PB1436 - PAR1 Knockout induced Pluripotent Stem Cell-derived Vascular Smooth Muscle Cells display higher Tissue Factor-dependent Thrombin Generation compared to Wild Type.

Background: Vascular smooth muscle cells (VSMCs) undergo phenotypic switching that influences vascular remodeling. Activation of protease-activated receptors (PARs) may influence phenotypic switching of VSMCs. While absence of the thrombin receptor (PAR1) in mouse embryos results in partial lethality with cardiovascular failure, the precise role of PAR1 in vascular development and vascular remodeling is yet to be fully understood.

Aims: Primary aims of this study are to assess the effect of PAR1 deficiency on VSMCs differentiation as well as the effect of PAR1 on thrombogenicity and phenotype switching of VSMCs.

Methods: PAR1 was knocked out in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 technology. PAR1 KO and control iPSCs were differentiated into induced VSMCs (iVSMCs). Differentiated iVSMCs were characterized by immunocytochemistry of canonical VSMC markers. Calibrated Automated Thrombinoscope (CAT) was used to assess thrombin generation of PAR1 KO and control iVSMCs. Additionally, PAR1 KO and control iVSMCs were induced to phenotypic switching by PDGF-BB whereafter VSMCs marker expression was measured by immunocytochemistry and western blotting.

Results: PAR1 KO-iVSMCs displayed a fully mature phenotype comparable to control iVSMCs. However, PAR1 KO iVSMCs showed higher Tissue Factor (TF)-dependent thrombin generation compared to control. Furthermore, TF expression was significantly increased in PAR1 KO iVSMCs compared to control. While both control and PAR1 KO VSMC marker expression significantly decreased in response to PDGF-BB, TF expression only increased in control iVSMCs treated with PDGF-BB.

Conclusion(s): PAR1 neither has an effect on canonical iVSMC markers in differentiation from iPSCs, nor affect expression of VSMC markers during phenotype switching. However, increased TF expression in the PAR1 KO iVSMCs suggests that the absence of PAR1 may result in increased thrombogenicity.