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.