Tet2 clonal hematopoiesis enhances thrombotic pathologies, increases platelet reactivity, and alters megakaryote function in the context of aging

Abstract Cardiovascular diseases (CVDs)—including heart attacks, strokes, and thrombotic events—account for over 80% of deaths in individuals over the age of 65. Aging is the strongest risk factor for CVD and is associated with prothrombotic changes in platelets, such as increased reactivity (~8% per decade), faster clot formation, and enhanced aggregation. While these age-related platelet phenotypes parallel rising CVD risk, their mechanistic underpinnings lack resolution. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by the expansion of somatic mutations in hematopoietic stem cells, is as a causal driver of CVD in the elderly. Among CHIP-associated mutations, TET2 loss-of-function (LOF) is the second most common and is the most strongly associated with CVD. However, it remains unclear how TET2 mutations alter megakaryocyte (MK) or platelet function, and if this contributes to the cardiovascular pathologies observed in aging. To investigate this, we generated new murine models with lineage-specific Tet2 deletions: A PF4Cre-mediated (MK/platelet-targeted) and an Mx1Cre-mediated (multilineage) Tet2 knockout line. These were crossed with a vWF-EGFP reporter to differentiate mutant versus wild-type platelets upon transplant. To model the aging phenotypes of human CHIP, where mutant clones expand in the backdrop of healthy cells, we aged Tet2KO mice from each line (1.5–2 years) and transplanted their donor cells into adult recipients using a non-myeloablative, irradiation-free strategy. In Mx1Cre mice, Tet2 deletion was induced post-transplant with poly(I:C), mimicking the late-life acquisition of mutations. This approach preserves the native hematopoietic environment, vascular niches, and more closely models the clonal expansion dynamics in CHIP. Tet2-deficient platelets had increased surface expression of αIIbβ3 integrin in resting conditions indicating a primed activation state. Upon stimulation, these mutant platelets showed heightened responsiveness to both the P2Y12 agonist ADP and the GPVI ligand collagen-related peptide (CRP), revealing distinct functional differences compared to co-circulating wild-type platelets within the same host. Proteomic analysis of aged Tet2KO platelets revealed mutation-specific alterations in mitochondrial metabolism, while Tet2-deficient MKs exhibited changes in cell cycle regulatory and cytoskeletal proteins. In PF4Cre Tet2KO chimeric mice, the presence of 10–30% mutant platelets dramatically enhanced platelet aggregation. Accordingly, in a laser-induced arterial injury model, CHIP chimeras formed larger thrombi, with >40% increased platelet accumulation, highlighting the contribution of mutant platelets to thrombosis. To test the synergistic effects of mutant platelets and leukocytes, we employed an inferior vena cava (IVC) stenosis model on Mx1Cre Tet2KO chimeras. We observed increased incorporation of mutant platelets and leukocytes into the thrombi, suggesting that Tet2 deficiency may amplify local platelet recruitment and aggregation at sites of vascular injury. When subjected to R300-mediated platelet depletion, Mx1Cre Tet2KO chimeric mice exhibited accelerated platelet recovery, indicative of elevated platelet production. This suggests that loss of Tet2 function may amplify thrombotic potential under stress or injury conditions. Together, these findings highlight a critical role for TET2-mutant clonal hematopoiesis in driving age-related alterations in MK and platelet function as well as increasing thrombotic risk. Future work will utilize these models to identify novel therapeutic targets aimed at reducing cardiovascular complications in aging populations.