Background: Factor XIII (FXIII) is the final enzyme of the coagulation cascade. While the other enzymatic coagulation factors are proteases, FXIII belongs to the transglutaminase family. FXIIIa covalently crosslinks the fibrin clot and represents a promising target for drug development to facilitate fibrinolysis. However, no FXIII-inhibiting compound has entered clinical trials. Here, we introduce the features of a peptidomimetic inhibitor of FXIIIa (ZED3197) as a potential drug candidate. Methods:The potency of ZED3197 against FXIIIa and the selectivity against other human transglutaminases were characterized using transamidation and isopeptidase assays. The inhibition of fibrin crosslinking was evaluated by biochemical methods and thromboelastometry. Further, the pharmacology of the compound was explored in a rabbit model of venous stasis and reperfusion.Results: ZED3197 proved to be a potent and selective inhibitor of human FXIIIa. Further, the compound showed broad inhibitory activity against cellular FXIIIA from various animal species. Rotational thromboelastometry in whole human blood indicated that the inhibitor, in a dose-dependent manner, prolonged clot formation, reduced clot firmness, and facilitated clot lysis without affecting the clotting time, indicating minimal impact on hemostasis. In vivo, the novel FXIIIa inhibitor effectively decreased the weight of clots and facilitated flow restoration without prolongation of the bleeding time.Conclusions: ZED3197 is the first drug-like potent compound targeting FXIIIa, a yet untapped target in anticoagulation. Due to the function of FXIII downstream of thrombin the approach provides minimal impact on hemostasis. In vivo data imply that the inhibitor dissociates an antithrombotic effect from increased bleeding tendency.