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Abstract Introduction: Hemophilia A (HA) is a severe bleeding disorder complicated by arthropathy of the large joints, due to synovial iron deposition, inflammation, impaired bone remodeling, and loss of bone mineral density. Arthropathy is caused by repeated episodes of joint bleeding but can also occur in patients without clinically recognized hemarthrosis, likely due to subclinical bleeding. Factor VIII (FVIII) knockout (F8-/-) mice develop hemophilic arthropathy following injury and have been used to study its pathogenesis. However, this mouse model has not been suitable to study the effects of FVIII prophylaxis as mice develop anti-FVIII antibodies following repeated injections. Aims: To study effects of secondary FVIII prophylaxis after joint bleeding, we developed a novel HA mouse model that lacks inhibitor formation (F8-/-Ighm-/-, or DKO). Methods: DKO mice were randomized into 3 experimental groups (Gr.): 1) no treatment, 2) single rFVIII injection (simoctocog alfa, 200 IU/kg, day 0), 3) initial rFVIII injection (200 IU/kg, day 0), followed by secondary prophylaxis with 100 IU/kg for 3 weeks (daily on days 1-4 and then 3 times per week). Mice were subjected to a unilateral joint injury in the left knee (day 1) and monitored for knee swelling and running behavior. Uninjured and untreated mice served as controls. Mice from Gr.1-3 were sacrificed at days 4, 11 or 21 for the collection of blood, spleen, and hind limbs. Thrombin generation assay and measurement of FVIII levels were performed on plasma to assess the pharmacodynamic effect of prophylaxis. Micro-computed tomography (µCT) imaging was performed on the tibia in which cortical thickness (Ct.Th), bone volume fraction (BV/TV), and trabecular thickness (Tb.Th) were evaluated. Histological analyses involved Hematoxylin & Eosin (H&E) and Safranin O staining to evaluate the Valentino joint score and the Osteoarthritis Research Society International (OARSI) score. Bulk mRNA-sequencing was performed on splenocytes to capture a systemic response profile to injury and treatment. Results: Mice receiving secondary rFVIII prophylaxis (Gr.3) exhibited increased thrombin generation compared to no treatment (Gr.1) or single rFVIII treatment (Gr.2), despite FVIII trough levels being near the detection limit in all groups. Prophylaxis prevented swelling of the injured knee on days 4, 11, and 21 and preserved normal running behavior. µCT analysis revealed distinct cortical and trabecular bone alterations in Gr.1 and 2, which were effectively prevented in Gr.3. Ct.Th ratios between injured/uninjured limbs showed time- and treatment- dependent changes, with Gr.1 and 2 exhibiting asymmetric thinning, increased cortical porosity, and extracortical bone growth compared to Gr.3 and control mice. Trabecular BV/TV progressively declined in Gr. 1 and 2, compared to Gr.3 across all timepoints. Tb.Th remained stable in Gr.3, but showed loss in Gr. 1 and 2. Injured knees showed higher Valentino and OARSI scores compared to the contralateral uninjured side. Prophylaxis prevented the deposition of erythrocytes and hemosiderin, while synovial hyperplasia and cartilage damage were not prevented. In mice receiving single rFVIII treatment (Gr.2), there was a trend to increased erythrocyte and hemosiderin deposition over time, which was not observed in Gr.3. Transcriptomic profiling demonstrated a systemic response to injury, particularly on day 4, with Gr.3 showing gene expression pattern similar to uninjured controls, while Gr.1 and 2 showed a strong response involving a broad range of biological pathways. More subtle differences were observed comparing only mice receiving secondary prophylaxis (Gr.3) to uninjured control mice on day 21, involving genes implicated in systemic inflammatory responses. Conclusion(s): Joint bleeding in HA results in a multifactorial pathological response involving deposition of hemosiderin, synovial inflammation, and changes in bone architecture. Secondary FVIII prophylaxis, but not a single FVIII treatment around the time of injury, prevented overt and microscopic bleeding and preserved bone structural changes in response to injury. Synovial hyperplasia and cartilage damage were not completely prevented. This study highlights the utility of the DKO mouse model for prophylactic FVIII therapy evaluation and testing next-generation therapies, offering robust evaluation of physical performance, joint pathology, inflammation, bone health recovery and systemic response.