The 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. Increased activated protein C (APC) formation in response to thrombin formation has been observed in asymptomatic FVL carriers in vivo. Here we further explored this association using a recently developed endothelial colony forming cell (ECFC)-based ex-vivo model. Methods ECFCs and citrated plasma were obtained from FVL carriers with/without previous venous thromboembolism (VTE+/-, n=7 each) and seven healthy controls. Coagulation was activated by 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 (AUC) calculated. Additionally, inhibition kinetics of exogenously added APC and APC sensitivity of the prothrombinase complex were measured in plasma. Expression of thrombomodulin and endothelial protein C receptor (EPCR) on ECFCs was assessed using cell-based enzyme-linked immunosorbent assays. Results In autologous plasma on ECFCs, the APC response to thrombin formation (AUC APC/AUC thrombin), was higher in FVL VTE-than FVL VTE+ patients (0.138 versus 0.028, P =0.026). APC inactivation kinetics, APC sensitivity, and thrombomodulin/EPCR expression on ECFCs did not differ between these cohorts and compared to healthy controls. Cross-over experiments with plasma from FVL VTE- and FVL VTE+ patients on non-FVL ECFCs yielded indistinguishable results. In contrast, in normal plasma on FVL VTE-ECFCs the APC response remained significantly higher than on FVL VTE+ ECFCs (0.052 versus 0.022, P =0.011). Conclusions Consistent with results from previous in vivo experiments, APC response rates to thrombin formation were higher in asymptomatic FVL carriers compared to those with previous VTE. Our observations suggest that this increased APC response is driven by the endothelium. Further studies are warranted to elucidate yet unknown endothelial mechanisms that might modulate the clinical expressivity of FVL.