Engineered microsystems to assess endothelial contribution to myeloproliferative neoplasm thrombosis

PROJECT ABSTRACT
Thrombosis is the leading cause of mortality among patients with myeloproliferative neoplasms (MPNs). MPNs
are characterized by excessive production of red blood cells, platelets, and/or leukocytes. Thrombosis risk in
MPNs is thought to be primarily secondary to excess clonal MPN cells. However, at present, the interaction
between the vascular endothelium and clonal MPN cells is poorly characterized. Clonal MPN growth is driven
by dysregulated Janus kinase-signal transductor and activator of transcription (JAK-STAT) signaling. The
JAK2V617F+ mutation occurs in up to 70% of MPN patients and increases the risk of thrombosis 6-fold.
Additionally, MPN patients have a higher risk of VTE in slow-flow splanchnic vasculature. Several in vitro and
in vivo studies demonstrate that endothelial cells (EC) with the JAK2V617F+ mutation express pro-adhesive and
thrombotic proteins, suggesting that EC signaling may contribute to increased thrombosis. My primary
objective is to define how EC activation contributes to MPN thrombosis. My central hypothesis is that within
the EC vascular, the JAK2V617F+ mutation evokes a pro-inflammatory and thrombotic cascade. In preliminary
studies, I evaluated blood outgrowth endothelial cells (BOEC) isolated from JAK2V617F+ patients. In JAK2V617F+
BOECs and in TNF-α-activated JAK2WT ECs, ruxolitinib and fedratinib (JAK1/2 inhibitors approved for use in
MPN) reduced tissue factor (TF) expression and activity. Additionally, Compared to JAK2WT ECs, JAK2V617F+
BOECs express higher levels von Willebrand factor (VWF), and growth arrest specific 6 (Gas6) protein. Gas6
is a vitamin-K dependent protein S homolog, which promotes both TF expression and triggers platelet and
monocyte activation after binding to receptors Axl, MERTK, and Tyro3. Interestingly, in preliminary studies,
JAK2V617F+ individuals had significantly higher plasma levels of Gas6, Axl, and MERTK than controls.
Importantly, recent work has shown that blockade of the Gas6-Axl pathway kills JAK2V617F+ hematopoietic stem
cells in vitro and reduces spleen size and prolongs survival in JAK2V617F+ mice. However, these studies did
not evaluate whether the Gas6-Axl-MERTK axis contributes to MPN thrombosis. Phenotypic variability
limits use of JAK2V617F animal models to assess hemostasis and thrombosis. Therefore, I propose to use
endothelialized microfluidics models to assess how JAK2V617F expression increases EC activation. Using an
endothelialized microfluidics model, I will culture JAK2V617F+ EC under physiologic shear to assess for changes
in pro-coagulant and adhesive function. Furthermore, I will assess pro-adhesive and thrombotic interactions
between JAK2V617F+ EC and whole blood. I will also explore how Gas6-Axl-MERTK signaling in JAK2V617F+ ECs
increases the pro-coagulant and pro-adhesive environment. Collectively, the proposed research will establish
the contribution of shear to JAK2V617F+ EC activation and evaluate Gas6-Axl-MERTK signaling in JAK2V617F+
pro-thrombotic activation.

Grant Number: 5K08HL159289-04
NIH Institute/Center: NIH
Principal Investigator: Joan Beckman