Supplementary Material for: Irradiation with 265-nm ultraviolet light-emitting diodes to plasma: Alterations of hemostatic parameters and von Willebrand factor

Introduction: Among pathogen inactivation methods for blood products, an ultraviolet C (UVC) irradiation has been attracting attention. We previously reported that 265 nm UV-LED (UVC-LED) effectively prevents bacterial infection. However, the effect of UVC-LED on plasma was not considered. This study aimed to explore the effects of high-dose UVC-LED irradiation, required for viral inactivation, on plasma hemostatic potential. Methods: After irradiation of plasma with high-dose UVC-LED for 60 min, the activities of coagulation factors and coagulation inhibitors and von Willebrand factor (VWF) antigen levels were determined. VWF multimer assay and global hemostatic analysis using reconstituted blood under flow were also conducted. Results: UVC-LED irradiation prolonged PT and APTT in a fluence-dependent manner. Fibrinogen levels and factors V and XI activities decreased to 40–47% of pre-irradiation levels. VWF multimer analysis revealed a striking reduction in high-molecular-weight VWF multimers, irrespective to ADAMTS13 activity, while VWF antigen levels showed a slight decrease. Electrophoresis under reducing conditions indicated minimal changes in the 250 kDa-VWF subunit band, suggesting that UVC-LED irradiation disrupts inter-subunit disulfide bonds in VWF multimers. Global hemostatic analysis using reconstituted blood with irradiated plasma showed a marked prolongation in primary hemostasis, indicating impaired platelet adhesion to collagen, reflecting reduced high-molecular-weight VWF multimers. Conclusions: We demonstrated for the first time that a high-dose UVC irradiation to plasma cleaves the inter-subunit disulfide bonds of VWF multimers and reduces the molecular size. In plasma products, the balance between maintaining hemostatic activity and inactivating various pathogens by UVC irradiation is a challenge for the future.

引言：在血液制品的病原体灭活方法中，紫外线C（UVC）照射法逐渐受到关注。我们此前的研究表明，265纳米紫外线发光二极管（UV-LED，即UVC-LED）可有效预防细菌感染。然而，UVC-LED对血浆的影响尚未被考虑。本研究旨在探讨病毒灭活所需的高剂量UVC-LED照射对血浆止血潜能的影响。
方法：用高剂量UVC-LED照射血浆60分钟后，测定凝血因子（coagulation factors）、凝血抑制剂（coagulation inhibitors）的活性及血管性血友病因子（von Willebrand factor，VWF）抗原水平。同时进行了血管性血友病因子多聚体分析（VWF multimer assay）及流动条件下重组血液的整体止血分析（global hemostatic analysis）。
结果：UVC-LED照射以剂量依赖的方式延长了凝血酶原时间（Prothrombin Time，PT）和活化部分凝血活酶时间（Activated Partial Thromboplastin Time，APTT）。纤维蛋白原水平及因子V和XI的活性降至照射前水平的40%至47%。血管性血友病因子多聚体分析显示，高分子量血管性血友病因子多聚体显著减少，且与ADAMTS13蛋白酶（A Disintegrin And Metalloproteinase with Thrombospondin Motifs 13，ADAMTS13）活性无关，而血管性血友病因子抗原水平仅轻微下降。还原条件下的电泳表明，250 kDa血管性血友病因子亚基带变化极小，提示UVC-LED照射破坏了血管性血友病因子多聚体中的亚基间二硫键（inter-subunit disulfide bonds）。使用经照射血浆的重组血液进行的整体止血分析显示初级止血（primary hemostasis）显著延长，表明血小板对胶原蛋白的黏附受损，这反映了高分子量血管性血友病因子多聚体的减少。
结论：我们首次证实，高剂量UVC照射血浆会裂解血管性血友病因子多聚体的亚基间二硫键并减小其分子大小。在血浆制品中，通过UVC照射维持止血活性与灭活多种病原体之间的平衡是未来的挑战。