Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach

Abstract

Background: The endothelial cell–dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. Methods: Endothelial colony–forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of thrombomodulin expression, ECFCs were stimulated with IL-1β (interleukin 1β). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). Results: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1β stimulated ECFCs and 3.66 nmol/L without IL-1β. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P =0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. Conclusions: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

The Endothelium Modulates the Prothrombotic Phenotype of Factor V Leiden: Evidence from an Ex Vivo Model

“…Indeed, using an ECFC-based ex vivo model of the PC pathway, we have recently shown that the APC response to thrombin formation was increased in FVL carriers, the majority of whom had no history of VTE. 19 In the present study, using the same model but distinguishing between symptomatic and asymptomatic FVL carriers, we observed an increased APC response only in the latter cohort. Of note, while homozygous FVL carriers showed the highest AUCs of thrombin in their respective cohorts (VTE+ and VTE-) and the highest AUCs of APC overall, the ratio of AUC APC/AUC thrombin in homozygous FVL carriers lay near the median of their respective cohorts.…”

“…The evaluation of thrombin and APC formation in plasma or in a purified system on ECFCs was performed as described previously 19 , with modifications. For the variant using plasma, cells were washed twice with DPBS in 24-well plates and 400 µL citrated platelet-poor plasma, previously defibrinated using Batroxobin, was added to the cell culture.…”

“…[16][17][18] Endothelial variables include, in particular, EPCR and TM, since their downregulation or variants in their encoding genes (PROCR respectively THBD) have been suggested as thrombotic risk factors. [19][20][21][22][23] We have recently introduced an ex vivo model of the PC system, in which endothelial colony forming cells (ECFCs) and autologous plasma are utilized for personalized assessment of the functionality of the PC pathway. 19 In the present study this experimental approach amongst others was used to further investigate plasmatic and endothelial factors that potentially modulate the APC response to thrombin in FVL carriers with or without previous VTE, in order to better understand the variable clinical expressivity of FVL.…”

“…16–18 Endothelial variables include, in particular, EPCR and TM, since their downregulation or variants in their encoding genes ( PROCR respectively THBD ) have been suggested as thrombotic risk factors. 19–23…”

Pentosan Polysulfate Affords Pleotropic Protection to Multiple Cells and Tissues

“…The endothelial-cell-dependent PC (protein C) pathway critically regulates coagulation, anti-inflammatory, and cytoprotective signaling [ 173 ] and is a major control system for thrombosis, and it limits inflammatory responses, and may also decrease endothelial cell apoptosis that may result from inflammatory cytokines and ischemia [ 174 ]. Essential components in this pathway include thrombin, thrombomodulin, endothelial cell protein C receptor (EPCR), protein C and protein S [ 174 ].…”