Unravelling the Role of Coagulation Factors in Hemophilia A-Associated Bone Metabolism

Introduction: Patients with hemophilia A not only experience spontaneous bleeding into joints or soft tissues, but also face an increased risk of low bone mineral density (BMD), which appears to be independent of commonly accepted risk factors [1] [2]. Emerging evidence suggests that coagulation factor VIII (FVIII) may play a role beyond the coagulation system, potentially influencing bone metabolism through direct or indirect mechanisms. However, the relationship between FVIII and bone health remains poorly understood. Our cell culture experiments using human osteoblasts and osteoclasts aim to elucidate these mechanisms, providing insight into the pathophysiology of bone metabolism in hemophilia A.

Method: In this study, human osteoclasts were differentiated from monocytes isolated from peripheral blood. These osteoclasts, along with a human SaOs-2 osteoblastic cell line, were incubated with various coagulation factors (FVIII, FIX, FX, vWF, vWF-FVIII, thrombin and FVIII-thrombin; 1 U/ml each) to assess their effects on cell viability and specific cellular functions. Osteoblast mineralization was evaluated using the Alizarin red S assay [3], and xylenol orange fluorescent staining. Tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts was quantified to evaluate osteoclast function. Cell viability was assessed using the cell counting kit 8 (CCK-8).

Results: Cell viability of human SaOs-2 osteoblasts slightly increased in the presence of FVIII or FIX (by 13% and 11% respectively), suggesting a potential role in supporting osteoblast survival. However, among all tested coagulation factors, only coagulation factor X significantly reduced mineralization relative to cell viability by 57%. Dose-response experiments with FX confirmed this finding, with mineralization in SaOs-2 osteoblasts decreasing by 31% at 0.25 U/ml to 96% at 4 U/ml. These findings were corroborated using xylenol orange fluorescent staining. In contrast, none of the coagulation factors influenced the cell viability or TRAP activity of human osteoclasts, indicating a selective effect on osteoblast function.

Conclusion: These findings indicate that FX exerts a dose-dependent inhibitory effect on osteoblast mineralization, highlighting a previously unrecognized role of this factor in bone metabolism. Since FX must be activated to FXa during the coagulation cascade - a process disrupted in patients with hemophilia A – this pathway may play a critical role in bone health. The significant influence of FX on osteoblast mineralization highlights the need for further investigation into the relationship between coagulation and bone metabolism in hemophilia A patients in order to optimize treatment options.

Publication History

Article published online:
13 February 2025

• References

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