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J. Biol. Chem., Vol. 275, Issue 48, 37340-37346, December 1, 2000

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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, and Mary F. Knauer

From the Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, California 92627

Received for publication, May 9, 2000, and in revised form, September 1, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 × 10⁴ M¹ s¹ in the absence of heparin. In the presence of heparin, this rate was accelerated to 1.7 × 10⁶, M¹ s¹, 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The protease, Factor XIa, plays an important role in the contact activation blood-clotting pathway in vitro and is believed to do so in vivo (1, 2). Factor XIa is a dimer consisting of two identical subunits and is found in plasma in low microgram concentrations (3-6). When activated by factor XIIa, each of the subunits of Factor XIa is cleaved and consists of a heavy and light chain held together by disulfide bonds (4, 7, 8). The serine protease enzymatic activity of Factor XIa resides within the light chain, and thus, each dimer of Factor XIa has two active sites (9).

Since Factor XIa activation is an irreversible process, the regulation of Factor XIa proteolytic activity is achieved through the action of specific inhibitors from at least two known classes of proteins. In the first class, four different members of the serine protease inhibitor (SERPIN) family have been characterized as irreversible inhibitors of Factor XIa (10-13). These include C1 inhibitor, 1-antitrypsin, 2-antiplasmin, and antithrombin III, with second order rate constants of 1.8, 0.1, 0.43, and 0.32 × 10³ M¹ s¹, respectively (14). Of these four inhibitors, the second order rate constants for C1 inhibitor and antithrombin III are improved by approximately 100-fold in the presence of heparins in vitro. On the basis of rate constants, C1 inhibitor was thought to be the best SERPIN candidate as a physiological regulator of Factor XIa in plasma.

In addition to the SERPIN inhibitors, Factor XIa is also potently inhibited by protease nexin 2 (PN2),¹ which refers to the subset of the several differentially spliced forms (including the 751-residue isoform) of the amyloid precursor protein (APP), which contain a kunitz-type inhibitor domain. Although the inhibition of Factor XIa by APP/PN2 is reversible, with a k_on of 2 × 10⁶ M¹ s¹ and a k_off of 8.0 × 10⁴ M¹ s¹, it is still a more efficient inhibitor of Factor XIa than any of the SERPIN inhibitors (15, 16). In addition, the k_on is improved by a factor of 20 in the presence of the heparin, probably through a template mechanism (17). Since APP/PN2 is abundant in platelets, it was thought to be the most probable primary physiological inhibitor of Factor XIa in plasma (18).

In addition to being inhibited by APP/PN2, FXIa also 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. As a consequence, the cell adhesive activities of APP/PN2 as well as its neuro-protective activities are abolished (19). Thus, the leakage of FXIa into extravascular spaces, especially in neuronal tissue, may have severe consequences.

In the present studies we discovered that Factor XIa is potently inhibited by PN1, a member of the SERPIN family. While screening a peptide library for sequences that would inhibit the low density lipoprotein receptor-related protein (LRP)-mediated cellular uptake of ¹²⁵I-Factor XIa-APP/PN2 complexes, we observed a radioactive band on SDS-PAGE that was the right size for a complex between Factor XIa and PN1. Further investigation revealed that indeed this was a complex between proteolytically active Factor XIa and PN1 present in the conditioned media of cells used in the screening assay that were overexpressing APP/PN2. We proceeded to characterize the interaction between PN1 and Factor XIa for several reasons. First, the best plasma SERPIN inhibitor of Factor XIa, C1 inhibitor, has a relatively slow reaction rate, even in the presence of heparin (14). Second, although PN1 is not as abundant as APP/PN2 in platelets (18), PN1 is clearly present on the platelet surface and in  granules and may contribute to FXIa inhibition in plasma (20, 21). Third, 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. The LRP and several of its ligands have been implicated as genetic factors in Alzheimer's disease (22-24).

