SERPIN Regulation of Factor XIa: THE NOVEL OBSERVATION THAT PROTEASE NEXIN 1 IN THE PRESENCE OF HEPARIN IS A MORE POTENT INHIBITOR OF FACTOR XIa THAN C1 INHIBITOR

Daniel J Knauer,Devdoot Majumdar,Pei-Chieh Fong,Mary F Knauer

doi:10.1074/jbc.m003909200

Daniel J Knauer, Devdoot Majumdar + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m003909200

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Abstract

In the present studies we have made the novel observation that protease nexin 1 (PN1), a member of the serine protease inhibitor (SERPIN) superfamily, is a potent inhibitor of the blood coagulation Factor XIa (FXIa). The inhibitory complexes formed between PN1 and FXIa are stable when subjected to reducing agents, SDS, and boiling, a characteristic of the acyl linkage formed between SERPINs and their cognate proteases. Using a sensitive fluorescence-quenched peptide substrate, the K(assoc) of PN1 for FXIa was determined to be 7.9 x 10(4) m(-)(1) s(-)(1) in the absence of heparin. In the presence of heparin, this rate was accelerated to 1.7 x 10(6), M(-)(1) s(-)(1), making PN1 a far better inhibitor of FXIa than C1 inhibitor, which is the only other SERPIN known to significantly inhibit FXIa. FXIa-PN1 complexes are shown to be internalized and degraded by human fibroblasts, most likely via the low density lipoprotein receptor-related protein (LRP), since degradation was strongly inhibited by the LRP agonist, receptor-associated protein. Since FXIa proteolytically modifies the amyloid precursor protein, this observation may suggest an accessory role for PN1 in the pathobiogenesis of Alzheimer's disease.

Highlights

In the present studies we have made the novel observation that protease nexin 1 (PN1), a member of the serine protease inhibitor (SERPIN) superfamily, is a potent inhibitor of the blood coagulation Factor XIa (FXIa)
Factor XIa Forms Complexes with a Second Protease Inhibitor, PN1, Present in the Media of 293 Cells Overexpressing APP751/protease nexin 2 (PN2)—In our recent studies we have shown that the heparin binding site in PN1 plays an important role in the cell-surface binding and catabolic rate of thrombin-PN1 complexes [30]
The heparin-binding peptide from PN1, which has been shown to inhibit the catabolism of PN1thrombin complexes, and GST-receptor associated protein (RAP), an lipoprotein receptor-related protein (LRP) agonist, were tested in parallel. 125I-FXIa was pre-incubated with serum-free 293/751ϩ-conditioned medium to allow complexes to form

Summary

SERPIN Regulation of Factor XIa

THE NOVEL OBSERVATION THAT PROTEASE NEXIN 1 IN THE PRESENCE OF HEPARIN IS A MORE POTENT INHIBITOR OF FACTOR XIa THAN C1 INHIBITOR*. In addition to being inhibited by APP/PN2, FXIa proteolytically cleaves APP/PN2 within the RHDS sequence of the amyloid ␤-peptide sequence associated with Alzheimer’s disease [19] This cleavage occurs at low nanomolar concentrations of FXIa, which are likely to be physiological. APP/PN2 and PN1 share Factor XIa as a target protease, and PN1 may play an important regulatory role by preventing the proteolytic cleavage of APP/PN2 by FXIa, which results in the loss of APP/PN2 biological activity as a cell adhesion promoter and neuro-protective agent. Fourth, both PN1 and APP/PN2 protease-inhibitor complexes are endocytosed via the LRP. These studies are the first to demonstrate that PN1 is a potent inhibitor of Factor XIa and suggest that extravasated Factor XIa may be another physiologically important target of PN1

EXPERIMENTAL PROCEDURES

Kassoc ϭ

RESULTS

Rate constant Kassoc

DISCUSSION