Abstract
Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.
Highlights
Factor Xa2 is a vitamin K-dependent serine protease that is responsible for generation of thrombin from prothrombin in the coagulation cascade [1,2,3,4]
This is derived from the observation that the reactivity of the autolysis loop mutants and heparin binding exosite mutants of Factor Xa (fXa) with Z (PZ)-dependent protease inhibitor (ZPI) was altered
It appears that Arg-143 directly interacts with ZPI since no reactivity for the mutant was observed in the absence of protein Z (PZ), but the cofactor enhanced the reaction, yielding a k2 of 1.2 ϫ 102 MϪ1 sϪ1 for the ZPI inhibition of the mutant (TABLE ONE)
Summary
Construction, Mutagenesis, and Expression of Recombinant Proteins —ZPI cDNA (obtained from Open Biosystems, Huntsville, AL) was subcloned into the StuI and XbaI restriction enzyme sites of the RSVPL4 expression/purification vector system and expressed in human embryonic kidney cells (HEK-293) as described [24]. Competitive Inhibition Studies—The competitive effect of the inactive S195A mutant of fXa on the wild-type fXa inhibition by the ZPI-PZ complex, and the non-serpin inhibitors TFPI and rTAP was studied using the same amidolytic activity assay described above. In this case, the inhibition of fXa (1 nM) on PC/PS vesicles (25 M) by each inhibitor (5 nM TFPI and rTAP, and 10 nM ZPI) was monitored in the presence of increasing concentrations of fXa S195A (0 –200 nM) in TBS/Ca2ϩ. Following 10 min of incubation at room temperature, 50 l of SpFXa (0.5 mM in EDTA) was added to each well, and the remaining activity of fXa was measured as described above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
