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DOI: 10.1055/s-0041-1728110
Aptamer-based ex vivo monitoring of thrombin and activated protein C generation on blood outgrowth endothelial cells for individualized assessment of the protein C pathway
Objective The endothelium plays a crucial role in hemostasis and its regulation, as is demonstrated by the thrombotic risk in disorders affecting the endothelial protein C (PC) pathway, such as PC deficiency and the factor V Leiden (FVL) mutation. Blood outgrowth endothelial cells (BOECs) are endothelial progenitor cells with endothelial character, but a high proliferative capacity. Aim of this study was to develop an assay to monitor the generation of thrombin and activated protein C (APC) in human plasma on BOECs and demonstrate its applicability to assess the PC pathway on an individual basis using cells originating from FVL carriers.
Material and Methods Isolation and characterization of BOECs was performed following standard protocols. All data were obtained at least in triplicate using cells in passage 7 cultured on 24-well plates. First, the APC generation assay was established in a buffer system, to which thrombin, PC, and thrombomodulin (as a substitute for the cells in the control approach) were added. Thrombin formation in defibrinated, recalcified, citrated plasma was induced by 1 pM of tissue factor. During a follow up period of 60 minutes the concentration of thrombin and APC in buffer or plasma on the BOECs was monitored using oligonucleotide-based enzyme capture assays (OECAs).
Results In buffer containing thrombin and PC (1 and 100 nM final concentration, respectively) the peak APC concentration after 60 minutes was 0.034±0.001 nM without cells and 0.124±0.001 nM on BOECs from healthy controls. In normal pooled plasma, the APC peak concentration was about 10fold higher on BOECs than without cells (1.02±0.062 versus 0.110±0.004 nM). Conversely, the thrombin peak concentration without cells (47.2±2.8 nM) was higher than on BOECs (27.1±2.8 nM), indicating the endothelial anticoagulant effect. In plasma of three FVL carriers on their individual cells, both thrombin and APC peak levels were higher than in the pooled healthy controls, with 44.6±17.6 and 1.78±0.09 nM, respectively.
Conclusion The observed increased thrombin and APC generation rates on BOECs from FVL carriers, a finding that is consistent with previously obtained in vivo data, demonstrate the applicability of the presented approach to assess the functionality of the PC pathway on an individualized basis. The obtained data warrants further studies in a larger population including patients with other disorders affecting the PC pathway.
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Publikationsverlauf
Artikel online veröffentlicht:
18. Juni 2021
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