Our studies demonstrate that Factor XIa inhibition by PN1 has a K_assoc of 2.0 × 10⁶ M¹ s¹, making it a 10-fold faster inhibitor of Factor XIa than C1 inhibitor when both reactions are carried out in the presence of heparin. The inhibitory complexes consist of SDS, heat, and reducing agent-resistant complexes between the light chain of Factor XIa and PN1. Initial studies on human fibroblasts indicate that Factor XIa-PN1 complexes are internalized by the LRP and eventually degraded in lysosomes. 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Purified Factor XIa and unfractionated heparin (160 units/mg, with an average molecular mass of 12 kDA) were purchased from Calbiochem. Size-classed heparin (15 kDa) was purchased from Neoparin, Inc., CA. Human protease nexin 1 was purified from serum-free medium conditioned for 48 h by human fibroblasts as described previously (25). The fusion protein GST-receptor associated protein (RAP), was purified from Escherichia coli extracts as described previously (26). Boc-EAR-AMC was purchased from Peptides International. Na¹²⁵I was from Amersham Pharmacia Biotech. All other common shelf reagents were from either Sigma or Calbiochem.

Protein Radioiodination and Characterization-- After purification, PN1 was active site-titrated with thrombin using a chromogenic substrate as described previously (27). Factor XIa was radioiodinated with [¹²⁵]iodine using the Iodogen method as described previously (27). Specific activities of individual preparations ranged from 6,000 to 12,000 cpm/ng.

Electrophoresis and Imaging-- Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and fixed in methanol:acetic acid:water (5:1:5). Gels were dried and exposed to a Bio-Rad phosphoimaging screen overnight. Digitized images were developed using a Bio-Rad GS-250 Molecular Imager.

Cell Culture-- A stably transfected line of 293 cells overexpressing APP751 (designated 293/751+) were maintained and sub-cultured as described previously (28). Human foreskin fibroblasts were also cultured as described previously (27). For cell culture experiments, both types of cells were trypsinized and re-grown to confluence in 24-well plates. Experimental cultures were grown in serum-free medium for at least 24 h.

Fluorescence-based Enzymatic Assay-- Enzymatic assays were conducted in Tyrode's buffer, pH 7.4, containing 0.1% bovine serum albumin (16). The amount of Factor XIa was constant in all of the assays, 0.3 nM, as was the concentration of the synthetic fluorogenic substrate, Boc-EAR-AMC, 100 µM. Other additions varied depending on the particular experiment (see Fig. 5). The assays were conducted in a final reaction volume of 2.0 ml at a regulated temperature of 37 °C with constant stirring. The change in relative fluorescence was monitored over a time course with a sampling interval of 10 s. The samples were excited at 375 nM, and the emission was quantified at 445 nM. K_assoc constants were calculated as described previously (29) using the following equation.

(Eq. 1)

