Endothelium-Dependent Protein C Activation in Hereditary Protein C Deficiency

Abstract

Background

Protein C (PC) activation on endothelial cells is a critical antithrombotic mechanism. Hereditary PC deficiency (PCD), which is caused by mutations in the PROC gene, can predispose affected individuals to thrombophilia. Previous studies investigated activated protein C (APC) generation in PCD patients without including endothelial cells, which are essential for physiological PC activation. This study aimed to assess APC generation in PCD patients using a novel endothelial cell-based assay.

Methods

Plasma samples from 21 patients with 19 heterozygous PROC mutations (median PC level 58%) and 24 healthy controls were analyzed. Endothelium-dependent APC generation was initiated by overlaying plasma on human umbilical vein endothelial cells (HUVECs) and adding tissue factor (1 pmol/L). APC levels were quantified using an oligonucleotide-based enzyme capture assay. The area under the curve (AUC) was calculated to monitor cumulative APC formation over time. A calibration curve generated from wild-type PC in PC-deficient plasma established reference ranges.

Results

Mean peak levels of APC were significantly lower in PCD patients than in healthy controls (0.75 vs. 1.83 nmol/L, p = 2 × 10⁻¹⁰). The AUC APC was below the reference range in 8 of 21 (38%) patient samples, indicating disproportionately severe impairment in APC generation. The observed variability in APC generation suggests that endothelial contributions may identify functional differences undetected by standard PC activity or antigen assays.

Conclusion

This study introduces a novel endothelial cell-based APC generation assay, demonstrating the functional consequences of PROC mutations and providing insights into the regulation of APC generation, with potential applications in thrombosis risk assessment and personalized therapy.

Keywords

endothelial cells - endothelial protein C receptor - protein C deficiency - thrombomodulin - thrombophilia

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' Contribution

H.L.M and N.S. contributed equally to this study. H.R. and N.S. conceived and designed the study; H.L.M., B.Pe., H.R., S.R., and N.S. performed the experiments and collected data; H.L.M., H.R., and N.S. analyzed the data; and H.L.M, J.M., J.O., B.Pe., B.Pö., H.R., S.R., and N.S. drafted and edited the manuscript. All authors revised the manuscript, agreed with its content, and approved of submission.

^* These authors contributed equally to this study.

Publication History

Received: 28 January 2025

Accepted: 09 April 2025

Accepted Manuscript online:
10 April 2025

Article published online:
01 May 2025

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