Assessment of Protein C Anticoagulant Pathway by Thrombin Generation Assay in the Presence of Endothelial Cells

Abstract 3394

The generation of thrombin is the pivotal event in the process of blood coagulation. In vivo, thrombin generation is regulated by cooperation between the vascular endothelium and the pro- and anti-coagulant systems in blood, such as the thrombin/endothelial cell-dependent activation of the protein C anticoagulant pathway that ultimately leads to inactivation of factors Va and VIIIa.

In vitro, thrombin generation is assessed most commonly in platelet-poor plasma by a fluorogenic substrate-based thrombin generation assay (TGA) in microtiter plates. While this assay can accurately measure the kinetics of thrombin generation in plasma (including Lag time [LT], Peak thrombin [PT], and endogenous thrombin potential [ETP]), it does not assess the influence of the endothelial cell-dependent protein C pathway on thrombin generation. As a result, the assay has limited value in the assessment of the hypercoagulable patient.

In the present study we introduced a surrogate endothelium to the TGA, thus including activated protein C-induced inactivation of factors Va and VIIIa in the assay system. Wells of flat-bottomed microtiter plates were coated with 3 × 10⁴quiescent EA.hy926 endothelial-like cells which consistently express thrombomodulin (TM) as well as the endothelial protein C receptor (EPCR). The concentration of active TM associated with EA.hy926 in the assay well was determined by a chromogenic assay and found to be ∼0.5 nM. Tissue factor (TF)-initiated thrombin generation was evaluated in normal pooled plasma (NP), and in protein C-deficient (PCd), protein S-deficient (PSd), and heterozygous factor V Leiden (fVL) plasmas, in the presence or absence of endothelial cells.

Thrombin generation in NP was reduced in the presence of endothelial cells as evidenced by a 55% reduction in PT, and a 40% reduction in ETP. LT was prolonged by 83% in the presence of endothelial cells. However, in PCd, PSd and fVL plasmas, endothelial-induced suppression of thrombin generation was blunted. Specifically, only relatively small reductions were observed in PT (26% in PCd, 28% in PSd, 20% in fVL) and ETP (12% in PCd, 6% in PSd, and 14% in fVL). Furthermore, only in the presence of endothelial cells, addition of protein C to PCd plasma dose-dependently reversed the effect of protein C deficiency on PT and ETP. In separate experiments in the absence of endothelial cells, we noted that while similar results were obtained in the presence of the soluble form of thrombomodulin, the concentration required (30 nM) was 60 times higher than that expressed on the endothelial cell monolayer. This suggests that protein C activation occurs more efficiently in the presence of EA.hy926 cells.

In summary, by introducing an endothelial cell monolayer to the TGA, and measuring thrombin generation kinetics in the presence or absence of these cells, we have adapted the assay to assess the contribution of the protein C anticoagulant system to thrombin generation in a physiologically relevant manner. This novel approach not only enables the functions of the endothelial-dependent PC pathway by expressing TM and EPCR, but also may provide other endothelial components relevant to thrombin generation (such as tissue factor pathway inhibitor [TFPI]). This approach to thrombin generation assessment may therefore have both research and clinical applicability.