Abstract Number: PB0853
Meeting: ISTH 2020 Congress
Background: Increased rate of fragility fracture and decreased bone mineral density are now well-recognized comorbidities of hemophilia. In osteoblasts from FVIII-deficient mice, this pathology is reversible via in vivo or ex vivo factor replacement. Decreased thrombin generation was first suspected to cause this pathology; however, our work suggests thrombin impairs differentiated function in osteoblasts in vitro.
Aims: We seek to demonstrate decreased active protein C (APC) generation, not thrombin, is a driver of bone disease in hemophilia.
Methods: Bone marrow stromal cells are isolated from FVIII deficient mice and wild-type controls and enriched for an osteoblast phenotype. Human osteosarcoma-TE85 (HOS-TE85) cells are acquired from continuous cultures. Cells are cultured with α-MEM containing 10% FBS, penicillin, streptomycin, ß-glycerophosphate, and ascorbic acid. Calcified deposits are stained with alizarin red. DNA, protein, and alkaline phosphatase (ALP) in cell lysate are measured via spectrophotometric assays. Statistical comparisons are made using Student’s T-test.
Results: In murine In HOS TE85, thrombin causes a ~70% decrease in mineralization compared to controls (p < 10-4, Figure 1) while APC addition results in a ~30% increase (p < 0.05). APC also results in a ~100% increase in ALP activity (p < 10-4, Figure 1). Thrombin addition to murine osteoblast-like cells in vitro results in a ~70% decrease in ALP activity (p < 10-5, Figure 2).
Conclusions: Thrombin causes HOS TE85 and murine osteoblasts to fall behind controls in proliferation and ALP expression, ultimately failing to mineralize. APC, however, appears to upregulate osteoblast differentiated function, namely ALP expression and mineralization in HOS TE85. This suggests decreased APC generation is a cause of bone disease in hemophilia and a potential therapeutic target. These experiments are ongoing and new findings will be presented. Understanding this mechanism is vital to preventing bone loss in hemophilia without raising the risk of inhibitor formation or other complications.
To cite this abstract in AMA style:Goldscheitter G, Taylor JA, Klein RF. APC: A New Suspect in the Hunt for a Mechanism of Bone Disease in Hemophilia [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/apc-a-new-suspect-in-the-hunt-for-a-mechanism-of-bone-disease-in-hemophilia/. Accessed January 23, 2022.
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