Degradation Assays-- Confluent cultures of HF cells were incubated with ¹²⁵I-Factor XIa-PN1 complexes at a concentration of 100 ng/ml in binding medium at 37 °C. Binding medium consisted of bicarbonate-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin buffered to pH 7.4 with 20 mM Hepes. Samples of the medium were withdrawn at hourly intervals for precipitation with 10% trichloroacetic acid. Trichloroacetic acid-soluble radioactivity (degraded ¹²⁵I-Factor XIa) was quantified using a  counter (30). Parallel samples of binding reactions were incubated in the absence of cells to determine background that has been subtracted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Factor XIa Forms Complexes with a Second Protease Inhibitor, PN1, Present in the Media of 293 Cells Overexpressing APP751/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 experiment shown in Fig. 1 was originally designed to determine if one of the heparin binding sites in APP751/PN2 might analogously play a role in the catabolism of ¹²⁵I-FXIa-APP751/PN2 complexes. In Fig. 1A, a synthetic peptide (pep12) corresponding to one of the heparin binding sites in APP751/PN2 (residues 96-110) (31) was tested for its ability to inhibit the catabolism of ¹²⁵I-FXIa-APP751/PN2 complexes by 293/751+ cells, a 293 cell line overexpressing APP751/PN2. As controls, the heparin-binding peptide from PN1 (residues 70-87), which has been shown to inhibit the catabolism of PN1-thrombin complexes, and GST-RAP, an LRP agonist, were also tested in parallel. ¹²⁵I-FXIa was pre-incubated with serum-free 293/751+-conditioned medium to allow complexes to form. Each peptide was added at a final concentration of 50 µg/ml, and then the reaction mixtures were incubated with HF cells for 3 h at 37 °C. Aliquots of the medium were precipitated in 10% trichloroacetic acid to assay for increases in trichloroacetic acid soluble radioactivity (¹²⁵I-FXIa degradation products, primarily iodo-tyrosine). The APP751/PN2 heparin-binding peptide, pep12, had no significant effect on the rate of ¹²⁵I-FXIa degradation (Fig. 1A). Unexpectedly, however, the degradation rate of ¹²⁵I-FXIa was markedly reduced to approximately 7.5 fmol by the PN1 heparin-binding peptide.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Factor XIa forms complexes with a second protease inhibitor, PN1, present in the media of 293 cells overexpressing APP751/PN2. Panel A, serum-free conditioned medium from 293/751+ cells was incubated with 100 ng/ml of 125I-FXIa for 30 min at 37 °C. At the end of the incubation, the peptides (50 µg/ml) and GST-RAP (25 µg/ml) were added before addition to HF cells. At the end of a 3-h incubation with triplicate cultures of HF cells at 37 °C, aliquots of the media were removed, and trichloroacetic acid-soluble radioactivity was quantified as a measure of degradation. Error bars indicate S.D. from the mean of the triplicate samples. Panel B, 125I-FXIa alone (lane 1), after incubation with conditioned media from 293/751+ cells (lane 2) or after the addition of 30 ng of purified PN1 (lane 3), was analyzed by SDS-PAGE on 10% polyacrylamide gels. The dried gels were exposed for 2 h to a phosphorescent screen, and a digitized image was prepared using a Bio-Rad Molecular Imager. The migration positions of molecular weight standards are indicated. Arrows indicate the heavy and light chains of 125I-FXIa, and the migration position of 125I-FXIa-PN1 complexes is also indicated.

We hypothesized that these data might be explained by formation of complexes between ¹²⁵I-FXIa and another protease inhibitor, potentially PN1, if 293/751+ cells also were making significant quantities of the other inhibitor. Indeed, subsequent SDS-PAGE analysis of the conditioned media incubated with ¹²⁵I-FXIa before addition to the HF cells revealed the presence of two high molecular weight complex bands (Fig. 1B). The larger of the complex bands had an expected size of approximately 98 kDa, which corresponds to the size of a complex formed between the light chain of FXIa and secreted APP751/PN2 (Fig. 1B, lane 2). However, the lower band had not been previously characterized, and we suspected it could correspond to complexes between ¹²⁵I-FXIa and another protease inhibitor secreted by the 293/751+ cells. PN1 was a likely candidate for the second inhibitor due to the size of the complexes in the lower band and the data in Fig. 1A. To test this directly, 30 ng of purified PN1 were incubated with 30 ng of ¹²⁵I-FXIa at 37 °C and electrophoresed in a parallel lane (Fig. 1B, lane 3). Indeed, higher molecular weight complexes formed between these purified components were observed in lane 3, and they co-migrated with the lower molecular weight band of complexes observed in 293/751+ cell-conditioned medium (lane 2). The observed size (71 kDa) of these complexes between purified PN1 and FXIa corresponds to the molecular mass of PN1 (43 kDa) added to the molecular mass of the catalytically active light chain of FXIa (28 kDa). The formation of an inhibitory complex between FXIa and PN1 has not been previously reported, and the presence of this inhibitory complex in the conditioned media of the 293/751+ cells explains the inhibitory effect of the PN1 heparin-binding peptide on Factor XIa catabolism in Fig. 1A. In fact, the inhibitor effect observed in Fig. 1A was due to the PN1 heparin-binding peptide specifically inhibiting the internalization and catabolism of ¹²⁵I-FXIa that had formed complexes with endogenously produced PN1 in the 293/751+ cell-conditioned medium.

