title: Factor seven activating protease (FSAP): does it activate factor VII?
abstract: BACKGROUND
  Factor seven activating protease (FSAP) was initially reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources.
  
  
  OBJECTIVE
  Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis.
  
  
  RESULTS
  Expression of wild-type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg(313) at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAP(R313Q) in its intact form. Thermolysin-activated FSAP displayed the same reactivity toward the substrate S-2288 as plasma-derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased V(max) by 2-3-fold, without affecting K(m) (62 nm) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation.
  
  
  CONCLUSIONS
  While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.
authors.affiliation: Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Stavenuiter
authors.forename: I
authors.initials: I
authors.lastname: Dienava-Verdoold
authors.forename: M G
authors.initials: MG
authors.lastname: Boon-Spijker
authors.forename: H J M
authors.initials: HJ
authors.lastname: Brinkman
authors.forename: A B
authors.initials: AB
authors.lastname: Meijer
authors.forename: K
authors.initials: K
authors.lastname: Mertens
mesh_terms: D001777:Blood Coagulation / Q000187:drug effects; D002308:Cardiolipins / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D015942:Factor VIIa / Q000378:metabolism*; D005342:Fibrinolysis; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011122:Polyphosphates / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D002308:Cardiolipins; D011122:Polyphosphates; D011994:Recombinant Proteins; D006493:Heparin; C433550:HABP2 protein, human; D012697:Serine Endopeptidases; D015942:Factor VIIa; D014568:Urokinase-Type Plasminogen Activator; D013820:Thermolysin