Feys H B, Van Aelst B, Devreese K, Devloo R, Coene J, Vandekerckhove P, Compernolle V
Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium.
Vox Sang. 2014 May;106(4):307-15. doi: 10.1111/vox.12106. Epub 2013 Oct 29.
Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).
Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen.
Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg).
RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.
用于个体输血产品的光化学病原体灭活技术（PCT）通过不可逆地损伤核酸来抑制复制。人们对PCT对血液成分完整性的附带影响感到担忧，这导致在常规实践中不愿引入该技术。本研究旨在揭示核黄素病原体灭活技术（RF-PRT）对血浆成分的损伤机制。
在有或无溶解分子氧存在的情况下，对RF-PRT处理后的血浆成分活性和抗原进行测定。
以血浆中的ADAMTS13作为哨兵分子，我们的数据显示其活性和抗原分别降低了23±8%和29±9%（n = 24），这与其他凝血因子平均降低25%的情况相符。ADAMTS13的蛋白质印迹显示分子完整性降低，没有明显的额外蛋白水解迹象，核黄素也不能直接抑制该酶。然而，在RF-PRT之前物理去除溶解氧可保护ADAMTS13以及FVIII和纤维蛋白原免受损伤，表明活性氧具有直接作用。氧化还原染料测量表明，在RF-PRT过程中特异性产生了超氧阴离子。作为弥散性不可逆生物分子损伤标志物的蛋白质羰基含量在RF-PRT后显著增加（3.1±0.8对1.6±0.5 nmol/mg蛋白质），但在没有溶解分子氧的情况下未增加（1.8±0.4 nmol/mg）。
单个血浆单位的RF-PRT产生活性氧，对相关血浆成分的生物分子完整性产生不利影响，这种副作用可通过在病原体灭活过程中应用缺氧条件来避免。
