Characteristics and Consequences of Platelets Derived from JAK2-Mutated Megakaryocytes

A myeloproliferative neoplasm (MPN) with the JAK2-V617F mutation frequently presents with thrombosis. Although blood cell proliferation is one cause of thrombotic events, individuals with JAK2-V617F clonal hematopoiesis and near-normal blood counts are also at higher risk of thrombosis than those without the mutation. This suggests that platelets with the JAK2-V617F mutation are functionally different from their wild-type (WT) counterparts. Platelets from Jak2-V617F mice (Jak2VF-Plts) show increased reactivity to ADP and thrombin (Blood:143;1539-1550,2024). However, the specific characteristics and functional consequences of Jak2VF-Plts need clarification. We prepared pools of platelet-rich-plasma (PRP) from Jak2VF and WT mice, adjusted their platelet counts to 5x105/mL, and compared their aggregation profiles with a CN3000 (Sysmex). Aggregation of Jak2VF-Plts was significantly enhanced by ATP and inhibited by collagen, when compared to WT-Plts. Also, as reported last year (ASH 2023, Abstract #740), Jak2VF-Plts form platelet-neutrophil complexes (PNCs) and initiate neutrophil extracellular traps (NETs) more strongly than WT-Plts. Remarkably, Jak2VF mice manifested NET-mediated thrombosis in both venous and arterial occlusion models. In search of mechanisms to account for irregular Jak2VF-Plts function, we performed proteome assays (LC-MS/MS) of WT and Jak2VF platelets. These assays showed differential expression of numerous glycoproteins that are broadly expressed on cellular membranes. Among them, we focused on decreased expression of collagen receptor GP6 and increased expression of PODXL, a member of the CD34 family of sialomucins. Reduced expression of GP6 leads to hypo-aggregation by collagen (Br J Hematol.1995;89:124-130), and may partially explain bleeding events in MPN. Platelet-specific overexpression of PODXL has been reported to provoke hyperactivation of platelets, especially by enhancing intercellular communication (Thrombosis Research:125; e300-e305,2010). Therefore, abundance of PODXL on Jak2VF-Plts may explain hyper-aggregation by ADP and formation of PNCs followed by NETs. Our FACS analysis of platelets and Western blotting of membrane extracts confirmed that PODXL is overexpressed on the cellular membranes of Jak2-mutated human platelets and two different Jak2-mutated murine models (Jak2-V617F transgenic mice and Jak2V617F/+ knock-in mice). Presently, we are generating megakaryocyte-platelet specific PODXL knock-out mice to see if the absence of PODXL normalizes the function of Jak2VF-Plts. How is the overexpression of PODXL induced? First, we hypothesized that PODXL is a direct target of the JAK-STAT pathway, which is sustainably activated by JAK2-V617F. However, administration of JAK1/2 inhibitor Ruxolitinib did not change the expression level of PODXL on platelets of Jak2VF mice, and exogenous overexpression of JAK2-V617F in MEG-1 (a human megakaryocytic leukemia cell line) did not elevate the expression level of PODXL. These findings suggest that overexpression of PODXL is not directly caused by JAK-STAT activation. Next, we hypothesized that a skewed differentiation pathway of megakaryocytes changes protein expression on platelet membranes. As previously reported (Blood:137;2139-2151,2021), CD41-positive megakaryocyte-biased hematopoietic stem cells (MK-biased-HSCs) were increased in Jak2VF mice compared with WT mice whose megakaryocytic progenitors (MKPs) were derived from megakaryocyte-erythroid-progenitors (MEP-derived-MKPs). We separated MK-biased-HSCs and MEP-derived-MKPs from Jak2VF mice and cultured them with thrombopoietin. Fourteen days later, both cell lines differentiated to megakaryocytes and produced platelets. We found that megakaryocytes and platelets derived from MK-biased-HSCs presented higher expression of PODXL compared with those from MEP-derived-MKPs. Thus, it is reasonable to conclude that JAK2 mutations affect megakaryopoiesis and platelet function by altering expression of PODXL and other membrane proteins.