title: Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).
abstract: FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.
authors.affiliation: Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Fumie
authors.initials: F
authors.lastname: Nakazawa
authors.forename: Christian
authors.initials: C
authors.lastname: Kannemeier
authors.forename: Aya
authors.initials: A
authors.lastname: Shibamiya
authors.forename: Yutong
authors.initials: Y
authors.lastname: Song
authors.forename: Eleni
authors.initials: E
authors.lastname: Tzima
authors.forename: Uwe
authors.initials: U
authors.lastname: Schubert
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Koyama
authors.forename: Michael
authors.initials: M
authors.lastname: Niepmann
authors.forename: Heidi
authors.initials: H
authors.lastname: Trusheim
authors.forename: Bernd
authors.initials: B
authors.lastname: Engelmann
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Preissner
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003067:Coenzymes / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D011485:Protein Binding; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012697:Serine Endopeptidases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D017077:Culture Media, Conditioned; D012313:RNA; D012259:Ribonuclease, Pancreatic; C433550:HABP2 protein, human; D012697:Serine Endopeptidases