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

Janus激酶JAK2 （JAK2V617F）的散发性突变Val617Phe与Bcr/ abl阴性骨髓增殖性疾病（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，其特征是特定造血谱系的增殖（PV中的红系，ET中的巨核细胞，PMF中的粒细胞和巨核细胞），而细胞成熟过程中没有阻塞。JAK2V617F是一种导致生长因子过敏的功能突变，在大多数PV患者和约60%的ET和PMF患者中检测到。血栓形成显示约30%的患者患有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。缺血性中风比急性冠状动脉综合征和外周动脉疾病更常见。静脉血栓的发生率也随着内脏静脉血栓（即消化血管床）的特别高的频率而增加。MPD血栓形成的机制在很大程度上仍然难以捉摸。怀疑血小板和内皮细胞特性受损，但迄今尚未证实。临床研究发现高龄、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异质延伸和血栓预防治疗的频繁使用的限制。由于缺乏组织样本，很难对mpd中的内皮细胞进行广泛的研究。最近描述的小鼠模型，其中JAK2V617F表达是可诱导的和组织特异性的，为确定这种突变对止血功能的影响提供了一个有吸引力的选择。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。该策略基于使用表达JAK2V617F的培养细胞、来自JAK2V617F患者的血液和血管样本以及几种转基因小鼠模型。已经确定了五个任务：(1)确定JAK2V167F在患者中的细胞定位和表型后果；(2)分析JAK2V617F在体外的功能影响；(3)利用不同的小鼠体内血栓模型评估JAK2V617F的功能后果，并确定其确切机制；(4)评价JAK2V617F在凝血活化中的潜在作用；(5)通过确定最近发现的钙网蛋白（CALR）突变在肝静脉或门静脉血栓形成患者中的流行程度，以及培育含有这种突变的新转基因小鼠，为未来的研究做准备。这可能会导致确定新的治疗方法和标志物的血栓形成风险的MPD患者。

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.