PB1080 - From clotting to calcification: Gla-domains as guardians of vascular remodeling

Background: The small family of vitamin K dependent (VKD) proteins, also known as Gla-proteins, play an essential role in human physiology and pathology. VKD proteins regulate coagulation, mineralization and cell proliferation. Their importance is illustrated by the fact that mouse knockout models or human deficiencies lead to embryonic lethality, accelerated ageing, or premature death. Surprisingly, while coagulation and mineralization have little in common, VKD proteins from both subfamilies affect the critical pathological process of vascular calcification. Mechanisms by which they inhibit calcification, however, are largely unknown.

Aims: Elucidate how Gla-containing VKD proteins regulate vascular calcification.

Methods: Uncarboxylated and carboxylated variants of VKD proteins were synthesized using chemical protein synthesis. Proteins were next tested for their ability to inhibit in vitro precipitation of calcium phosphate. Cellular calcification was evaluated by adding VKD proteins to human vascular smooth muscle cells (hVSMC) cultured under calcifying conditions. In addition, protein structure was analyzed using circular dichroism and NMR. Finally, bulk RNA sequencing was performed of cells treated with different VKD proteins.

Results: Three different VKD proteins were synthesized; matrix Gla protein (MGP), osteocalcin, and the Gla-domain of protein S (PSGla). We could show that carboxylation influenced the secondary structure of the VKD proteins. Additionally, uncarboxylated variants of these proteins were unable to inhibit in vitro calcium phosphate precipitation and calcification of hVSMCs. While all carboxylated variants inhibited (osteocalcin > protein S Gla > MGP) in vitro biophysical calcium crystal precipitation, significant differences were observed in biological calcification inhibition of hVSMCs. Both PSGla and MGP could fully inhibit calcification, yet RNA sequencing showed distinct differences in gene expression of hVSMCs treated with PSGla and MGP.

Conclusion(s): VKD proteins from the mineralization and coagulation subfamily interfere with vascular calcification. Differences in inhibitory activity may point to different modes of action. This is complemented by the differences in gene expression observed.