JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation
Bärbel Edelmann, Nibedita Gupta, Tina M. Schnoeder, Anja M. Oelschlegel, Khurrum Shahzad, Jürgen Goldschmidt, Lars Philipsen, Soenke Weinert, Aniket Ghosh, Felix C. Saalfeld, Subbaiah Chary Nimmagadda, Peter Müller, Rüdiger Braun-Dullaeus, Juliane Mohr, Denise Wolleschak, Stefanie Kliche, Holger Amthauer, Florian H. Heidel, Burkhart Schraven, Berend Isermann, Andreas J. Müller, Thomas Fischer
Bärbel Edelmann, Nibedita Gupta, Tina M. Schnoeder, Anja M. Oelschlegel, Khurrum Shahzad, Jürgen Goldschmidt, Lars Philipsen, Soenke Weinert, Aniket Ghosh, Felix C. Saalfeld, Subbaiah Chary Nimmagadda, Peter Müller, Rüdiger Braun-Dullaeus, Juliane Mohr, Denise Wolleschak, Stefanie Kliche, Holger Amthauer, Florian H. Heidel, Burkhart Schraven, Berend Isermann, Andreas J. Müller, Thomas Fischer
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Research Article Hematology

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation

Abstract

JAK2-V617F–positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1– (VCAM1-) and intercellular adhesion molecule 1–coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti–VLA-4 and anti–β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

Authors

Bärbel Edelmann, Nibedita Gupta, Tina M. Schnoeder, Anja M. Oelschlegel, Khurrum Shahzad, Jürgen Goldschmidt, Lars Philipsen, Soenke Weinert, Aniket Ghosh, Felix C. Saalfeld, Subbaiah Chary Nimmagadda, Peter Müller, Rüdiger Braun-Dullaeus, Juliane Mohr, Denise Wolleschak, Stefanie Kliche, Holger Amthauer, Florian H. Heidel, Burkhart Schraven, Berend Isermann, Andreas J. Müller, Thomas Fischer

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Figure 4

PI3K and CalDAG-GEFI are involved in Rap1 activation.

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PI3K and CalDAG-GEFI are involved in Rap1 activation. Static adhesion on...
Static adhesion on immobilized VCAM1 in the presence of (A) wortmannin (10 nM, 30 minutes) or (B) LY294002 (25 μM; 3 hours) in 32D JAK2-V617F and JAK2-WT cells. Minus symbol (–) indicates DMSO control. Representative Western blots (of 3 independent experiments) of phospho-Akt and Akt demonstrate the efficient blockade of signaling. (C) Inhibition of Rap1 activation by LY294002 treatment (25 μM; 3 hours) in 32D JAK2-V617F cells. Lower panel shows quantitative analysis of Rap1 activation given as fold compared with controls and corrected for loading variations using total Rap1; minus symbol (–) indicates DMSO control. (D) Rap1 pull-down and static adhesion on immobilized VCAM1 in the absence and presence of Akt inhibitor VIII treatment (0.5 μM; 4 hours) in 32D JAK2-V617F and JAK2-WT cells; minus symbol (–) indicates DMSO control. Representative Western blots (of 3 independent experiments) of phospho-Akt and Akt demonstrate the efficient blockade of signaling. (E) Static adhesion of 32D JAK2-V617F and JAK2-WT cells on immobilized VCAM1 in the absence and presence of BAPTA/AM (10 μM; 1 hour). (F) Static adhesion on immobilized VCAM1 of 32D JAK2-V617F cells transfected with scrambled (scr) or CalDAG-GEFI shRNA. (G) Rap1 pull-down after knockdown of CalDAG-GEFI results in reduced Rap1 activity. Data are shown as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (1-way ANOVA with Bonferroni’s post test and unpaired, 2-tailed Student’s t test). Three independent experiments were performed each.