Treatment of endothelium with the chemotherapy agent vincristine affects activated protein C generation to a greater degree in newborn plasma than in adult plasma
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INTRODUCTION: Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and alpha(2)macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. MATERIALS AND METHODS: APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-alpha(1)AT were assayed by ELISA. APC-alpha(2)M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. RESULTS: Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-alpha(1) AT and APC-alpha(2) M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. CONCLUSIONS: The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.
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