Factor XIa Forms Complexes with PN1 in a Concentration-dependent Manner-- Since it has not previously been shown that PN1 is able to form an inhibitory complex with FXIa, we next tested complex formation over a broad range of PN1 concentrations to establish the range for completeness and kinetics of the reaction. ¹²⁵I-FXIa, at a concentration of 2.5 nM, was incubated with the indicated concentrations of PN1 for 15 min at 37 °C (Fig. 2A). The reactions were terminated by the addition of sample buffer and resolved by SDS-PAGE to separate free ¹²⁵I-FXIa from ¹²⁵I-FXIa·PN1 complexes. The light and heavy chains of FXIa are resolved under the reducing conditions of the sample buffer (1% mercaptoethanol) into bands of approximately 28 and 51 kDa. The catalytic reactivity of the FXIa resides solely in the light chain, and so, although it forms a higher molecular weight complex with PN1, approximately 70 kDa, the intensity of the heavy chain of FXIa, remains unchanged. Approximately 50% of the ¹²⁵I-FXIa was in complex at a PN1 concentration of 13.2 nM, and the reaction was essentially complete at a PN1 concentration of 33 nM. These data clearly show a dose dependence of FXIa inhibition, and based on the molarity of PN1 and FXIa in the reactions, suggests a rapid association rate. The PN1 used in these experiments was active site-titrated against human thrombin, and the molarities of PN1 presented are based on the active PN1 fraction. The amount of PN1 required to achieve full complex formation was in excess to the FXIa in the reaction, and this may be due to several factors including partitioning between cleaved PN1 acting as a substrate for FXIa and PN1 in complex with FXIa. Since this could call into question the potential role of PN1 as a physiological inhibitor of FXIa, we next tested the inhibitory activity of PN1 toward Factor XIa in the presence of heparin, which is abundant in the physiological environment at endothelial cell surfaces and in the subintima.

View larger version (65K): [in this window] [in a new window]   Fig. 2.   125I-FXIa forms SDS-resistant complexes with PN1 in a concentration-dependent manner. Panel A, 125I-FXIa, at a concentration of 2.5 nM per reaction, was incubated with the indicated concentrations of PN1 at 37 °C for 30 min in a final volume of 35 µl. At the end of incubation, 35 µl of SDS-PAGE-reduced sample buffer was added to each reaction. The samples were analyzed by SDS-PAGE on 10% polyacrylamide gels. The digitized image was prepared as described under "Experimental Procedures." Panel B, 125I-FXIa at a concentration of 2.5 nM per reaction, was incubated with the indicated concentrations of PN1 at 37 °C for 15 min in the presence of 50 nM heparin (20 kDa). The reactions were terminated and analyzed as described in panel A.

To address whether PN1 might act as a substrate for FXIa, we next performed the titration experiment in the presence of 50 nM fractionated heparin (20 kDa) with reaction conditions and PN1 concentrations adjusted appropriately (Fig. 2B). We chose the 20-kDa size class of heparin based on its ability to act as a template for the acceleration of FXIa inhibition by APP/PN2 (17). Importantly, these data show that at a PN1 concentration of 5 nM, nearly all of the FXIa was sequestered into a high molecular weight complex. Since each FXIa has two active sites for each 160,000 kDa dimer, then the number of active sites is also at 5.0 nM. Thus, when placed at equimolar concentrations in the presence of heparin, all of the FXIa and PN1 are partitioned into a high molecular weight complex. Although we cannot rule out that some of the PN1 might be cleaved non-productively by acting as a substrate for FXIa in the absence of heparin, this is clearly not the case in the presence of heparin. The data strongly suggest that PN1 may indeed act as a physiological inhibitor of FXIa.

