Assessment of cellular and plasmatic determinants of the APC response to thrombin in factor V Leiden carriers using an endothelial cell-based ex vivo model

Background

The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Aim of this study was to investigate potential modulating factors of the APC response in an ex vivo model of the PC pathway using endothelial colony forming cells (ECFCs) and autologous plasma.

Methods

ECFCs were isolated from FVL carriers with (VTE+) or without (VTE-) previous VTE and healthy controls (n=7 each). After being overlaid with autologous defibrinated plasma, thrombin formation was initiated by addition of tissue factor (1 pmol/L). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs). Additionally, cross-over experiments with FVL and non-FVL cells/plasma were conducted as well as measurements of ECFC-dependent APC formation in HEPES buffer containing thrombin and the zymogen protein C (PC). Thrombomodulin and endothelial protein C receptor (EPCR) expression were quantified in cell-ELISAs. Inactivation kinetics of exogenously added APC were monitored in plasma by OECA.

Results

Thrombin formation rates did not differ, but APC formation rates in plasma were higher in the FVL VTE- cohort than in the FVL VTE+ cohort (P=0.011) and healthy controls (P=0.003) as indicated by the ratio between the area under the curve (AUC) of APC generation to the thrombin AUC. Cross-over experiments with non-FVL cells and FVL VTE+ plasma yielded increased APC formation in comparison to the autologous approach (P=0.009), indistinguishable from FVL VTE- plasma. The APC AUC of all other combinations and the thrombin AUC did not differ. APC formation in the purified system, APC inactivation kinetics, thrombomodulin and EPCR expression as well as plasma levels coagulation factors and inhibitors did not differ significantly between cohorts.

Conclusion

Consistent with previous in vivo experiments, the APC response to thrombin formation was impaired in FVL carriers with a history of VTE in comparison to asymptomatic FVL carriers. However, this difference disappeared when FVL cells were replaced by non-FVL cells in cross-over experiments, suggesting that endothelial cell-specific factors contribute to the APC response and thus to thrombotic risk in FVL carriers. Further studies are warranted to identify these factors.

Offenlegung Interessenkonflikt:

J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co., Ltd., CSL Behring, Freeline, Grifols, LFB, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd., Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. The other authors declare no competing financial interests.