The Mechanism by Which Heparin Promotes the Inhibition of Coagulation Factor XIa by Protease Nexin-2

Abstract

Previous kinetic studies have shown that protease nexin-2 is a potent, reversible, and competitive inhibitor of factor XIa. Here we show that high molecular weight heparin potentiates the ability of protease nexin-2 to inhibit factor XIa with a parabolic concentration dependence, predominantly because of an increase of the association rate constant with little perturbation of the dissociation rate constant. No effect on factor XIa inhibition by protease nexin-2 was observed with heparin preparations of 6 -22 saccharide units (0.1 nM-10 M), whereas heparin preparations with 32-64 saccharide units potentiated factor XIa inhibition by protease nexin-2 in a size-and concentration-dependent manner. We propose a model wherein heparin exerts this effect by providing a template for the assembly of factor XIaprotease nexin-2 complexes, and only heparin polymers consisting of greater than 32 saccharide units (M r ϳ10,000) are sufficiently long to provide a template to which factor XIa and protease nexin-2 molecules can bind simultaneously. Heparin-mediated enhancement of factor XIa inhibition by protease nexin-2 was partially abrogated by high molecular weight kininogen, suggesting that high molecular weight kininogen may play a role in regulating factor XIa activity. Protease nexin-2 (PN-2)1 is a ϳ120-kDa soluble fragment of Alzheimer amyloid ␤-protein precursor isoforms containing the 56-amino acid Kunitz-type (or Kunin) 2 protease inhibitor domain (1-4). PN-2 has been found to be an abundant platelet ␣-granule protein that is secreted from platelets upon their activation with physiological agonists (5-8). Factor XIa (FXIa) is a homodimeric serine protease (ϳ160 kDa) involved in the intrinsic pathway of blood coagulation.We have previously conducted detailed kinetic analyses to characterize the inhibition of FXIa by PN-2 using a fluorogenic substrate that is ϳ1,000-fold more sensitive for FXIa than the most sensitive and specific chromogenic substrates (8). The mechanism of FXIa inhibition by PN-2 is best described as a simple, one-step binding interaction during which PN-2 reversibly inhibits FXIa. Our studies have shown that PN-2 is a slow, tight binding inhibitor of FXIa, with an association rate constant (k on ) of 2.1 Ϯ 0.2 ϫ 10 6 M Ϫ1 s Ϫ1 , a dissociation rate constant (k off ) of 8.5 Ϯ 0.8 ϫ 10 Ϫ4 s Ϫ1 , and a K i of 400 pM, a value in good agreement with previous reports (6 -8).Further, we found that high molecular weight kininogen (HK) protects FXIa from inhibition by PN-2 with a saturable concentration dependence (the EC 50 for HK was essentially identical to the K d for the binding interaction between FXIa and HK) and that this protection results from the decreased ability of PN-2 to inhibit the FXIa⅐HK complex (8). Zinc ions, which are known to affect several functions of both FXIa (9, 10) and PN-2 (11), are known to increase the affinity of the FXIa⅐PN-2 complex (9, 12). We found that Zn 2ϩ was able to abolish the protection afforded to FXIa by HK and hypothesized the existence of a zinc-dependent confor...

A Binding Site for Heparin in the Apple 3 Domain of Factor XI

“…Data from our laboratory (22) demonstrated that heparin functions by a template mechanism to increase the association rate of FXIa and PN-2 with a resultant decrease in the inhibition constant. It can be concluded from these observations that there are heparin-binding sites within FXI and FXIa; however, it is not clear whether the heparin-binding site utilized by the zymogen is the same or different from that utilized by the enzyme.…”

“…Another important function of heparin is to promote the inhibition of FXIa by protease nexin II (PN-2) by increasing the association rate of FXIa binding to PN-2 (22). This increased association rate results in a decrease in the K i from 300 pM, in the absence of heparin, to 30 pM in the presence of heparin (22).…”

“…This increased association rate results in a decrease in the K i from 300 pM, in the absence of heparin, to 30 pM in the presence of heparin (22). The effect of heparin and glycosaminoglycans on the binding of FXI to cell surfaces and other proteins is, therefore, an important area of study.…”

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

“…Thus, it has been observed that normal human plasma contains very little (Ͻ60 pM) PN2 (i.e. concentrations well below the reported K i value) (300 -500 pM) for FXIa inhibition by PN2 (19,20,(22)(23)(24). However, the protein is secreted from platelet ␣-granules in sufficient quantities (2-30 nM) to result in rapid and complete inhibition of FXIa in solution (20,23).…”

“…The S19A mutant resulted in a marginal decrease of 1.5-fold (IC 50 1.86 nM) inhibitory activity against FXIa compared with WT PN2KPI. This meager reduction in inhibitory function suggests that the P4Ј site Ser 19 residue has a minor role in FXIa inhibition.…”