Complexes Formed between PN1 and FXIa Are Stoichiometric and Apparently Covalent-- To test the stability of ¹²⁵I-FXIa-PN1 complexes, the experiment shown in Fig. 2 was repeated with ¹²⁵I-FXIa at a final concentration of 2.5 nM and PN1 at 26 nM. This time the reaction was equally divided into four samples. Half of the samples received an equal volume of SDS-PAGE sample buffer without reducing agent, whereas the other received sample buffer with reducing agent. One of each sample type was then boiled for 2 min before electrophoresis, whereas the other was left at room temperature (Fig. 3). As expected, in the absence of reducing agent, the complexes migrated at a molecular mass of 120 kDa, the mass of one ¹²⁵I-FXIa subunit (80 kDa) plus the molecular mass of one PN1 molecule. Also as expected, in the presence of reducing agent, the complexes migrated as a stoichiometric complex with a molecular mass of 70 kDa, the mass of one ¹²⁵I-FXIa light chain plus the mass of one PN1 molecule. Under both reducing and non-reducing conditions, the complexes were in an apparent stoichiometry of 1:1 and were stable to boiling. This strongly suggests a covalent linkage between the two. Whether this covalent linkage is due to an SDS-induced acyl-linked complex from a stable tetrahedral intermediate or is the natural progression of the reaction remains unresolved, as it does for all other SERPIN-protease complexes.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Complexes formed between 125I-FXIa and PN1 are stoichiometric and apparently covalent. 125I-FXIa (2.5 nM) was incubated with a 10-fold molar excess of PN1 for 30 min at 37 °C to form 125I-FXIa-PN1 complexes. The reaction, 80 µl total, was divided into four equal aliquots. An equal volume of reduced SDS-PAGE sample buffer was added to two of the samples, whereas the other two received unreduced SDS-PAGE sample buffer. One of each sample type was heated to 100 °C for 2 min, creating a total of four different sample conditions. All of the samples were analyzed by SDS-PAGE on a 10% polyacrylamide gel. The digitized image was developed and visualized as described under "Experimental Procedures." Lane 1, 125I-FXIa-PN1 complexes non-reduced, not heated; lane 2, 125I-FXIa-PN1 complexes non-reduced, heated; lane 3, 125I-FXIa-PN1 complexes reduced, not heated; lane 4, 125I-FXIa-PN1 complexes reduced and heated.

Complex Formation between FXIa and PN1 Is Heparin-accelerated-- Since complex formation between FXIa and APP751/PN2 is heparin-accelerated (17) and PN1 is a heparin-activated SERPIN, we tested whether size-classed 20-kDa heparin (which corresponds to 64 saccharide units) could also accelerate inhibitory complex formation between FXIa and PN1. The activation of PN1 by heparin toward thrombin, another PN1 cognate protease, has been reasonably well characterized (32). We bracketed the optimal heparin concentration for PN1 activation toward thrombin, to look for activation of PN1 toward FXIa. PN1 (5 nM) and ¹²⁵I-FXIa (2.5 nM) were reacted for 1 min at 37 °C in the presence of the indicated concentrations of fractionated heparin (Fig. 4). The reactions were terminated and analyzed for complex formation by SDS-PAGE under reducing conditions as described in Fig. 3. In the absence of heparin and at heparin concentrations of 5 nM and below, no complex formation was observed during the 1-min incubation. At a heparin concentration of 20 nM and above, complex formation was driven to completion during the 1-min incubation. This narrow window between 5 and 20 nM for heparin activation is characteristic of the high affinity heparin-mediated acceleration of SERPIN-protease complex formation (33). Interestingly, the concentration-dependent heparin-activated reactions between PN1 and FXIa and between PN1 and thrombin (data not shown) display nearly identical profiles, and both peak at a heparin concentration between 5 and 25 nM.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Complex formation between FXIa and PN1 is heparin-accelerated. Constant concentrations of 125I-FXIa (2.5 nM) and of PN1 (5 nM) were incubated for 1 min at 37 °C in the presence of the indicated concentrations of size-classed 20-kDa heparin in a final reaction volume of 30 µl. The reactions were terminated by the addition of 30 µl of SDS-PAGE-reduced sample buffer. The samples were resolved by SDS-PAGE on a 10% polyacrylamide gel, and the digitized image was prepared as described under "Experimental Procedures."

