PB0964 - Endothelial activation of protein C is disproportionately impaired in hereditary protein C deficiency

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Nadine Schwarz, MSc

PhD candidate
Institute of Experimental Haematology and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

Hannah L. McRae

PhD Candidate
Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany
JM

Jens Müller

Professor
Institute of Experimental Haematology and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg, MD

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
SR

Sara Reda

Researcher
Faculty of Medicine and University Hospital Cologne
Köln, Nordrhein-Westfalen, Germany
BP

Bernd Pötzsch

Professor
Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany
HR

Heiko Rühl

Privatdozent
Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany

Background: Hereditary protein C deficiency (PCD) is a thrombophilia characterized by reduced functional protein C (PC) levels in plasma. As a consequence, activated protein C (APC) formation, which takes place on the endothelial surface, is impaired. However, it is not clear to what degree different PROC gene mutations affect endothelial PC activation.

Aims: To evaluate endothelial cell-dependent APC formation in plasma from PCD patients.

Methods: PROC variants were identified by Next Generation Sequencing. In PCD patients (n=22) and healthy controls (n=12), plasma levels of coagulation factors and inhibitors were measured. Human umbilical vein endothelial cells (HUVECs) were cultured in 48-well plates and overlaid with citrated and defibrinated patient plasma. APC formation in the supernatant was induced by addition of 1 pmol/L tissue factor, 16.6 mmol/L CaCl2, and 4 µmol/L phospholipids and measured over 120 minutes via oligonucleotide-based enzyme capture assay (OECA). A dilution series of purified PC in PC-deficient plasma was assessed to serve as reference.

Results: There were fourteen unique PROC variants amongst patients, four carried 512C>T and in four patients, no mutation could be identified. Functional PC levels ranged from 35.5-74.6% and were significantly lower as compared to controls. Levels of protein S, antithrombin, and coagulation factors in plasma did not differ between cohorts. Endothelial APC formation was significantly lower in PCD patients than in healthy controls (Figure 1). The area-under-the-curve of endothelial-dependent APC formation was compared against the reference curve. While none of the healthy controls laid below 25% of the reference curve, 11/22 PCD patients showed less APC formation (Figure 2).

Conclusion(s): In 50% of PCD patients, APC formation was disproportionately impaired. This indicates that the assessment of residual PC levels might underestimate the prothrombotic potential in hereditary PCD. Further studies are warranted to understand the interplay between PROC variants and APC formation in PCD.