Abstract Introduction: Myeloproliferative neoplasms (MPN) are associated with both a higher risk of thrombosis and bleeding. Interestingly, both hyper- and hypo-reactivity in platelets is characteristic for MPN patients, the latter being the result of cellular exhaustion. The majority (90%) of MPN have activating mutations in JAK2, a kinase operating downstream of the platelet/megakaryocyte receptor for thrombopoietin (TPO), c-Mpl. JAK2 mutations or TPO binding to c-Mpl lead to intracellular signaling in platelets, including phosphoinositide-3-kinase (PI3K). However, TPO is a platelet “primer”, i.e. it does not cause platelet aggregation itself, but synergizes with other agonists in converting the main platelet integrin, αIIbβ3, from a bent, low affinity (B+) to an extended, high affinity state (E+H+). If and how altered JAK2 signaling affects platelet adhesive function and thrombotic plug formation are significant knowledge gaps in our understanding of MPN. Aims: Here, we aimed to determine whether JAK2 mutations or TPO binding to c-Mpl can enhance platelet adhesiveness via transition of αIIbβ3 to an intermediate affinity state (E+H-), and whether altered platelet adhesive function mediated by E+H- αIIbβ3 contributes to increased VT response in a murine MPN model. Methods: Platelet function was investigated in blood samples from a well-characterized mouse model with a 13 copy human JAK2V617F transgene driven by the vav1 promoter, and from JAK2V617F MPN patients. The inferior vena cava stenosis and femoral vein electrolytic injury models were used to study venous thrombosis in JAK2V617F or control mice. Microfluidics studies were performed to assess platelet adhesive behavior under flow conditions on fibrin or captured VWF. Platelet rolling, stable adhesion, and thrombus formation were analyzed using a custom machine learning model. A novel flow cytometry-based Förster Resonance Energy Transfer (FRET) assay was established to monitor extension of murine αIIbβ3 integrin. E+H+αIIbβ3 was detected using JON/A-PE antibody. Platelets positive for αIIbβ3 extension but negative for JON/A-PE staining were classified as expressing αIIbβ3 in the E+H- state. Results: In vivo, a significant increase in venous thrombosis was observed in JAK2V617F expressing mice when compared to controls. JAK2V617F platelets also showed increased, αIIbβ3 -dependent adhesiveness when perfused over fibrin (2.7 fold increase), suggesting that the integrin is present in a pre-activated form on circulating platelets in these mice. A novel FRET assay to assess αIIbβ3 extension revealed αIIbβ3-integrins in an intermediate affinity conformation (E+H-) in circulating JAK2V617F platelets, but not WT platelets. Mechanistic studies were conducted to elucidate how the JAK2V617F mutation induces E+H-αIIbβ3 in platelets. Pre-incubation of WT platelets with the TPO-mimetic, romiplostim, induced E+H-αIIbβ3 and adhesion to fibrin under flow (6-fold increase in comparison to vehicle). Similarly, increased platelet adhesion to fibrin under flow was observed in whole blood from MPN patients (4-fold increase) and in romiplostim-treated whole blood from healthy volunteers (3.6 fold increase). Romiplostim treatment of WT blood also led to increased platelet adhesion to immobilized-VWF under flow. Romiplostim-induced E+H-αIIbβ3 and platelet adhesiveness under shear conditions were sensitive to the PI3 kinase inhibitor, wortmannin (75% decrease of adhesiveness). In line with this observation, the induction of E+H-αIIbβ3 is controlled by the RAP1 GTPase-activating protein, RASA3, a PI3K-regulated inhibitor of platelet integrin activation. Conclusion: Together, these findings for the first time demonstrate that platelet priming in MPN causes increased adhesiveness due to surface expression of αIIbβ3 in an intermediate affinity state, mediated by PI3K/RASA3 signaling. Our studies further suggest that platelet priming may contribute to increased venous thrombosis risk observed in MPN, while impaired platelet response to agonist stimulation and alterations in coagulation proteins may explain the increased bleeding risk.
Wolfgang Bergmeier (Mon,) studied this question.