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Editorials
boses at unusual sites including the hepatic, portal and splenic veins, the cerebral sinuses and the mesenteric arter- ies. The mechanism(s) underlying this pro-thrombotic ten- dency in MPN are incompletely understood and have been the subject of speculation for almost seven decades. Over the last 24 months, several reports have appeared which have shed new light on the mechanisms underlying this thrombotic tendency. They implicate a pro-inflammatory MPN milieu as well as interactions between excessive num- bers of qualitatively abnormal blood cells and the vessel endothelium in the generation of these thrombotic events (Figure 1). In this issue of Haematologica, Guy et al.1 demon- strate a role for integrins in the development of thromboses using endothelial cells (EC) engineered to overexpress the MPN driver mutation JAK2V617F in vivo and in vitro.
Our initial understanding of the MPN pro-thrombotic state was largely influenced by the seminal observations of Pearson and Wetherley-Mein.2 They demonstrated that the incidence of thrombotic events in PV patients was directly related to the degree of hematocrit elevation. Red cells are the primary determinant of blood viscosity, which increases non-linearly with increasing hematocrit levels at both arte- rial and venous shear rates. Numerous studies have also suggested that increased red cell numbers increase the mar- gination of platelets along the vessel walls. Recently, Walton et al.,3 using a transfusion-based polycythemia model in healthy mice, showed that polycythemic mice had accelerated rates of arterial thrombus formation and shortened clotting times due to a platelet-dependent increase in thrombus formation. Their data collectively reflect the manner in which red cells independently pro- mote the development of arterial but not venous thrombo- sis. Klatt et al.,4 however, provided further data indicating that red cells trigger additional events beyond biophysical interactions that accelerate venous thrombosis. They showed that platelet/red cell interactions lead to increased platelet FAS ligand (FASL) exposure which then activates the death receptor (FASR) present on red cells. This ligand/receptor interaction ultimately results in further externalization of red cell phosphatidylserine which pro- motes the assembly of coagulation factor complexes lead- ing to thrombin generation and the formation of occlusive thrombi. Klatt et al. reported that these events could occur on a collagen surface with low shear rates which resembles a venous system. The consequences of excessive numbers of red cells in MPN patients was validated by Marchioli et al.5 who showed that sustained normalization of hemat- ocrit levels (<45%) in high-risk PV patients was associated with reduced numbers of thrombotic events. Furthermore, Alvarez-Larran et al.6 demonstrated that PV patients with higher phlebotomy requirements were at the highest risk of developing thrombotic events. However, several lines of evidence strongly suggest that additional mechanisms beyond hematocrit elevation are required to explain a num- ber of observed clinical manifestations including: (i) the occurrence of thrombotic events in over a third of patients prior to the diagnosis of PV; (ii) the occurrence of splanchnic vein thromboses, frequently in patients with a JAK2V617F mutation with normal blood counts; (iii) the increased inci- dence of thrombotic events in normal individuals found to have clonal hematopoiesis of indeterminate potential with a JAK2 mutation; (iv) the persistent rate of thrombosis fol-
lowing normalization of the hematocrit in PV patients; and (v) the increased rate of thrombosis in ET and MF patients without polycythemia. Intuitively, physicians have linked MPN-associated thrombocytosis to the high incidence of thrombotic events, however, the thrombotic risk in ET patients does not seem to be related to the degree of throm- bocytosis7 and those patients with extreme degrees of thrombocytosis (>1.5 million) are ironically at a higher risk of bleeding rather than clotting due to the development of a secondary form of von Willebrand disease.
The conclusion that additional factors beyond excessive numbers of blood cells contribute to the MPN pro-throm- botic tendency was bolstered by the more recent observa- tion that patients with a JAK2V617F mutation, particularly those individuals with a high variant allele burden, were at a greater risk of developing thrombotic events than those with calreticulin mutations.8 Several groups have provided evidence that mutated JAK2 might affect not only hematopoietic cells but also EC, which raises the possibility that MPN might actually arise in some patients in a primi- tive cell that resembles the hemogenic endothelium.9 In this issue of Haematologica Guy et al.1 report the construction of several murine models which can be used to evaluate the contribution of EC to the MPN pro-thrombotic state. They demonstrate that mice that were genetically engineered to express JAK2V617F in EC but not hematopoietic cells had a predilection to develop thrombotic events in spite of having normal blood counts and normal rates of thrombin genera- tion. Importantly, this thrombotic tendency was accentuat- ed by the creation of a pro-inflammatory milieu through the administration of low doses of tumor necrosis factor alpha. Using both in vitro and in vivo approaches they next showed that JAK2V617F+ human and murine EC were capable of promoting both leukocyte rolling and adhesion. Although the most common integrins associated with leukocyte adhesion to EC were not upregulated in these mutated EC, Guy et al. did demonstrate increased surface expression of P-selectin (CD62P) and von Willebrand factor (VWF), both of which are contained within Weibel-Palade bodies in EC. Importantly the pro-adhesive properties of the JAK2V617F+ EC were reversed by treatment with either a P-selectin blocking antibody or hydroxyurea, a drug that remains the standard of care for treating high-risk PV and ET patients. The authors concluded that hydroxyurea did not block the effects of P-selectin but rather decreased the release of P-selectin and VWF from Weibel-Palade bodies. The upregulation of P-selectin by mutated EC was attrib- uted to increased STAT3 phosphorylation which is a down- stream event of JAK/STAT signaling. Importantly, earlier this year, Guadall et al.10 generated data that supported the findings of Guy et al. using a totally different experimental system. They developed wild-type and JAK2V617F+ EC from immortalized human pluripotent stem cells and showed that JAK2V617F+ EC promoted the adherence of leukocytes and were characterized by increase phosphorylation of STAT3 and overexpression of both VWF and P-selectin. The availability of large numbers of JAK2V617F+ human EC from immortalized human pluripotent stem cells allowed these investigators to document gene expression analyses, demonstrating increased expression of genes associated with inflammation and cell adhesion in JAK2V617F+ human EC. P-selectin has been previously implicated by the
haematologica | 2019; 104(1)