Phänotypische Charakterisierung von Protein-C-Mangel verursachenden PROC-Mutationen mittels Untersuchung der Bildung von aktiviertem Protein C in einem endothelialen Ex-vivo-Modell
Nadine Schwarz (Bonn / DE), Hannah L. McRae (Bonn / DE), Jens Müller (Bonn / DE), Behnaz Pezeshkpoor (Bonn / DE), Johannes Oldenburg (Bonn / DE), Sara Reda (Bonn / DE; Köln / DE), Bernd Pötzsch (Bonn / DE), Heiko Rühl (Bonn / DE)
A broad variety of PROC gene mutations is known to cause hereditary protein C deficiency (PCD), a classical type of thrombophilia. In routine diagnostics, PCD is tested for by measuring functional protein C (PC) levels in plasma. These methods, however, do not allow for an assessment of how PROC mutations affect activated PC (APC) formation, which takes place on the endothelial surface. This study aimed to assess endothelium-dependent APC formation ex vivo in patients with PROC mutations.
Next Generation Sequencing was applied to identify PROC mutations in patients with PCD (n=25). Healthy blood donors (n=12) served as controls. Apart from reduced PC levels in the patient cohort, all study participants were required to have normal levels of other coagulation factors and inhibitors. For the ex vivo model, human umbilical vein endothelial cells (HUVECs) were cultured in 48-well plates and overlaid with defibrinated, citrated plasma obtained from patients and healthy controls. APC formation in the supernatant was induced by tissue factor, CaCl2, and phospholipids and monitored over 120 minutes via oligonucleotide-based enzyme capture assay. A dilution series of purified PC in PC-deficient plasma was assessed as a reference.
Endothelial APC formation was significantly reduced in PCD patients than in healthy controls (Fig. 1). PC plasma levels correlated strongly with the area under the curve of APC formation (AUC APC) in the ex vivo model (r=0.824). When comparing the AUC APC in study participants against the reference curve obtained with purified PC, it did not exceed a range of +/-25% in healthy controls. In the PCD cohort, the AUC APC lay below this range in 7/15 (47%) patients with missense mutations, 4/7 (57%) with nonsense mutations, and in all patients with deletions (n=2) or splice-site mutations (n=1) (Fig. 2). No patient showed a relative increase of the AUC APC when compared against the reference curve.
In the majority of patients with PCD, APC formation was disproportionately impaired in the ex vivo model. These data indicate that the assessment of residual PC levels might underestimate the prothrombotic potential in hereditary PCD. Moreover, different mutations might affect APC formation at different extents despite similar residual PC levels in routine tests. Further studies are warranted to understand the interplay between PROC variants and APC formation in PCD.
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