Joint damage in hemophilia results from repeated intra-articular bleeds (IAB). With no treatment available to reduce blood-induced synovitis, bleeding prevention is crucial to maintain joint health, but a more targeted therapy is needed. We reported that genetic ablation of protein S (PS) protects hemophilic mice against bleeding, particularly IAB, and that PS reduction by a small interfering RNA prevents acute hemarthrosis in mice with hemophilia A (HA) and improves hemostasis in non-human primates with acquired hemophilia. Further, PS reduction enhanced thrombin generation in human models of HA ex vivo. Here, we characterized the effect of GA of PS on joint health using a mouse model of chronic hemarthrosis (CH).

HA (F8-/-), HA with genetic ablation of PS (F8-/-Pros1-/-) and WT mice were subjected to CH by 3 joint bleeds induced at 7 days apart. Histology, immunostaining, electron microscopy (EM), joint lavage analyses and RNA sequencing of the synovium were performed in steady-state (SS) and CH.

During CH, joint diameters increased in F8-/- but not in F8-/-Pros1-/- and WT mice. Histology confirmed this result with neither bleeding nor synovitis in F8-/-Pros1-/- mice (Fig.1A). Iron deposition was minimal in F8-/-Pros1-/- with no cartilage erosion. The lining layer (LL) integrity was preserved in F8-/-Pros1-/- with CH as shown by histology and EM. In CH, macrophages were present in the LL of F8-/-Pros1-/- and WT mice, and the subLL of F8-/- mice (Fig.1B). Inflammatory cytokines level was lower in F8-/-Pros1-/- than in F8-/- joint lavage (Fig.1C). In SS, mRNA coding for proteins involved in bone resorption were decreased while those coding for cell communication and bone formation were increased in F8-/-Pros1-/- versus F8-/-mice. Post-CH, F8-/-Pros1-/- mice displayed a positive regulation of genes involved in macrophage activation, bone regeneration and tissue remodelling, and a negative regulation of genes involved in angiogenesis and fibroblast growth. Differently to F8-/- mice, F8-/-Pros1-/- mice synovium displayed increased expression of pleiotrophin (PTN) (Fig.1D), a secreted growth/differentiation factor involved in bone development and tissue repair. PTN was strongly expressed in synovium LL of F8-/-Pros1-/- compared to F8-/- and WT mice (Fig.1E).

These data identify PS as an important modifier of joint health in HA, at least partly via PTN.