Kinetic Analysis of the Inhibition of FXIa by PN1 in the Presence and Absence of Heparin-- To more precisely determine the rate of the inhibitory reaction between PN1 and FXIa and to accurately measure the effect of heparin on the reaction rate, we employed a fluorometric assay using the fluorogenic substrate, Boc-EAR-AMC (17). At sub-micromolar concentrations of reactants, the simple equation

(Eq. 2)

provides a good measure of the association rate between PN1 and its target proteases (29). Factor X Ia (0.3 nM) and PN1 (6.6n M) were incubated for the indicated times at 37 °C in a reaction volume of 100µl of Tyrode's buffer. The reactions were then diluted to a final volume of 2.0 ml, and residual protease activity was measured by the addition of Boc-EAR-AMC. Shown in Fig. 5A are the progression curves of Boc-EAR-AMC cleavage by residual FXI a proteolytic activity over a 400-s time interval after the indicated pre-incubation time in the absence of heparin. Cleavage was monitored by the relative fluorescence increase at 445 nM after excitation at 375 nM. Relative to controls, FXI a activity was decreased approximately 90% by a 40-min pre-incubation with PN1. At all time points the data were linear, and these slopes were used to calculate K_assoc of PN1 for FXIa using each of the time points. The average K_assoc of PN1 for FXIa, 0.08 × 10⁶ M¹ s¹, is shown in Table I. Also shown in the table for comparison is the K_assoc of C1 inhibitor with FXIa and the K_on of the reversible inhibitor, APP/PN2, with FXIa.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Kinetic analysis of the inhibition of FXIa by PN1 using a fluorogenic substrate, Boc-EAR-AMC. Panel A, PN1 inhibition of FXIa proteolytic activity as a function of time in the absence of heparin. PN1 (6.6 nM) and FXIa (0.3 nM) were incubated at 37 °C for the times indicated on the figure. At the indicated time, 100-µl samples of the reaction were diluted into a final volume of 2.0 ml of Tyrode's buffer containing Boc-EAR-AMC (100 µM). The change in fluorescence was quantified at 10-s intervals for approximately 400 s. Pre-incubation times: open circles, 0 min; filled squares, 10 min; filled triangles, 20 min; filled circles, 30 min; filled diamonds, 40 min. Panel B, PN1 inhibition of FXIa proteolytic activity is rapidly accelerated in the presence of heparin. All conditions in B were identical to those described in A, except that the reactions were done in the presence of soluble unfractionated heparin. The preincubation times were shortened to a minimum of 30 s and to a maximum of 4.0 min before the assay for FXIa activity using Boc-EAR-AMC. Preincubation times in the presence of 50 units/ml unfractionated heparin: open circles, 0 s; filled diamonds, 30 s; filled circles, 60 s; filled triangles, 120 s; filled squares, 240 s. Panel C, initial velocity as a function of preincubation of FXIa with PN1 in the presence and absence of heparin. For a direct comparison, the data shown in panels A and B were re-plotted as the ratio of initial velocity at each of the preincubation times (V) to the initial velocity in the absence of inhibitor (Vo). Filled squares, in the presence of heparin; filled circles, in the absence of heparin.

To quantitatively assess the effect of heparin on the rate of FXIa inhibition by PN1, the experiments shown in Fig. 5, panel A, were repeated in the presence of 50 units/ml unfractionated heparin at the considerably shortened preincubations times indicated (Fig. 5, panel B). Unfractionated heparin was intentionally used in these experiments, so that the rate constants obtained could be directly compared with other studies where the inhibitory activities of antithrombin III, C1 inhibitor, and antitrypsin toward FXIa were investigated (14). In the presence of heparin, FXIa inhibition was nearly 70% complete by 4 min, as compared with 40 min in the absence of heparin (Figs. 5, A and B). A plot of V/V₀ versus time of pre-incubation of inhibitor with protease in the presence and absence of heparin is shown in Fig. 5C to directly compare the data in panels A and B. This graph clearly demonstrates an approximate 20-fold acceleration in the rate of inhibition of FXIa by PN1 in the presence of heparin.

