Endothelium Modulates the Prothrombotic Phenotype of Factor V Leiden: Evidence From an Ex Vivo Model.

Abstract

Background: Clinical expressivity of the thrombophilic factor V Leiden (FVL) mutation is highly variable. Recently, we demonstrated an increased APC (activated protein C) response in asymptomatic FVL carriers compared with FVL carriers with a history of venous thromboembolism (VTE) after in vivo coagulation activation. Here, we further explored this association using a recently developed ex vivo model based on patient-specific endothelial colony-forming cells (ECFCs).

Methods: ECFCs and citrated plasma were obtained from FVL carriers with previous VTE (VTE+, n=9), FVL carriers without previous VTE (VTE-, n=8), and 7 healthy controls. Coagulation was activated by TF (tissue factor) in defibrinated recalcified plasma added to confluent cell cultures. Thrombin and APC concentration were measured over time, and the respective areas under the curve were calculated. Additionally, inhibition kinetics of exogenously added APC, APC inhibitor levels, and APC sensitivity ratios were measured in plasma. Expression of TM (thrombomodulin) and EPCR (endothelial protein C receptor) on ECFCs was assessed using cell-based ELISAs.

Results: In autologous plasma on ECFCs, the APC response (area under the curve APC/area under the curve thrombin) was higher in FVL VTE- than in FVL VTE+ patients (0.138 versus 0.028; P=0.026). APC inhibitor levels, APC inactivation kinetics, and APC sensitivity ratios did not differ between cohorts. Crossover experiments, which combined pooled plasma from FVL VTE- patients with FVL VTE+ ECFCs in the ex vivo model, and vice versa, showed increased APC response rates when FVL VTE- ECFCs were used, regardless of the plasma component. In cell-based ELISAs, TM and EPCR expression showed no significant difference.

Conclusions: Although the FVL gene product induces an almost identical APC resistance phenotype in plasma, the endothelial cell-dependent APC response rates differ significantly, with a higher APC response in asymptomatic FVL carriers. Further studies are warranted to elucidate the disease-modulating role of the endothelium in FVL carriers at the molecular level.