Hemophilia A and B are inherited bleeding disorders that result in impaired thrombin generation. Correction of bleeding in hemophilia patients is best achieved by the therapeutic administration of factor VIII (FVIII) or factor IX (FIX), the respective missing coagulation factors. Replacement therapy is however complicated by the short half-lives and immunogenicity of the therapeutic molecules. Alternatives to replacement therapy include bypassing agents such as activated factor VII or Emicizumab, a bispecific monoclonal antibody (mAb), or molecules that neutralize natural anticoagulant proteins such as TFPI, antithrombin or activated protein C. These molecules are delivered systemically and do not accumulate at the site bleeding. Hence, they do not fully compensate for the missing coagulation factor and may trigger potentially life-threatening thrombotic events. AAV-based gene therapy has brought encouraging results for hemophilia but is restricted to patients who have not developed anti-drug antibodies (ADA) to the therapeutic coagulation factor or to AAV, and may be associated with liver toxicity, at least in HA patients. There is thus a need for novel therapeutic molecules that specifically reinforce thrombin generation at the very bleeding site and only at the time of vessel injury. Our previous work demonstrates the importance of the natural anticoagulant protease nexin-1 (PN-1, or serpinE2) as a major but somewhat ignored regulator of thrombin. We demonstrated that blocking PN-1 corrects the hemophilia phenotype. PN-1 is expressed ubiquitously including by platelets. Our working hypothesis proposes that the targeted neutralization of platelet-released PN-1 should reinforce thrombin generation at the very bleeding site and only when it is required. To validate our hypothesis, we will develop an array of bispecific chimeric molecules made of different nanobodies (VHH), which we refer to as Nanochimeras: a pole of the Nanochimeras will neutralize PN-1 activity; the second pole will bind GPVI, a transmembrane glycoprotein protein specifically expressed by platelets and megakaryocytes. The Nanochimeras will thus concentrate PN-1-neutralizing VHHs at the platelet surface. Some of the Nanochimeras will be fused to the Fc portion of the human (hu) IgG1 to confer increased in vivo half-life, and potentially longer therapeutic efficacy. Our preliminary data include 3 VHHs that neutralize hu and mouse PN-1 to different extents, 6 VHHs that bind two different epitopes on huGPVI with nanomolar affinities and do not activate platelets or prevent collagen-induced platelet activation, and the proof that huPN-1 is functional in mouse plasma. We also started backcrossing knock-in huGPVI transgenic (Tg) mice on FVIII-deficient (HA) mice. Using these molecules and the technologies already in place in the two partner laboratories, we will pursue the following objectives: i) generate VHHs binding huGPVI with subnanomolar affinities that do not interfere with platelet functions, and VHHs with optimized PN-1 neutralizing activity, ii) generate and functionally validate recombinant bispecific Nanochimeras, iii) generate and validate a novel preclinical model of huPN-1 Tg huGPVI Tg HA mice, and iii) determine the in vivo half-life of the Nanochimeras, confirm their therapeutic potential in correcting thrombin generation, delineate the extent of thrombus formation and predict their immunogenicity. While the proof of concept will be obtained in a preclinical model of HA, our Nanochimeras will be suitable to patients with HA or HB, irrespective of whether they have developed ADA to FVIII or FIX, patients with other bleeding disorders characterized by insufficient thrombin generation, as well as HA patients who experience breakthrough bleeds while treated with Emicizumab.