When the K_assoc was calculated using the same method as in panel A, we arrived at a K_assoc of 1.72 ± 0.2 × 10⁶ M¹ s¹ in the presence of heparin (Table I). This represents a 20-fold increase in the K_assoc of PN1 for FXIa in the presence of heparin. Table I also compares PN1 to C1 inhibitor, the best SERPIN inhibitor of FXIa characterized to date. Significantly, PN1 inhibits FXIa 20 times faster than C1 inhibitor in the presence of heparin, which is thought to be a physiological inhibitor of FXIa in plasma. For another comparison, PN1 is considered to be a physiological inhibitor of both plasmin and urokinase, with K_assoc constants for these proteases of 0.13 × 10⁶ M¹ s¹ and 0.15 × 10⁶ M¹ s¹, respectively (32), and these constants are 10 times slower than the K_assoc of PN1 for FXIa in the presence of heparin. It is also worth noting that the inhibition of neither urokinase nor plasmin by PN1 is accelerated by heparin (32). Although the reversible inhibitor, APP/PN2, has a K_on for FXIa 15 times faster than K_assoc of PN1, the interaction is non-covalent and occurs through the kunitz domain in APP/PN2 (Table I). This is a conceptually important difference, since FXIa in complex with PN1 is part of a suicide reaction that is not reversible, whereas FXIa incomplex with APP/PN2 has the potential to regain its proteolytic activity. Since PN1 has been shown to be present on the surface of platelets, which is the preferred site of FXI activation, this raises the possibility that PN1 may act as a threshold modulator of FXIa (34).

¹²⁵I-FXIa-PN1 Complexes Are Internalized by the LRP in Human Fibroblasts-- Since the rationale underlying the present study is that PN1 may act as an important regulator of extravasated FXIa into tissues, then tissues should have an adequate mechanism for the catabolism of FXIa-PN1 inhibitory complexes. In the case of thrombin-PN1 and uPA-PN1 complexes, cellular internalization is mediated by the LRP (35, 36). This results in the transport of the inhibitory complexes to the lysosomes, where they are degraded. To determine if ¹²⁵I-FXIa-PN1 complexes were also internalized via the LRP, we conducted the degradation experiment shown in Fig. 6. Confluent cultures of normal HF cells were incubated with ¹²⁵I-FXIa-PN1 complexes (100 ng/ml) in the absence and in the presence of the LRP inhibitory protein, RAP. RAP blocks the binding of all known LRP ligands and is routinely used to assess the role of the LRP in the endocytosis of LRP ligands (37). At the indicated times, samples were removed from the cultures and analyzed for the appearance of trichloroacetic acid-soluble radioactivity (low molecular mass iodo-tyrosine products resulting from ¹²⁵I-FXIa-PN1 complex degradation). By the 4-h time point the HF cells degraded approximately 75 fmol of the ¹²⁵I-FXIa-PN1 complexes in the absence of RAP. In the presence of RAP, complex degradation was reduced to 10 fmol over the same time period. Since HF cells have little or no very low density lipoprotein receptor (which is also inhibited by RAP), these data strongly suggest that ¹²⁵I-FXIa-PN1 complex internalization by HF cells is LRP-dependent. Whether the LRP interacts with the FXIa or the PN1 moiety of the complex needs to be addressed in future studies.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Degradation of 125I-FXIa-PN1 complexes is inhibited by RAP-GST. Confluent HF cells in a 24-well plate were incubated at 37 °C in 400 µl of binding medium containing 125I-FXIa-PN1 complexes at a concentration of 100 ng/ml in the absence (circles) or presence of 200 nM RAP-GST (squares). Aliquots from triplicate wells were taken at the indicated times and subjected to precipitation in 10% trichloroacetic acid. Soluble radioactivity in the samples was quantified by -counting at 70% efficiency. Error bars indicate 1 S.D. from the mean of the triplicate samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present studies we initiated a series of experiments designed to investigate the role of the heparin binding site in APP/PN2 in FXIa-PN2 complex internalization and catabolism. During the course of these studies, we observed that FXIa not in complex with APP/PN2, probably generated through the dissociation of FXIa-APP/PN2 complexes (17), formed complexes with cell-secreted PN1 that were stable to SDS-PAGE. The 70-kDa complexes formed between PN1 and FXIa were further characterized and found to be stable to SDS-PAGE after boiling in the presence of reducing agents. This suggests that the complexes most likely resemble classic 1:1 stoichiometric, covalent SERPIN-protease complexes.

