Zeerleder Sacha, Zwart Bas, te Velthuis Henk, Stephan Femke, Manoe Rishi, Rensink Irma, Aarden Lucien A
Department of Immunopathology, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
FASEB J. 2008 Dec;22(12):4077-84. doi: 10.1096/fj.08-110429. Epub 2008 Aug 27.
Plasma proteins such as early complement components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleosome-releasing factor (NRF) activity. However, recalcified plasma (r-plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size-exclusion and anion-exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS-PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII-activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to completely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single-chain (sc) FSAP from r-plasma. Purified scFSAP efficiently removes nucleosomes from sn cells. We report that factor VII-activating protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.
血浆蛋白如早期补体成分和IgM参与晚期凋亡或继发性坏死（sn）细胞的清除。我们最近描述了一种可被蛋白酶抑制剂抑肽酶抑制的血浆蛋白酶对于从sn细胞中清除核小体至关重要。一个明显的候选者，纤溶酶，确实具有核小体释放因子（NRF）活性。然而，在纤溶酶原耗尽后，重新钙化的血浆（r-血浆）仍保留其NRF活性，这表明血浆中存在另一种负责NRF活性的蛋白酶。在本研究中，我们使用尺寸排阻色谱和阴离子交换色谱来纯化负责血浆中NRF活性的蛋白酶。对含有NRF活性的色谱馏分进行SDS-PAGE分析，发现一条与NRF活性相对应的蛋白带。序列分析表明这条带是因子VII激活蛋白酶（FSAP）。我们开发了针对FSAP的单克隆抗体，并且能够用针对FSAP的单克隆抗体完全抑制血浆中的NRF活性。使用亲和色谱，我们能够从r-血浆中纯化单链（sc）FSAP。纯化的scFSAP能有效地从sn细胞中清除核小体。我们报告因子VII激活蛋白酶可能通过催化从继发性坏死细胞中释放核小体而在细胞内稳态中发挥作用。
