Abstract 534: Aging is Associated with Peroxide Mediated Increased Thrombin Generation

Abstract

Human aging is associated with increased incidence of deep vein thrombosis (DVT), but the mechanisms are not fully elucidated. Using experimental models of DVT, we and others have demonstrated that mice also display increased susceptibility to thrombosis during aging. Further, we established that aged mice overexpressing antioxidant glutathione peroxidase (Gpx1; that converts peroxides to water) are protected from age associated increased susceptibility to DVT. However, the mechanistic pathway for peroxide-dependent thrombosis remains elusive. Recent studies have suggested that hydrogen peroxide mediated activation of neutrophils causes release of neutrophil extracellular traps (NETs). NETs are known to activate coagulation and contribute to the development of DVT. We hypothesized that aging cause peroxide-dependent increased release of NETs and increased thrombin generation. We studied Gpx1 Tg mice and their wild type (WT) littermates at 4 (young) and 20 months (old) of age. Cell-free DNA (cfDNA), a circulating marker of NETs was measured in the plasma using Qubit assay. We observed a significant increase in cfDNA in old WT mice compared to young WT mice (28.2±2.2 vs. 11.4±0.8 ng/μl, P<0.001), but there was no difference in cfDNA between old WT and old Gpx1 Tg mice. Next, we assessed thrombin generation using a Calibrated Automated Thrombogram (Daignostic Stago). The aged WT mice showed significant increase in endogenous thrombin potential (ETP, 430.2±26.7 vs. 277.9±42.1 nM.min, P<0.05) as well as peak amplitude, compared to young WT mice (65.4±4.3 vs. 40.8±7.1 nM, P<0.05). Interestingly, ETP was significantly decreased in aged Gpx1 Tg mice (P<0.05 vs. aged WT), suggesting a peroxide mediated effect on thrombin generation with aging. To examine how the findings in aged mice translate to human aging, we examined plasma samples from healthy young (18-39 years, n=11) and older (50-79 years, n=17) human subjects. We observed a significant increase in cfDNA and ETP in older compared to younger humans (13.4±.9 vs. 12.3±.4 ng/μl, p<0.05 for cfDNA and 1917.6±141.2 vs 1475.9±66.3 nM.min, p<0.05 for ETP). These data suggest that both mouse and human aging is associated with increased release of cfDNA and higher ETP, and that in mice peroxide mediates effects of aging on thrombin generation.