Tissue Factor Pathway Inhibitor Modulates Endothelial and Vascular Smooth Muscle Cell Functions By Drifting Cellular Phenotypes

Background. Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway by inhibiting the TF/FVIIa/FXa complex. Beyond its established role, recent studies have suggested non-canonical functions of TFPI, including protection against atherogenesis in animal models. However, the underlying molecular mechanisms remain unclear.

Objective. This study aims to elucidate the impact of TFPI on proliferation and phenotypic switching of vascular smooth muscle cells (VSMC) and endothelial cells (EC) using a novel, genetically modified variant of TFPI.

Methods. Pluripotent stem cells were genetically modified to express a partially functional TFPI variant (NP_006278.1:(Tyr137_Arg140del)) and differentiated into endothelial and vascular smooth muscle cells. In silico models were employed to elucidate the binding of this modified TFPI to FXa. TFPI expression and secretion were quantified via Western blot and ELISA. Functionality of the mutant TFPI was assessed using chromogenic assays for FVIIa, FXa, and prothrombinase inhibition, as well as calibrated automated thrombography. Cellular proliferation in VSMC was quantified with an EdU- and an impedance-based assay. Extracellular vesicle secretion by contractile and synthetic VSMC was measured using a particle tracer device. VSMC were analyzed for the expression of contractility markers and vascular calcification in response to high calcium stimuli.

Results. EC-derived TFPI exhibited partial functionality, with Kunitz domain 1 and the C terminus remaining effective in inhibiting FVIIa and prothrombinase, respectively, while the functionality of Kunitz domain 2 was impaired. Inhibition of thrombin formation was also reduced with the mutant TFPI variant. Mutant EC displayed aberrant expression of EC markers CD31 and CD144, along with reduced TFPI secretion. VSMC with the TFPI mutation showed decreased TFPI expression and secretion, significantly reduced expression of contractility markers, and increased proliferation, which was reversible upon repair of the mutation or treatment with the FXa inhibitor rivaroxaban. Mutant, synthetic VSMC did not increase TFPI secretion, unlike wild-type cells, but had a 4.4-fold increase in vesicle secretion compared to wild-type cells. Calcification was significantly higher and faster in mutant VSMC, a process that could be partially reversed with extracellular recombinant TFPI.

Conclusion. Our findings demonstrate that a Kunitz domain 2-deficient TFPI variant affects the proliferation and phenotypes of endothelial and vascular smooth muscle cells in a FXa-dependent manner. Aberrant marker expression in EC could possibly lead to cell activation or endothelial-to-mesenchymal transition. The altered TFPI and vesicle secretion patterns in mutant VSMC indicate a stronger synthetic phenotype, corroborated by lower expression of contractile markers. These molecular changes could explain the enhanced calcification rate and increased vascular remodeling observed in previous animal studies. This study highlights the critical role of TFPI in maintaining vascular integrity and underscores the need for further research into its non-canonical functions.