PB0335 - Characterization of a novel partially functional variant of tissue factor pathway inhibitor deficient in Kunitz type domain 2

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Vanessa Bröker

PhD student
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands

Co-Author(s)

AA

Asim C. Akbulut

Post Doc
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
ST

Stella Thomassen

Scientist
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
Elisabetta Castoldi

Associate Professor
Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands
Maastricht, Limburg, Netherlands
Kanin Wichapong, PhD

Senior Researcher
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands
DW

David Weber

Scientist
TRON – Translational Oncology University Medical Center of the Johannes-Gutenberg-University Mainz, Mainz, Germany
Mainz, Rheinland-Pfalz, Germany
Phillip Wenzel, Professor, MD

Deputy Director
University Medical Center Mainz
Mainz, Rheinland-Pfalz, Germany
Leon J. Schurgers, PhD

Professor
Department of BIochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
Tilman M. Hackeng, PhD

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Netherlands

Background: Tissue factor pathway inhibitor (TFPI) acts as a constitutive inhibitor of the extrinsic coagulation pathway by inhibition of the tissue factor/FVIIa/FXa complex. Mutations in TFPI are rare, but several SNPs have been identified that correlate with an increased risk of cardiovascular disease. TFPI also regulates cell signaling by protease-activated receptors. Here, we characterize an engineered TFPI variant deficient in Kunitz type domain 2 (KD2) function and identify possible non-canonical pathways affected by TFPI.

Aims: The aim of this project was to characterize a novel TFPI variant deficient in KD2 function expressed by endothelial cells in vitro.

Methods: The TFPI mutation NP_006278.1:p.(Tyr137_Arg140del) was introduced into induced pluripotent stem cells using CRISPR/Cas9 and cells were differentiated into endothelial cells. Intracellular and secreted protein was analyzed using Western blot and ELISA. In silico homology modelling elucidated the potential protein structure, while molecular dynamics modelling probed KD2 and FXa interaction stability. FVIIa inhibition (via KD1), FXa inhibition (via KD2), and prothrombinase inhibition (via C-terminus) were assessed using chromogenic assays. In addition, wild-type and mutant cells were analyzed for differential gene expression using bulk RNA sequencing.

Results: The mutant TFPI, characterized by a four amino acid deletion in KD2, was detectable in Western blots and ELISA. In silico analysis showed a modified FXa binding site in KD2, impeding stable binding to FXa. Functional assays demonstrated no FXa inhibition, while inhibitory activity for FVIIa and prothrombinase complex were retained. RNA sequencing showed differential expression of genes involved in diverse signaling pathways such as blood vessel remodeling and extracellular matrix organization.

Conclusion(s): We introduce a novel, partially functional TFPI variant expressed by cultured endothelial cells. While functionality of KD1 and C-terminus are retained, binding to FXa is abolished. RNAseq revealed KD2 mutation’s impact on diverse signaling pathways which indicates other, non-canonical functions of TFPI.