Enhancing joint and bone health outcome in Hemophilia A by protein S inhibition

Abstract

Abstract Background: Hemophilia A (HA) is a genetic disorder caused by deficient coagulation factor VIII, leading to impaired clotting. Current treatments improve outcomes but do not fully prevent spontaneous or subclinical bleeds, especially in patients on prophylaxis. These unresolved bleeds can cause progressive joint damage and arthropathy, highlighting the need for alternative therapies. We previously identified protein S (PS), a key coagulation regulator, as a target to restore hemostatic balance in HA. We developed a liver-targeted small interfering RNA (PS-siRNA) therapy that selectively reduces hepatic PS while preserving extrahepatic levels to minimize thrombotic risk. In HA mouse models, this approach restored hemostasis and prevented joint bleeding. Synovial analysis from people with HA showed lower PS levels in those on prophylaxis compared to on-demand therapy. Beyond coagulation, PS also modulates immune signalling, inflammation, and bone remodelling via Mertk and Tyro3 receptors tyrosine kinase. Aim: To explore how reduced protein S (PS) levels help protect joints from blood-induced inflammation and support bone health in HA mice. Methods: Male and female mice aged 16–24 weeks were used, including wild-type (WT), HA (F8-/-), and PS-deficient HA mice (F8-/-Pros1-/- and F8-/-Pros1+/-). A recurrent hemarthrosis (RH) model was established by inducing three knee joint injuries at 7-day intervals. In a separate cohort, 20 µL of autologous blood was injected into the intra-articular space of the knee, and joint diameter and morphological changes were assessed 7 days post-injection. Phagocytosis assays were performed using bone marrow-derived macrophages (BMDMs) exposed to senescent red blood cells (RBCs). Synovial tissue from knee joints was collected for RNA sequencing. Bone health was evaluated using whole-body dual-energy X-ray absorptiometry and micro-computed tomography (µCT) of proximal tibia. Additionally, a cohort of F8-/- mice was treated with PS-siRNA or vehicle control prior to µCT analysis. Results: RNA sequencing of injured synovium revealed that F8-/-Pros1-/- mice showed upregulation of gene subsets involved in phagocytosis and efferocytosis compared to F8-/-mice. In vitro, phagocytosis of senescent RBCs by BMDMs was enhanced in F8-/-Pros1-/- (18±9%) compared to F8-/- (7±2%) but remained lower than WT (35±6%; p=0.03, n=6). Seven days after intra-articular blood injection, joint swelling and persistent RBCs deposition were observed in F8-/- mice but not in WT or F8-/-Pros1-/- mice, suggesting improved clearance and reduced inflammation with PS deficiency. Following RH, genes associated with inflammation resolution (e.g., Mrc1, Trem2, Cx3cr1, Socs3, CD81) were upregulated in WT and F8-/-Pros1-/- but not in F8-/- mice. At baseline, F8-/- synovium showed increased expression of bone remodeling and osteoclast differentiation markers (RANKL, Csf1, Ccl3, IL20, Ifng), indicating imbalanced osteogenesis. At 16 weeks of age, F8-/-Pros1-/- mice displayed significantly higher bone mineral density (BMD: 0.089±0.002 vs 0.085±0.002 g/cm²; p<0.0001) and bone mineral content (BMC: 0.85±0.08 vs 0.73±0.08 g; p=0.009, n=8–11) compared to F8-/- mice. µCT analysis showed increased trabecular connectivity density (Conn.D: 157±38 vs 104 ± 13 mm³; p = 0.02) and reduced trabecular spacing (Tb.Sp: 0.194±0.01 vs 0.233±0.01 mm; p=0.0057, n=4–7). Conn.D remained consistently higher in F8-/-Pros1-/- mice at 22 weeks (102±26 vs 72±20 mm³) and 24 weeks (70±6 vs 49±8 mm³; p=0.02, n=3–4). F8-/-Pros1+/- mice also showed improved BMD (0.08±0.002 g/cm²; p=0.04) and BMC (0.84±0.05 g; p=0.01, n=7), with a trend toward increased Conn.D (145±22 mm³; p=0.1) and reduced Tb.Sp (0.199 ± 0.009 mm; p=0.01). Preliminary µCT data in F8-/- mice treated with PS-siRNA (10 mg/kg subcutaneously, 10 weeks) showed improved bone microarchitecture compared to vehicle-treated controls (Tb.Sp: 0.31±0.07 vs 0.37±0.08 mm; Conn.D: 59±55 vs 41±27 mm³), supporting the therapeutic potential of PS inhibition in preserving joint and bone health in HA. Conclusion: Our findings show that partial reduction of PS levels (~50%) improves joint integrity and bone health in HA mice. Ongoing studies are evaluating the therapeutic potential of PS-siRNA treatment to enhance skeletal outcomes in HA, laying the foundation for future clinical translation.