PROJECT SUMMARY Factor XIa (fXIa), the protease form of the plasma protein factor XI (fXI), contributes to thrombin generation primarily by catalyzing activation of factor IX (fIX). This appears to reflect an ancillary role in the host hemostatic response to injury, as fXI deficiency causes, at most, a relatively mild bleeding disorder. Despite its limited role in hemostasis, mounting evidence from human population studies and animal models support the premise that fXI contributes substantively to venous and arterial thrombosis. This has lead to considerable interest in therapeutic inhibition of fXIa, with the hope that such a strategy will produce a useful antithrombotic effect, with a smaller impact on hemostasis than currently used anticoagulants such as heparin, warfarin and newer direct oral anticoagulants. FXI is structurally distinct from the vitamin K-dependent coagulation proteases that form the core of the thrombin generation mechanism. Indeed, fXI arose from a duplication of the gene for prekallikrein (PK), the precursor of the protease ?-kallikrein. PK, along with factor XII (fXII) and high molecular weight kininogen (HK) form the kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory peptides in response to injury. As a homolog of PK, fXI retains activities of the parent molecule. However, fXI has acquired unique features that facilitate its interactions with the thrombin generation mechanism. Our current working model is that fXI functions as a bi-directional interface between thrombin generation and the KKS, and that this places it in a position to influence the effects of both systems on thrombotic and inflammatory processes. Work in our laboratory is directed at establishing a better understanding of the biochemistry, molecular biology and pathophysiology of fXI, and its relationships with thrombin generation and the KKS. We take a broad approach to this problem, which is reflected in the three Focus Areas described in this application. Focus Area 1 investigates important structure-function relationships in the fXI molecule that are relevant to its activity in flowing blood. Focus Area 2 will investigate the contributions of fXI, fXII, PK and HK to thrombus formation and sepsis in mouse models. We will also investigate the role of fXI in modulating bleeding tendency in mice lacking factor IX (a model of hemophilia B), and pursue a recent observation that the majority of fXI in the vasculature forms a non-circulating pool associated with the blood vessel wall. Finally, work in Focus Area 3 is directed at a better understanding of fXII, the precursor of a protease (fXIIa), that activates fXI and PK, and that contributes to thrombo-inflammatory processes. We strongly feel that the expertise in our laboratory at Vanderbilt University, and the stellar group of collaborators in academia and industry that have worked with us for over a decade, place us in a unique position to make important contributions to the field of thrombosis research, with the goal of better informing efforts to develop novel antithrombotic therapies.