Factor seven activating protease (FSAP): does it activate factor VII?

Abstract

Summary. 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. Objectives: 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.

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

“…To confirm that the observed proteolytic activity was not the result of an impurity in our plasmapurified FSAP preparation, we used rFSAP, which is inactive and resistant to autoactivation; however, the mutation allows for in vitro activation of FSAP with thermolysin. 39 Active rFSAP, but not inactive rFSAP, efficiently proteolyzed histones. For comparison, we incubated histones with rAPC, because it was previously found to cleave histones.…”

“…28 The production and purification of recombinant FSAP-R313Q (rFSAP) have been described in detail. 39 Nucleosomes were purified from Jurkat cells as previously described. 40 All histones including H1 were present in our preparation.…”

New Fundamentals in Hemostasis

“…TF largely resides on the cell surface in an inactive (cryptic) configuration, but under certain conditions it is readily decrypted as described below. It is not precisely understood how FVII is cleaved into FVIIa, but a proteolytic role is suggested for either the minute amounts of FVIIa that circulate in the blood (100 pM) (198) or the factor VII-activating protein (FSAP), but recent data argue against the latter (276). At physiological concentrations, FVIIa without TF shows little activity, because of a unique sequence characteristic that retains the FVIIa in a zymogenlike conformation (230).…”

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2

“…This, however, proved to be a misnomer, because coagulation factor VII is a remarkably poor substrate for FSAP/HABP2 (3). In contrast, HABP2 does effectively activate single-chain urokinase-type plasminogen activator (scuPA), 2 suggesting a role in fibrinolysis (3)(4)(5). HABP2 has further been reported to cleave tissuefactor pathway inhibitor (6).…”

“…In view of the numerous substrates that have been proposed for HABP2 (3)(4)(5)(6)(7)10), it remains difficult to predict the phenotype associated with Gly-221 substitution. It remains plausible that HABP2 is a promiscuous enzyme that activates more substrates than scuPA alone and thus may have more roles than stimulation of fibrinolysis.…”