in vitro platelet function, in particular aggregation, is impaired in MPD patients, 19-22 a finding difficult to reconcile with the increased thrombotic risk. Although it is generally accepted that platelets contribute to the increased occurrence of thrombosis in patients with MPDs, very little is known how the JAK2V617F mutation changes intracellular platelet signaling pathways, subsequent platelet function and how this contributes to the MPD phenotype.In this study, we therefore aimed to identify the role of JAK2 in platelet function and how the JAK2V617F mutation affects platelet function in ET patients. We studied agonist-stimulated intracellular signaling pathways and related these findings to integrin ␣ IIb ␤ 3Submitted May 20, 2012; accepted November 26, 2012. Prepublished online as Blood First...

Is JAK2V617F finally off the hook?

“…This is consistent with patient studies, where impaired in vitro platelet function in ET patients was unrelated to JAK2 V617F status. 5 Thus, although JAK2 V617F in platelets may not directly affect platelet function and the prothrombotic phenotype, the possibility that it contributes at a later stage of the disease cannot be completely ruled out. These findings are also interesting in the light of recent publications describing a role for erythrocytes, leukocytes, and endothelial cells in hemostatic changes in MPN mouse models and the potential impact of JAK2 V617F on the prothrombotic potential of erythrocytes and endothelial cells.…”

“…2 Molecular changes in platelets, erythrocytes, leukocytes, and endothelial cells have all been suggested to contribute to the phenotype. [2][3][4][5] However, the mechanism underlying MPN-associated hemostatic changes still remains largely elusive. As the risk of thrombosis is strongly associated with the JAK2 V617F gene load in both ET and PV patients, the question that has fascinated many scientists in recent years is whether the JAK2 V617F mutation is the direct underlying cause of thrombosis and/or bleeding in MPN patients.…”

JAK2V617F leads to intrinsic changes in platelet formation and reactivity in a knock-in mouse model of essential thrombocythemia

“…16,[19][20][21] This dichotomy is also found in studies of intracellular signaling pathways relevant to platelet function: one group has described increased activation of Src kinases in ET platelets, 22 and another recently reported a dysfunction of the phosphatidylinositol 3-kinase (PI3K) pathway. 23 This bewildering variation in results is potentially due to the fact that the study of platelet function using samples from patients with PV and ET has been hampered by a number factors, including the following: (1) Clonality: Peripheral blood cells from patients with MPN have been shown to originate from an abnormal hemopoietic clone, 24 but in a significant proportion of patients, normal polyclonal hemopoiesis coexists alongside the abnormal clone. 25 This means that studies of peripheral blood platelets investigate the compound activities of platelets originating from both normal and malignant clones in varying ratios between patients.…”

“…[46][47][48] In keeping with these studies, a recent publication confirmed that TPO potentiation of platelet response to SFLLRN (a PAR-1 thrombin receptor agonist) was mediated by PI3K, leading to an increase in fibrinogen binding to integrin aIIbb3 but without an effect on P-selectin expression (ie, a granule secretion). 23 Interestingly, when looking at samples from patients with ET (with or without JAK2V617F), the same group made the exact reverse observation (ie, a decrease in the function of the PI3K/Rap1/ integrin aIIbb3 pathway, which could be secondary to medication). In contrast, our findings in this mouse model show an increase in fibrinogen binding as well as P-selectin expression in response to various agonists, in keeping with the increased hemostasis observed in the functional assays.…”

Pathogenetic mechanisms of thrombosis in patients with myeloproliferative neoplasm

“…This pathway is important in regulating α 1 bb3 integrin activity and platelet activation. 26 Platelet response to ADP and thrombin has been shown to be more pronounced in patients with MPN, especially those associated with the JAK2 mutation. 27 Platelet receptors for adhesion and aggregation are less expressed in chronic myeloproliferative patients compared to the control lot.…”