©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protease Nexin-2/Amyloid -Protein Precursor Inhibits Factor Xa in the Prothrombinase Complex (*)

(Received for publication, August 3, 1995)

Fakhri Mahdi (1) William E. Van Nostrand (2) Alvin H. Schmaier (§)

From the  (1)Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, Ann Arbor, Michigan 48109-0724 and the (2)Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92717

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of coagulation factors XIa and IXa. Here we show that PN-2/AbetaPP and its KPI domain also inhibited isolated factor Xa with a K of 10M. On a solid phase binding assay, PN-2/AbetaPP formed a complex with factor Xa. Incubation of molar excess factor Xa to PN-2/AbetaPP produced a single cleavage within PN-2/AbetaPP's heparin binding domain liberating a 8.2-kDa amino-terminal peptide. PN-2/AbetaPP and its KPI domain equally inhibited factor Xa in the prothrombinase complex with a K of 1.9 times 10M and 1.3 times 10M, respectively. AbetaPP which does not contain the KPI domain was a substrate of factor Xa but did not inhibit it, indicating the PN-2/AbetaPP inhibition of factor Xa was not substrate inhibition. All of the factor Xa inhibition in the prothrombinase complex by PN-2/AbetaPP and its KPI domain on the chromogenic assay was accounted for by inhibition of release of prothrombin fragment F as determined on immunochemical assay. In the prothrombinase complex, PN-2/AbetaPP inhibited factor Xa with a k = 1.8 ± 0.7 times 10^6M min similar to antithrombin III and heparin inhibition (k of 3.0 ± 0.2 times 10^6M min). These studies indicated that PN-2/AbetaPP in the assembled prothrombinase complex inhibited factor Xa comparable to antithrombin III in the presence of heparin. PN-2/AbetaPP's factor Xa inhibitory activity along with its known inhibition of factors XIa and IXa suggest that this protease inhibitor and related proteins could be regulators of hemostatic reactions on membranes of cells in the intravascular compartment.


INTRODUCTION

Amyloid beta-protein precursor (AbetaPP), (^1)a multidomain protein, is the parent protein of amyloid beta protein, a 39-42 amino acid peptide that is deposited in senile plaques and in the walls of cerebral blood vessels of patients with Alzheimer's disease (Kang et al., 1987; Glenner and Wong, 1984). The single gene for AbetaPP found on chromosome 21 encodes at least three distinct mRNAs produced by alternative splicing that result in three different sized proteins (AbetaPP, AbetaPP, and AbetaPP) (Ponte et al., 1988; Tanzi et al., 1988; Kitaguchi et al., 1988). Two of these mRNAs code for proteins (AbetaPP and AbetaPP) which contain a domain homologous to Kunitz-type protease inhibitors (KPI) (Ponte et al., 1988; Tanzi et al., 1988; Kitaguchi et al., 1988). The secreted isoforms of AbetaPP containing the KPI domain are identical to protease nexin-2 (PN-2) (Van Nostrand et al., 1989; Oltersdorf et al., 1989).

PN-2/AbetaPP and its KPI domain have been recognized to be potent inhibitors of trypsin, chymotrypsin, epidermal growth factor binding protein, and the subunit of nerve growth factor (Van Nostrand et al., 1989, 1990b; Oltersdorf et al., 1989). PN-2/AbetaPP which is present in high concentrations in platelets is a potent inhibitor of factor XIa (Van Nostrand et al., 1990a, 1990b; Smith et al., 1990). PN-2/AbetaPP also is an inhibitor of factor IXa (FIXa) in the assembly of the tenase complex on phospholipid vesicles (PSPC), platelets, and endothelial cells (Schmaier et al., 1993, 1995). Similarly, a homologue of PN-2/AbetaPP, amyloid beta-protein precursor-like protein-2, has been shown to have inhibitory activity against hemostatic enzymes factors XIa, IXa, and Xa (Petersen et al., 1994; Sprecher et al., 1993; Van Nostrand et al., 1994). These studies suggest that this family of proteins may have a regulatory role in hemostasis. While examining the ability of PN-2/AbetaPP to inhibit factor IXa on non-biologic surfaces, we found in one assay that the degree of factor IXa inhibition could not be fully accounted for by its inactivation alone. The present investigation shows that PN-2/AbetaPP also is an inhibitor of factor Xa alone and when assembled on PSPC in the prothrombinase complex. Recognition that PN-2/AbetaPP's inhibits factor Xa enlarges its role as a regulator of hemostasis.


