Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Abstract

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 Å (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI Coagulation factor XI (FXI) 4 is a unique homodimeric coagulation protein, present in human plasma at a concentration of ϳ30 nM, that is essential for normal hemostasis, as evidenced by the fact that FXI deficiency is associated with a hemorrhagic disorder (1). The zymogen FXI can bind to receptors (2-4), consisting of the glycoprotein Ib-IX-V complex (5) on the plasma membranes of activated human platelets, where it can be incorporated into platelet membrane microdomains (i.e. lipid rafts) (6) for efficient activation by thrombin or by FXIIa (7-10). The resulting enzyme, FXIa, can then activate the vitamin K-dependent protein, FIX, to initiate the consolidation phase of blood coagulation (1).A variety of important control mechanisms exist for regulating the activity of coagulation proteases in plasma. Several members of the serpin family have been proposed as physiological regulators of FXIa activity in plasma, including C1 inhibitor (11, 12), ␣-1-protease inhibitor (13, 14), antithrombin III (15), ␣-2-antiplasmin (16), and protease nexin 1 (17). However, a platelet secretory protein and member of the class of Kunitz-type inhibitors, protease nexin 2 (PN2), has recently been shown to be a much more potent and physiologically relevant FXIa inhibitor based on detailed kinetics studies (18 -23). PN2 is a ϳ120-kDa isoform of the Alzheimer ␤-amyloid protein precursor (APP) that contains a Kunitz-type serine protease inhibitor (PN2KPI) domain (22). Platelets are an important source of several isoforms of APP, including the APP 751 isoform of PN2 (23, 24). Full-length APP is membraneassociated (25) and is processed by proteases in platelets (26, 27). Upon platelet activation by physiological stimulators, PN2 is secreted from ␣-granules into plasma and inhibits FXIa (22-24), suggesting a role for this protein in blood coagulation. PN2 is a slow, tight binding inhibitor of FXIa with K i of ϳ300 -500 pM (18,20,22). The KPI domain of PN2 is 57 amino acids in length (Glu 289 -Ile 345 in the 751-amino acid isoform of PN2) and is known to contain the entire FXIa inhibitory function of PN2 (19 -21, 28). Similarly, all of the information r...

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

“…Residual enzyme activity was measured under pseudo-first order kinetic conditions as previously reported (25,33), using the substrate t-butoxycarbonyl-Glu(␥-benzyl ester)-Ala-Arg-4-methylcoumaryl-7-amide (Peptides International), and the concentration at which 50% of activity remains (IC 50 ) was determined using Kaleidograph version 3.05 (Abelbeck Software, PCS, Inc., Reading, PA) non-linear least squares regression software. The IC 50 value was converted to an inhibition constant (K i ) using Equation 3,…”

“…Cationic trypsin and mesotrypsin concentrations were quantified by active-site titration using 4-nitrophenyl 4-guanidinobenzoate (Sigma) (32). Recombinant APPI was expressed in Pichia pastoris as a soluble secreted protein essentially as described previously (33). Expression cultures grown in BMMY medium (buffered medium containing methanol and yeast nitrogen base) at 30°C were harvested after 48 h. The supernatant was subjected to 95% ammonium sulfate precipitation at room temperature.…”

“…where [S] represents the substrate concentration, and K m is the Michaelis constant of FXIa for t-butoxycarbonyl-Glu(␥-benzyl ester)-Ala-Arg-4-methylcoumaryl-7-amide, determined to be ϳ275 M. To measure the effect of APPI or cleaved APPI* on clotting time, APTT assays were performed as described previously, using 25 l of APTT reagent (Kontact, Fisher) (33).…”

A Catalytic Domain Exosite (Cys527−Cys542) in Factor XIa Mediates Binding to a Site on Activated Platelets

“…Whereas plateletbound FXIa is protected from inhibition by protease nexin-2, secreted by activated platelets, unbound FXIa is efficiently inhibited, thereby localizing FIX-activation to the platelet surface. The isolated FXIa catalytic domain shown here is taken from the co-crystal structure of rFXIac complexed with PN2KPI (PDB:1ZJD) (44). The structure is displayed in the same orientation as that in which thrombin is conventionally shown.…”

Sequence and Conformational Specificity in Substrate Recognition

“…Recombinant Kunitz domains were expressed and purified from the methylotropic yeast P. pastoris under control of the alcohol oxidase (AOX1) promotor, following protocols described previously for BPTI (23,25) and APPI (15,26) with minor modifications. Briefly, expression cultures grown in BMMY medium (buffered medium containing methanol and yeast nitrogen base) at 30°C were harvested after 72 h. The supernatant was subjected to ammonium sulfate precipitation (95% saturation) at room temperature.…”