Tet2 Age-Associated Somatic Mutations Impair Megakaryopoiesis and Platelet Function in Clonal Hematopoiesis

Aging is accompanied by the accumulation of somatic mutations in hematopoietic stem cells (HSCs) and their differentiated blood cell progeny, a state referred to as clonal hematopoiesis of indeterminate potential (CHIP). Loss-of-function mutations of the epigenetic modulator Tet methylcytosine dioxygenase 2 (TET2) are common in elderly individuals with CHIP, accounting for nearly 50% of mutations, and large exome sequencing studies have demonstrated a clinical association between mutations in TET2 and altered platelet traits. While epidemiological data highlight the contribution of aging to cardiovascular disease and thrombosis, direct causality between TET2 mutations and changes to both platelets and platelet-producing megakaryocytes (MKs) are lacking. We generated and leveraged new mouse models to define the effects of TET2 on MK and platelet function, demonstrating that loss of TET2 phenotypically and functionally alters MKs, resulting in the production of dysfunctional platelets.

To investigate the role of Tet2 on megakaryopoiesis we generated novel mouse models with pan-hematopoietic (Mx1^Cre; referred to as Tet2^HSC-KO) or MK-lineage targeted (PF4^Cre; Tet2^MK-KO) knockout of Tet2 and crossed these lines to a Vwf-EGFP reporter mouse that labels MKs, platelets, and platelet-primed HSCs. To model Tet2 clonal expansion we employed an irradiation-free adoptive transfer approach of chimeric ‘CHIP’ reconstitution in which 5 × 10⁶ (CD45.2) whole bone marrow donor cells were intravenously injected for three days into recipient mice (CD45.1) and allowed to expand. In these mixed-chimeras, donor blood cells are marked by CD45.2 and donor MKs and platelets by EGFP positivity enabling the quantification and visualization of mutant and nonmutant MKs and platelets in the same host.

We observed that loss of Tet2 drove selective expansion of the mutant MK lineage and resulted in increased numbers of higher ploidy (16-32N) MKs with an enlarged cytoplasm, as quantified by flow cytometry and validated using in situ immunofluorescence staining of femoral cryosections, indicative of enhanced platelet production capacity. This phenotype was observed in chimeric mice reconstituted with either Tet2^HSC-KO or Tet2^MK-KO bone marrow donor cells, suggesting that TET2 intrinsically regulates MK phenotype and function. Tet2^MK-KO mice also exhibited an increase in newly generated, thiazole orange (TO)^high platelets as well as monocyte and neutrophil-platelet aggregates (MPAs, NPAs). Additionally, high-resolution confocal imaging revealed decreased proplatelet elongation and proplatelet extensions in Tet2-deficient MKs compared to controls suggesting cytoskeletal alterations. These data demonstrate that Tet2deficiency in the MK lineage alone is sufficient to phenotypically alter MKs and platelet production.

To test if Tet2-deficient platelets exhibit prothrombotic phenotypes, we compared platelet adhesion and aggregation from Tet2^MK-KOand Tet2^MK-WT mice using an ex-vivo dynamic adhesion assay under flow conditions. Platelets from Tet2^MK-KO mice had a significant increase in adhesion and surface area coverage (2.13 and 1.96-fold changes; P=0.0087 and P=0.054), respectively, suggesting that loss of Tet2 led to the production phenotypically altered platelets that may be prothrombotic. In ongoing work, these novel chimeric models will be used to assess the contribution of Tet2-mutated platelets to thrombosis and resistance to dual antiplatelet therapy in vivo.

In summary, these studies suggest a causative link between Tet2 loss-of-function mutations in MKs and the production of dysfunctional platelets in the setting of clonal hematopoiesis. Understanding how megakaryopoiesis and platelet production are altered by Tet2-deficiency is an important first step in designing effective and personalized interventions to counteract age-related platelet pathologies to alleviate the cardiovascular and thrombotic disease burden among the elderly.

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