PB1219 - Structure-function studies on the serine protease domain of Factor VII activating protease (FSAP) to characterize its interaction with substrates and inhibitors.

Background: Factor VII activating protease (FSAP) is a circulating serine protease in blood with homology to coagulation and fibrinolysis factors and is encoded by the HABP2 gene. Its substrates include, Factor VII, tissue factor pathway inhibitor (TFPI), pro-urokinase and fibrinogen. In relation to inflammation, FSAP cleaves histones to neutralize them, generates anaphylatoxin C5a and activates protease-activated receptor-2. Many circulating serine- and kunitz-type protease inhibitors inhibit FSAP. Substrate recognition in serine proteases depends on the heavy chain as well as various loops in the serine protease domain (SPD).

Aims: Characterize the role of various loops of SPD-FSAP in their interactions with substrates and inhibitors.

Methods: Based on homology modelling, we designed mutants of FSAP in its serine protease domain (SPD). Mutant SPD’s were expressed in E. coli and characterized for their proteolytic activity against small peptidic chromogenic substrate S2288, fibrinogen and histone as well as inhibition by TFPI, aprotinin and C1 inhibitor (C1Inh).

Results: Replacement of the basic stretch in the 60 loop (63-65, chymotrypsin numbering) with serine was resistant to inhibition with TFPI, but not other inhibitors. The R84S mutant was resistant to all inhibitors with normal cleavage of S2288, histones and fibrinogen. Mutating the acidic stretch in the 90 loop (96-99) to serine reduced the proteolysis of histones and fibrinogen as well as inhibition with all inhibitors but the cleavage of S2288 was unchanged. R170A, Q192D and 60-loop mutants were defective in cleaving fibrinogen but showed consistent activity towards histones. We did not identify any mutants with defective histone cleavage without also diminishing fibrinogen cleavage.

Conclusion(s): Mutants with disrupted substrate specificity will help to decipher the mechanisms of action of FSAP more precisely. Resistance to plasma inhibitors will enhance the stability of FSAP in vivo and augment its therapeutic effect in e.g., ischemic stroke.