Endothelial Mechanisms Modulate the Prothrombotic Phenotype of Factor V Leiden Carriers: Evidence from an Ex Vivo Model

Introduction: The factor V Leiden (FVL) mutation leads to activated protein C (APC) resistance and shows a highly variable clinical expressivity. Our previous in vivo studies demonstrated that asymptomatic FVL carriers exhibit higher APC formation rates in response to extrinsic coagulation activation compared to those with a history of venous thromboembolism (VTE) [1]. This study aimed to explore the potential role of endothelial cells in modulating the APC response in FVL carriers using an ex vivo model of the protein C (PC) pathway, incorporating endothelial colony-forming cells (ECFCs) and autologous plasma.

Method: ECFCs were isolated from FVL carriers with our without a history of VTE (VTE+, n=8; VTE-, n=9), or from healthy controls (n=7). Confluent monolayers of ECFCs were overlaid with defibrinated autologous plasma and coagulation was induced by addition of CaCl₂ and tissue factor (1 pmol/L). Thrombin and APC formation were measured over time using aptamer-based enzyme capture assays. To analyze endothelial cell-specific effects, cross-over experiments combining pooled plasma from FVL VTE- and VTE+subjects with each FVL VTE- and VTE+ECFCs were performed.

Results: In the cohorts of FVL VTE-, FVL VTE+, and non-FVL carriers, success rates of ECFC isolation and cell cultivation parameters were comparable and the endothelial phenotype was confirmed by expression of endothelial cell-specific surface markers measured by flow cytometry. In the autologous ECFC-based model of the PC pathway, the ratio between the area under the curve (AUC) of APC and thrombin formation (AUC APC/AUC thrombin) was significantly higher in FVL VTE- compared to VTE+(P=0.026) and to non-FVL carriers (P=0.007). In agreement with the autologous approach, the combination of FVL VTE- cells and VTE- plasma yielded a higher ratio of AUC APC/AUC thrombin than the combination of FVL VTE+cells and VTE+plasma (P=0.0161). When performed with interchanged plasma components, i.e. combining FVL VTE- cells and VTE+plasma, respectively FVL VTE+cells and VTE- plasma, the ratio of AUC APC/AUC thrombin in the FVL VTE- cohort and in the FVL VTE+cohort remained unchanged ([Fig. 1]).

Fig. 1 Cross-over experiments using individual ECFCs and pooled plasma from FVL VTE- and VTE+individuals. The formation of thrombin and APC was studied in the ex-vivo PC pathway model combining ECFC from FVL carriers with a history of venous thromboembolism (FVL VTE+, n=8, red symbols) and asymptomatic FVL carriers (FVL VTE-, n=9, blue symbols) with pooled plasma from FVL VTE+subjects (red columns) or FVL VTE- subjects (blue columns). The AUC APC / AUC thrombin ratio was compared using the Student’s t-test.

Conclusion: Consistent with previous in vivo experiments, the ex vivo APC response to thrombin formation was impaired in FVL carriers with a history of VTE in comparison to asymptomatic FVL carriers. Cross-over experiments suggest that endothelial cell-specific mechanisms contribute to the observed differential APC response and in FVL carriers rather than plasmatic factors. Further studies are warranted to identify these mechanisms that might modulate the clinical expressivity of FVL.

 

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Conflict of Interest:

None

• References

• 1 Rühl H, Berens C, Winterhagen FI, Reda S, Müller J, Oldenburg J, Pötzsch B.. Increased Activated Protein C Response Rates Reduce the Thrombotic Risk of Factor V Leiden Carriers But Not of Prothrombin 20210GA Carriers. Circ Res 2019; 125: 523-534

Publication History

Article published online:
13 February 2025