Heparin-induced thrombocytopenia/thrombosis (HIT/T), the most frequent drug-induced immune thrombocytopenia, is a common cause of life- and limb-threatening thrombosis. Although heparin/platelet factor 4 (PF4) antibodies are detected in many patients treated with heparin, there is little understanding of why only a subset of patients develops thrombosis. We recently produced and characterized the first mouse model that recapitulates the salient features of the disease. A second generation model, designated IIA/hPF4-mPF4KO, expresses human FcγRIIA and PF4 but lacks endogenous mouse PF4. These models allow systematic investigations of factors that contribute to pathogenic consequences of HIT/T antibodies. In the current study, we hypothesize that hypercholesterolemia, a known stimulus for atherosclerosis, endothelial dysfunction, and platelet hyperreactivity, would augment thrombosis in our mouse model of HIT/T. Age and sex-matched IIA/hPF4-mPF4KO mice were fed an atherogenic diet (Paigen diet) (AD; 15% cocoa butter, 1% cholesterol, 0.5% cholate) (n=10) or maintained on standard diet (SD; 4.5% fat, no cholate) (n=10). Mice fed the AD for only 4 weeks had significantly increased cholesterol levels (173 ± 29 mg/dl vs. 50 ± 18 mg/dl for SD-fed; p < 0.0001). Mice were then injected with 30 U heparin and KKO, a mouse monoclonal heparin/PF4 antibody. The mean nadir platelet count in AD-fed mice was 34.6% ± 9.1 lower than that in the SD-fed mice (p< 0.0001). Thrombin-anti-thrombin III (TAT) levels, which reflect thrombin generation in vivo, in the AD-fed mice increased from 32 ± 5 μg/at baseline to 79 ± 16 μg/l (p < 0.0001) coincident with the platelet nadir. In contrast, SD-fed mice, with a less profound fall in platelets, showed no increase in TAT. Histological examination showed multiple platelet-fibrin thrombi in lungs and livers of AD-fed mice, whereas the SD-fed mice showed no histological evidence of thrombosis. Thus, in our mouse model, short-term diet-induced hyperlipidemia significantly increases the severity of heparin/PF4 antibody-mediated thrombocytopenia and thrombosis. Our studies provide evidence that a specific host factor can enhance the pathologic effects of heparin/PF4 antibodies in vivo and contribute to thrombotic risk in patients with HIT/T. An increased understanding of the contribution of prothrombotic factors not only will facilitate identification of patients with heparin/PF4 antibodies who are at increased risk of thrombosis, but also provide novel approaches to treatment of HIT/T.