Abstract
Factor VIII (FVIII) has recently received increasing attention for its roles beyond the coagulation cascade. Indeed, FVIII has been shown to regulate the endothelial cell function, modulate the immune cell reactivity and influence bone mineral density (BMD). Patients with hemophilia A (HA) show reduced BMD, which correlates with increased fracture risk suggesting that FVIII may directly interact with the bone cells involved in the maintenance of the bone equilibrium, namely osteoblasts and osteoclasts. Consequently, the absence of FVIII might affect the differentiation and activity of these cells, and/or alter their interplay, ultimately affecting bone homeostasis. Here we investigated the effect of FVIII in modulating the in vitro differentiation of murine osteoblasts and how osteoclastogenesis is influenced by the altered activity of hemophilic osteoblasts.
Methods. Osteoblast precursors from 3-week-old wild-type (wt) and HA mice were obtained after collagenase digestion of bones while bone marrow (BM)-derived myelomonocytic progenitors (MMPs) were collected by flushing the bones of 8-week-old animals. Osteoblasts were differentiated with ascorbic acid and β-glycerophosphate while osteoclasts were treated with M-CSF and RANKL. During osteoblast differentiation, cells were treated with varying concentrations of simoctocog alfa (recombinant FVIII [rFVIII]), FIX, or hirudin (an inhibitor of thrombin activity). In vitro differentiation of osteoclasts from the MMPs was performed in co-culture with mature osteoblasts or in the presence of conditioned medium collected from their culture. Cell maturation and activity was assessed by specific staining (e.g. with Alizarin- Red-S [ARS] and tartrate-resistant acid phosphatase [TRAP]) and by quantification of cell-specific genes.
Results.In vitro differentiation and mineralization of HA osteoblasts displayed a basal deficiency compared with wt samples, which was restored by rFVIII supplementation, starting from the concentration of 10 ng/ml. Interestingly, rFVIII promoted differentiation of both HA and wt osteoblasts in a dose-dependent manner, with a proportionally stronger effect on HA cells. This improvement was independent of thrombin activity since co-treatment with hirudin did not affect the FVIII-induced differentiation. FIX alone showed no impact on osteoblast differentiation, confirming that the FVIII effect was not mediated by an in vitro activation of the coagulation cascade. The number of osteoclasts generated in vitro by both HA and wt MMPs was significantly higher in presence of HA osteoblasts than in co-culture with wt osteoblasts. The latter promoted similar osteoclastogenesis between HA and wt samples. Similar results were obtained using conditioned medium from osteoblast culture, suggesting that soluble factors secreted from HA osteoblasts were responsible for the increased osteoclast differentiationobserved in vitro.
Conclusions. FVIII promotes in vitro osteoblast differentiation independently of coagulation cascade activation and thrombin generation, confirming a non-hemostatic role for FVIII in bone cell biology. FVIII deficiency alters both osteoblast differentiation and activity which in turn promotes osteoclastogenesis. This imbalance may lead to increased bone resorption observed in patients with HA, contributing to skeletal fragility as a clinical comorbidity. These findings suggest that FVIII replacement therapy in HA patients may offer benefits beyond hemostatic control, potentially enhancing bone health and reducing fracture risk. Further investigation into the molecular mechanisms linking FVIII to bone homeostasis could inform new strategies for managing hemophilia-associated comorbidities.
