PB1087 - Beyond bleeding: Exploring coagulation factors and cytokines in bone health in vitro

Background: Hemophilia A (HA) patients exhibit low bone mineral density (BMD), independent of conventional risk factors [1]. Recent research suggests that coagulation factor VIII (FVIII) may influence bone health directly or indirectly [2], prompting the need for deeper understanding.

Aims: This study investigates the complex interplay among coagulation factors, cytokines and bone health in HA patients. Using cell culture experiments with osteoblasts and osteoclasts, the ultimate goal is to establish potential therapeutic strategies for patient well-being.

Methods: Human and murine osteoblast cell lines, along with human osteoclasts, were cultured with human coagulation factors (FVIII, FIX, FX, vWF, vWF-FVIII complex, thrombin and FVIII-thrombin; 1 U/ml each) and cytokines (IL-6 and TNF-α; 50 ng/ml each).
Assessments included cell viability (CCK8 assay), osteoblast mineralization (alizarin red S assay) and osteoclast activity (tartrate-resistant acid phosphatase (TRAP) activity measurement).
Student´s t-test was used, considering p-values < 0.05 as significant.

Results: Coagulation factors had distinct effects on human and murine osteoblasts: Compared to controls, thrombin reduced cell viability of murine osteoblasts by 18%, whereas vWF, vWF-FVIII, FIX, FVIII-thrombin, and FX increased viability in human cells by 33%, 24%, 26%, 23% and 31% respectively.
Mineralization of murine osteoblasts was unaffected, but mineralization of human osteoblasts decreased with vWF, vWF-FVIII, thrombin and FX by 44%, 37%, 35% and 56%. In human osteoclasts, FX led to increased TRAP activity by 16%.
TNF-α reduced viability in murine and human osteoblasts (29% and 53%) and mineralization of murine osteoblasts increased (119%). TNF-α and IL-6 reduced osteoclast TRAP activity by 50% and 60%.

Conclusion(s): This study reveals distinct responses of human and murine osteoblasts to human coagulation factors, emphasizing FX´s significant influence on osteoblasts and osteoclasts. Further investigations of these interactions and the impact of inflammatory cytokines are crucial. Understanding the interplay between coagulation, inflammation and bone metabolism is pivotal for future therapeutic approaches.