We decided to pursue the interaction between PN1 and FXIa for several reasons. First, the inhibitor that is considered to be most likely physiologically relevant for FXIa is APP/PN2, and that inhibitory complex is non-covalent and subject to dissociation. Second, the only other SERPINs that are known to inhibit FXIa with reasonable rate constants are C1 inhibitor and antithrombin III. In the absence of heparin, both of these could be described as relatively poor inhibitors (K_assoc, 1.8 and 0.32 × 10³ M¹ s¹, respectively). And third, these SERPIN inhibitors are primarily restricted to plasma, which raises the question of how extravasated FXIa is handled physiologically. PN1, which is restricted primarily to tissues, might be a good candidate molecule to serve this function in extra-vascular spaces.

In our more detailed kinetic studies on the interactions of FXIa with PN1 using the very sensitive fluorogenic substrate Boc-EAR-AMC, we determined the K_assoc to be 7.9 ± 2.0 × 10⁴ M¹ s¹. This is nearly equal to the K_assoc of PN1 for urokinase, for which PN1 is believed to be a physiological inhibitor. Unlike urokinase, PN1 inhibition of FXIa is accelerated in the presence of heparin, and the K_assoc was markedly enhanced to 1.7 × 10⁶ M¹ s¹. This makes PN1 a 10-times faster inhibitor of FXIa than C1 inhibitor in the presence of heparin. It is important to note that PN1 is most likely to encounter FXIa in a heparin-rich environment past the boundaries of endothelial cells in a situation where vascular integrity has been lost. Thus, the present data suggest that PN1 may play a significant role in the regulation of activated FXI. It is also worth noting that in the presence of heparin, PN1 acts exclusively as an inhibitor of FXIa and not as a substrate. When the products of the heparin-accelerated inhibitory reaction were analyzed, there was no evidence that any PN1 was partitioned into a cleaved form not in complex with FXIa. In the data shown in Fig. 2B, an amount of PN1 equimolar to FXIa was sufficient to entrap all of the FXIa in a high molecular weight inhibitory complex.

Finally, the present data suggest that PN1 may play an important physiological role in the regulation FXIa, which has been shown to proteolytically modify APP/PN2 and augment its biological activities. Factor XIa that escapes into nervous tissue has the potential to cleave the APP and, thus, abrogate its cell adhesion and neuroprotective and growth-promoting activities (17, 38-41). Given the abundance of PN1 in nervous tissue, it probably assumes the role of the primary inhibitor of FXIa under these conditions. Although the present data do not precisely explain the presence of PN1 in the brain, it places PN1 present at the site of -peptide generation along with FXIa that can proteolytically process APP751/PN2, altering its biological activities. The potent inhibition of FXIa by PN1 suggests that PN1 may play an important protective role against FXIa proteolytic activity in tissues. It will be very interesting in future studies to examine the interaction of FXIa-PN1 complexes with cells of neuronal origin and to determine how the complexes might be metabolized in neuronal tissues.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1GM34001-12 and by American Health Assistance Foundation Grant A1999044.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all correspondence should be addressed. Tel.: 949-824-4703; Fax: 949-824-4709; E-mail: mfknauer@uci.edu.

Published, JBC Papers in Press, September 5, 2000, DOI 10.1074/jbc.M003909200

	ABBREVIATIONS

The abbreviations used are: PN, protease nexin; FXIa, Factor XIa; APP, amyloid precursor protein; LRP, low density lipoprotein receptor-related protein; HF, human foreskin fibroblasts; PAGE, polyacrylamide gel electrophoresis; RAP-GST, receptor-associated protein-glutathione S-transferase fusion protein; Boc-EAR-AMC, Boc-Glu-(O-Bzl)- Ala-Arg-7-amino-4-methylcoumarin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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