Role of the JAK2V617F mutation in the pathophysiology of thrombosis in myeloproliferative diseases

Janus激酶JAK2(JAK2V617F)的零星突变Val617Phe与bcr/abl阴性的骨髓增生性疾病(MPDS)有关：真性红细胞增多症(PV)、原发性血小板增多症(ET)和原发性骨髓纤维化(PMF)。这些是克隆性造血干细胞疾病，其特征是特定造血系的增殖(PV中的红系、ET中的巨核细胞、PMF中的粒细胞和巨核细胞)在细胞成熟过程中没有受到阻碍。JAK2V617F是一种导致生长因子过敏的功能突变，已在大多数PV患者以及~60%的ET和PMF患者中检测到。血栓形成在大约30%的患者中表现为MPD，是发病率和死亡率的主要原因。高达40%的ET和PV患者发生血栓形成。缺血性卒中比急性冠脉综合征和外周动脉疾病更常见。静脉血栓的发生率也增加，尤其是内脏静脉血栓形成的频率特别高(即消化血管床)。MPD血栓性素质的潜在机制在很大程度上仍然难以捉摸。怀疑是血小板和内皮细胞特性受损，但到目前为止还没有得到证实。临床研究证实，高龄、既往血栓病史、白细胞增多和JAK2V617F状态是MPD患者血栓形成的危险因素。JAK2V617F对MPD患者血小板功能影响的直接评估受到突变克隆的异质性扩展和血栓预防治疗的频繁使用的限制。由于组织样本的稀缺，很难实现对MPDS中内皮细胞的广泛研究。最近描述的JAK2V617F可诱导表达的小鼠模型为确定该突变对止血功能的影响提供了一个有吸引力的替代方案。我们项目的目的是阐明JAK2V617F促进动脉、静脉和微循环血栓形成的具体机制。该策略基于表达JAK2V617F的培养细胞、JAK2V617F患者的血液和血管样本以及几个转基因小鼠模型。现已确定了五项任务：(1)确定JAK2V167F在患者体内的细胞定位和表型结果；(2)在体外分析JAK2V617F的功能后果；(3)使用不同的体内血栓形成小鼠模型评估JAK2V617F的功能后果并确定确切的机制；(4)评估JAK2V617F在激活凝血功能方面的潜在作用；(5)通过确定最近发现的钙网蛋白(CALR)突变在肝或门静脉血栓形成患者中的流行率以及开发含有该突变的新的转基因小鼠来准备未来的研究。这可能导致识别新的治疗方法和MPD患者血栓形成风险的标志物。

The sporadic mutation Val617Phe of the Janus kinase JAK2 (JAK2V617F) is associated with the Bcr/Abl-negative myeloproliferative disorders (MPDs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are clonal hematopoietic stem cell disorders characterized by the proliferation of particular hematopoietic lineages (erythroid in PV, megakaryocytic in ET, and granulocytic and megakaryocytic in PMF) without blockage in cell maturation. JAK2V617F is a gain of function mutation leading to growth factors hypersensitivity and has been detected in most PV patients and in ~60% of ET and PMF patients. Thrombosis reveals MPD in about 30% of patients and is a major cause of morbidity and mortality. Up to 40% of ET and PV patients develop thrombosis. Ischemic stroke is more frequent than acute coronary syndrome and peripheral artery disease. Incidence of vein thrombosis is also increased with a particularly high frequency of splanchnic vein thrombosis (i.e. of the digestive vascular bed). The mechanisms underlying the MPD thrombotic diathesis are still largely elusive. Impairment of platelet and endothelial cell properties is suspected but so far not demonstrated. Clinical studies identified advanced age, prior history of thrombosis, leukocytosis and the JAK2V617F status as risk factors for thrombosis in MPD patients. The direct assessment of the effect of JAK2V617F on platelet function in MPD patients is limited by the heterogeneous extension of the mutant clone and the frequent use of prophylactic treatment for thrombosis. An extensive study of endothelial cells in MPDs is difficult to achieve due to the scarcity of tissue samples. The recently described mouse models in which JAK2V617F expression is inducible and tissue-specific offer an attractive alternative to determine the impact of this mutation on haemostatic functions.The aim of our project is to elucidate the specific mechanisms by which JAK2V617F favours arterial, venous and microcirculatory thrombosis. The strategy is based on the use of cultured cells expressing JAK2V617F, of blood and vascular samples from patients with JAK2V617F and on several transgenic mouse models. Five tasks have been identified: (1) determine the cellular localization and phenotypic consequences of JAK2V167F in patients; (2) analyse functional consequences of JAK2V617F in vitro; (3) assess the functional consequences of JAK2V617F using different mouse models of thrombosis in vivo and determine the precise mechanisms involved; (4) evaluate the potential role of JAK2V617F in the activation of coagulation; (5) prepare future studies by determining the prevalence of the very recently discovered calreticulin (CALR) mutations in patients with thrombosis of hepatic or portal vein and development of new genetically modified mice harbouring this mutation. This could lead to the identification of new therapeutic approaches and of markers for thrombotic risk in MPD patients.