EXPERIMENTAL PROCEDURES

Proteins

PN-2/AbetaPP (AbetaPP) was purified from fibroblast culture media using techniques of heparin affinity chromatography and immunoaffinity chromatography as described previously (Van Nostrand et al., 1990b). The KPI domain of PN-2/AbetaPP, which was provided by Dr. Steven Wagner, Salk Institute Biotechnology/Industrial Associates, La Jolla, CA, was produced in a recombinant yeast expression system and purified as described previously (Wagner et al., 1992). The protease inhibitory activities of purified PN-2/AbetaPP and KPI domain were determined by neutralization with active-site titrated trypsin (Van Nostrand et al., 1990b; Wagner et al., 1992). AbetaPP was obtained and purified like PN-2/AbetaPP from culture media from human glioblastoma U-138 cells stably transfected to overexpress it (Davis-Salinas et al., 1994). Human factors IXa (FIXa), Xa (FXa), X, and II (FII) were purchased from Enzyme Research Laboratories, South Bend, IN. Human FIXa on nonreduced sodium dodecyl sulfate-13% polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands at 52 and 33 kDa, and when reduced with 2% beta-mercaptoethanol, four bands at 29, 25, 14, and 12 kDa. The 25- and 12-kDa bands seen on reduced SDS-PAGE represented only 5-10% of the total protein in all preparations. Human FXa was activated with Russel viper venom. On reduced 15% SDS-PAGE, FXa consisted of three majors bands at 36, 34, and 24 kDa. These bands account for 90% of the protein present. Prothrombin on nonreduced 8% SDS-PAGE was a single band at 70 kDa; upon reduction, the apparent molecular mass increased to 80 kDa. All FIXa and FXa used in these investigations were active-site titrated with antithrombin III (American Diagnostica, Greenwich, CT) using a modified procedure of Griffith et al.(1985) as previously reported (Schmaier et al., 1993). Purified thrombin-activated bovine factor Va (FVa) was purchased from Haematologic Technologies Inc., Essex Junction, VT. This protein on nonreduced 8% SDS-PAGE had two major bands at 100 and 68 kDa; upon reduction, two major bands were seen at 97 and 75 kDa, respectively, and two minor bands accounting for less than 10% of the protein present at 51 and 33 kDa. Human alpha-thrombin (3250 units/mg) was generously provided by Dr. John W. Fenton III, N. Y. State Department of Health, Albany, NY.

Phospholipid Vesicle Preparation

Phospholipid vesicles were prepared from a mixture of L-alpha-phosphatidylserine (Sigma) and L-alpha-phosphatidylcholine (Sigma) (25/75, mol/mol) that were dried in a glass test tube under a stream of nitrogen (Rawala-Sheikh et al., 1990). The dried material was resuspended in 0.05 M Tris-HCl, 0.175 M NaCl, pH 7.5, and sonicated for 30 s multiple times on ice over 60 min (Rawala-Sheikh et al., 1990). After sonication, some preparations were ultracentrifuged at 100,000 times g to produce a homogenous suspension free of large particles and multilamellar liposomes (Barenholz et al., 1977). No difference in the cofactor activity of the phosphatidylserine/phosphatidylcholine vesicles (PSPC) was noted whether they were ultracentrifuged or not. The PSPC were stored at 4 °C.

