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JAK2V617F endothelial cells are pro-thrombotic
try analysis did not reveal any GFP expression in granulo- cytes, platelets or red blood cells in Pdgfb- iCreERT2;JAK2V617F/WT;mT/mG mice 20 days after tamoxifen administration (Online Supplementary Figure S1F), confirm- ing that Cre recombination under the Pdgfb promoter is highly restricted to EC 15-20 days after tamoxifen admin- istration.
The expression of JAK2V617F by endothelial cells leads to increased thrombus formation
To investigate whether Pdgfb-iCreERT2;JAK2V617F/WT mice displayed a greater propensity to thrombosis, we examined pulmonary thrombus formation using experi- mental conditions that allowed assessment of EC involvement, without exposure of the sub-endothelium: (i) spontaneous thrombosis under basal conditions, (ii) a model of mild thrombosis with administration of low doses of collagen together with epinephrine to induce
weak activation of platelets and vasoconstriction and better demonstrate an intrinsic pro-thrombotic pheno- type of JAK2V617F EC, and (iii) a weak inflammatory trigger of thrombosis with injection of a low dose of TNF-α (250 ng/mouse). A dose of 500 ng TNF-α is commonly used to trigger inflammation25-27 but we chose the lower dose to reveal a potential hypersensitivity to inflammation. To quantify thrombi, we performed Carstairs staining which allows visualization of platelets, red blood cells, and fibrin.28 Small, spontaneously formed thrombi were observed in the lungs of Pdgfb-iCreERT2;JAK2V617F/WT mice, but not in littermate JAK2V617F/WT control mice (Figure 1A). In both models of mild induction of thrombosis, we observed a significant increase in thrombus formation in Pdgfb-iCreERT2;JAK2V617F/WT mice compared with that in controls (Figure 1A-C). Together, these results demon- strate that JAK2V617F EC have a pro-thrombotic pheno- type.
D
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A
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Figure 2. JAK2V617F-expressing endothelial cells have normal anticoagulant activity. (A,B) There was no statistical difference in either (A) the rate or (B) the extent of tissue factor (TF) -triggered thrombin generation at the surface of endothelial cells which were or were not activated with tissue necrosis factor (TNF)-alpha (n=3 for all conditions). (C-E) There was no statistical difference in (C) the rate of thrombin-triggered protein C activation at the surface of endothelial cells (n=3 for all con- ditions), (D) the production of nitrite or (E) the production of 6-keto prostaglandin F1a (n=3 for all conditions). Results are expressed as mean value ± SEM from three experiments. Statistical significance was determined by the Mann-Whitney test.
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