Abstract Cardiovascular disease (CVD) kills one in four of the people who die annually in the United States and is a major disease burden worldwide. Inflammation is the main underlying trigger of CVD and exacerbates the common ischemia-reperfusion injury (IRI). Recruitment of leukocytes to damaged tissues is a key step in inflammation and is dependent on the activation of β2 integrins. Circulating leukocytes in the bloodstream use β2 integrins to arrest on the endothelium in response to inflammatory signals. Neutrophils express two of the four β2 integrins, LFA-1 (αLβ2) and Mac-1 (αMβ2). Two proteins, kindlin-3 and talin-1, are required for neutrophil arrest. The molecular mechanism underlying neutrophil arrest and how kindlin-3 cooperates with talin-1 in integrin activation are poorly understood. It is unclear what signaling events lead to the kindlin-3 binding to and activation of β2 integrin. We hypothesize that kindlin-3, together with talin-1, induces integrin activation by breaking the inhibitory salt bridge within the heterodimeric α/β integrin subunits, and that blocking kindlin-3- dependent β2 integrin activation suppresses inflammation and reduces IRI. To test this hypothesis, we have generated reporter mouse lines for simultaneous detection of β2 integrin activation and imaging of kindlin-3 and talin-1 in mouse neutrophils. We show initial data that identify a novel salt-bridge dependent mechanism of β2 integrin activation in inflammation. We propose to use molecular and cellular engineering, flow cytometry, live cell imaging by quantitative dynamic footprinting, and intravital microscopy to address three specific aims: (1) We will assess how kindlin-3 promotes integrin activation by unclasping the inhibitory salt bridge within the heterodimeric α/β integrin subunits. (2) We will test the hypothesis that kindlin-3 permits talin-1-mediated integrin activation and neutrophil adhesion. (3) We will evaluate if blocking kindlin-3 mitigates IRI damage. The proposed research is innovative and significant because it will decode the conundrum of how high-affinity integrin activation contributes to IRI-induced inflammation. This proposal will establish molecular mechanisms of integrin activation and provide mechanistic insights for the development of new therapeutic drugs to reduce ischemia-reperfusion injury.