Measurement of Factor Xa Formation

The enzymatic activity of human FIXa was measured by its ability to activate human factor X using polylysine as an artifical surface (Schmaier et al., 1993, 1995; Lundblad and Roberts, 1982; Griffith et al., 1985; McCord et al., 1990). Factor IXa (4.45 nM) was incubated with factor X (400 nM) in 0.1 M triethanolamine, 0.1 M NaCl, pH 8.0, containing 0.1% polyethylene glycol (M(r) = 8000), 0.2% bovine serum albumin, and 60 nM polylysine for 40 min at 20-25 °C. At the end of the incubation, an aliquot of the activated factor X solution was added to a solution of 0.4 mM tosyl-Gly-Pro-Arg-p-nitroanilide (Sigma). Hydrolysis proceeded for 60 min at 20-25 °C and the reaction was terminated by the addition of 50% acetic acid after which the optical density reading was obtained at 405 nm. FIXa alone had no amidolytic activity on the chromogenic substrate. The FIXa used in this assay activated 0.5% of the added factor X. When inhibition studies were performed with PN-2/AbetaPP and its KPI domain, the inhibitor was incubated with FIXa for 5 min at room temperature prior to the addition of factor X. All inhibition constants determined from the results of the chromogenic assay were calculated from the residual activity at end point.

Measurement of Factor Xa Activity

Factor Xa activity (1-2.5 nM) was measured in 0.1 M triethanolamine, 0.1 M NaCl, pH 8.0, containing 0.1% polyethylene glycol (M(r) = 8000), 0.2% bovine serum albumin, and 60 nM polylysine or 0.02 M Hepes, 0.15 M NaCl, pH 7.4, containing 0.5 mg/ml bovine serum albumin, 2 mM Ca, and 0.1% polyethylene glycol using 0.4 mM tosyl-Gly-Pro-Arg-p-nitroanilide (Sigma) for 35 min at 20-25 °C. In certain experiments, the polylysine was removed from the buffer. In other experiments 25 µM PSPC, 4.8 units/ml thrombin-activated factor VIII (FVIIIa), or 5 nM thrombin-activated bovine factor Va (FVa) were added to the reaction mixture in the presence of 2 mM Ca. The reaction was terminated by the addition of an equal volume of 50% acetic acid after which the optical density was obtained at 405 nm. Hydrolysis of the substrate was linear over the time of the reaction. When inhibition studies were performed with PN-2/AbetaPP, its KPI domain, or antithrombin III, the inhibitor (2-10 nM) was incubated with FXa (1 nM) for 5 min at room temperature prior to the addition of the chromogenic substrate. All inhibition constants determined from the results of the chromogenic assay were calculated from the residual activity at end point. When investigations with antithrombin III were performed, 1 unit/ml heparin (Elkins-Sinn, Cherry Hill, NJ) was included in the reaction mixture.

Measurement of Factor X Activation Peptide

Simultaneous samples of human FIXa activation of factor X in the presence of polylysine were prepared for both chromogenic and immunochemical determination of factor X activation. Immunochemical determination of activation of factor X by FIXa was measured as nanomoles of factor X activation peptide liberated as detected by radioimmunoassay using an antiserum directed to the factor X activation peptide (Bauer et al., 1989). These assays were generously performed by Dr. Kenneth A. Bauer, Beth Israel Hospital, Boston, MA. The percent liberation of factor X activation peptide in PN-2/AbetaPP- or its KPI domain-treated samples was calculated from the ratio of nanomole activation peptide released when the inhibitor was present versus the nanomole activation peptide released when no inhibitor was present times 100. The percent liberated peptide in the inhibitor-treated sample was utilized as a measure of residual FIXa activity to calculate inhibition constants.

Measurement of Prothrombin Activation

Activation of human prothrombin (FII) (1000 nM) by FXa (1 nM) in the presence of 25 µM PSPC, 5 nM bovine factor Va, and 0.7 mM HD-Phe-Pip-Arg-p-nitroanilide-2HCl (S2238) (Kabi-Pharmacia, Franklin OH) was performed at 37 °C in 0.025 M Hepes, 0.175 M NaCl, pH 7.5, containing 5 mg/ml bovine serum albumin and 2 mM Ca (Rosing et al., 1980, 1993). After a 4-min hydrolysis, the reaction was stopped with an equal volume of 50% acetic acid and read in a microplate reader at 405 nm. Factor Xa itself did not hydrolyze this substrate. Preliminary studies revealed that at a 4-min activation time, there was sufficient substrate present such that hydrolysis was on an upward slope and there was no substrate exhaustion over the time of measurement. When investigations with inhibitors were performed, FXa (1 nM) and its inhibitor (PN-2/AbetaPP, KPI domain, or antithrombin III) (2-10 nM) were preincubated or not with PSPC and FVa for 5 min followed by starting the reaction with the addition of FII and the chromogenic substrate.

