Nucleosome‐releasing factor: a new role for factor VII‐activating protease (FSAP)

Abstract

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.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

“…It is remarkable in this context that the effects of histones were observed with concentrations that can be attained in vivo in Escherichia coli-treated baboons, 8 in which the plasma level of total histones was estimated to be approximately 70 g/mL, and in human patients, where similar levels were found (Xu J, unpublished observations, August 2010). Moreover, plasma concentrations may underestimate the amount of histones locally associated with cells 51 at sites of cellular release and/or altered blood flow, where they could be much higher. Nuclear proteins released from dying cells, including histones, have been implicated as mediators of inflammation and cell injury in a variety of severe human diseases such as sepsis, trauma, ischemia, autoimmune diseases, and cancer, all of which are known to be associated with thrombosis of the macro-and/or microcirculation.…”

Factor VII activating protease (FSAP) regulates the expression of inflammatory genes in vascular smooth muscle and endothelial cells

“…Activation: The zymogen form (pro-FSAP) is activated by polyanions such as DNA, RNA and heparin 46 and also by apoptotic and necrotic cells 47 , nucleosomes 48 and histones 49 . Histones can activate both, purified and plasma pro-FSAP …”

“…FSAP is activated by apoptotic and necrotic cells 47 and mediates the release of nucleosomes from late apoptotic cells 48 . In systemic lupus erythematous FSAP is not able to mediate nucleosome release due to autoantibody cross-linking to the nucleosomes 81 .…”

DNA and factor VII–activating protease protect against the cytotoxicity of histones

“…24,25 Extracellular histones and nucleosomes may derive from activated macrophages, 11 NETs, 18 or apoptotic or (secondary) necrotic cells. 12,[26][27][28][29][30] For apoptotic and necrotic cells, both passive and active mechanisms of chromatin release have been demonstrated. We previously established that the serine protease factor VII-activating protease (FSAP) is activated in serum upon incubation with late apoptotic or necrotic cells and that its activity is required to efficiently release chromatin from these cells into the extracellular environment.…”