Inactivation of Factor XIa by Plasma Protease Inhibitors

Abstract

A B S T R A C T Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, a1-protease inhibitor, antithrombin III, Cl-inhibitor, and a2-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with a1-protease inhibitor, antithrombin III, and Cl-inhibitor were 4.08, 10, and 14.6 M`min-' X 103, respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 X 10-1, 4.68 X 10-2, and 2.4 X 10-2 min-', respectively. These kinetic data predict that al-protease inhibitor should account for 68%, antithrombin III for 16%, and Cl-inhibitor and the equipotent a2-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions.Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (Kd) = 0.17 ,M. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by Cl-in-hibitor with a Kd of 0.08 AM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Fac-

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

“…6)-Proline is a structurally important amino acid. An Ala substitution at this location resulted in a 5-fold loss of inhibitory activity (IC 50 of 5.92 nM) compared with WT PN2KPI, whereas replacements with Arg (IC 50 of 1.71 nM) or Lys (IC 50 of 2.25 nM) for Pro 13 rescued the loss of function ( Fig. 6A), in agreement with the results of the APTT assay (Fig.…”

“…Other members of the KPI family, such as BPTI (32) and soybean protease inhibitor (36), also have proline at the P3 position and adopt the same backbone conformation as PN2KPI. PN2KPI forms a turn around Pro 13 and allows Lys 192 of FXIac to insert into PN2KPI and form two hydrogen bonds with carbonyl oxygen atoms of Cys 14 (P2) and Gly 12 (P4) (Fig. 4B).…”

“…Therefore, P1 of PN2KPI is important for its selectivity and potency in the inhibition of FXIa. P3 Site (Pro 13 ) of PN2KPI-There are two Pro residues in PN2KPI, one at position 13 (P3 site) and the other at position 32, each located at the beginning of characteristic loop regions that are involved in interaction with FXIac as observed in the PN2KPI-FXIac co-crystal structure. Pro 13 is a highly conserved residue among the Kunitz family of protease inhibitors.…”

“…Pro 13 is a highly conserved residue among the Kunitz family of protease inhibitors. The ␤ carbon in Pro 13 is oriented toward a solventexposed region; therefore, this site could accept a variety of amino acids (34). A study utilizing phage display mutagenesis, where a large number of random mutations are generated within the PN2KPI domain, showed that amino acids found at position 13 were highly dependent upon the amino acid at position 39 (43).…”

“…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).…”