Measurement of Prothrombin Fragment F

Simultaneous samples of FXa activation of FII in the presence of PSPC and FVa were prepared for both chromogenic and immunochemical determination of FII activation. Prothrombin fragment F (Lau et al., 1979) was measured using a commercial kit from Baxter Diagnostics, Inc., graciously provided by Viola Sotomayer of Baxter Diagnostics. The percent liberation of prothrombin fragment F in PN-2/AbetaPP- or its KPI domain-treated samples was calculated from the ratio of nanomole of activation peptide released when the inhibitor was present versus the nanomole of activation peptide released when no inhibitor was present times 100. The percent liberated peptide in the inhibitor-treated sample was utilized as a measure of residual FXa activity to calculate inhibition constants.

Calculation of Kinetic Parameters and Constants

The K(m) and V(max) of human FXa activation of FII in the presence of PSPC (25 µM) and FVa (5 nM) were determined by measuring the rate of FII activation (20-1600 nM) by 1.0 nM FXa in four independent experiments. The mean ± S.E. of each point performed in triplicate in four individual experiments were analyzed on a double reciprocal plot by linear regression. The K(m) and V(max) were determined from a substrate/velocity plot and the negative reciprocal of the x- and y-intercepts of the double reciprocal plots, respectively. The nanomolar alpha-thrombin formed was determined by comparing the level of hydrolysis seen in the present assay with the level of hydrolysis measured by known concentrations of human alpha-thrombin under identical assay conditions. The turnover numbers for factor IIa formation (k) were determined by the ratio of the maximum concentration of factor IIa formed (V(max)) divided by the concentration of the forming enzyme (FXa). The stoichiometry of FXa inhibition by PN-2/AbetaPP and its KPI domain was determined by nonlinear regression as previously reported (Schmaier et al., 1993). Briefly, FXa at 1 nM was added to 2-100 nM inhibitor (PN-2/AbetaPP or its KPI domain) and the residual FXa activity was determined. The plotted x-intercept of the inhibitor concentration versus the % inhibition of FXa activity indicated the concentration of added inhibitor to the known amount of added FXa.

The equilibrium inhibition constants (K(i)) presented for PN-2/AbetaPP and its KPI domain were calculated as previously reported (Van Nostrand et al., 1990b) by the procedure of Bieth(1984) for tight-binding inhibitors using the following equation: K(i) = {[(I)/(1-a)] - (E)}/(1/a), to yield an apparent K(i) where (I) is the inhibitor concentration, (E) is the factor Xa concentration, and a is the residual factor Xa activity after incubation with the inhibitor. The actual K(i) was calculated using the subsequent equation: K(i) = K(i)/1 + ([S]/K(m)), where [S] is the concentration of the substrate, factor II, and K(m) is the Michaelis constant for the factor Xa-factor II (protease-substrate) reaction (Bieth, 1984). The second-order association rate constants (k) for each of the inhibitors were calculated using the integrated second-order rate equation: k" = [(1/I-E)bulletln E(I - EI)/I(E - EI)]/t, where E is the FXa concentration, I is the inhibitor concentration, EI is the concentration of the FXa-inhibitor complex, and t is the time of incubation in minutes (Gigli et al., 1970).

Determination of complex formation between Factor Xa and PN-2/AbetaPP

Complex formation between PN-2/AbetaPP and FXa was demonstrated by solid phase binding assay. Microtiter plates were coated with PN-2/AbetaPP (250 ng) in 0.1 M Na(2)CO(3), pH 9.6, and then blocked with 1% radioimmunoassay grade bovine serum albumin (Sigma). After washing, the wells were incubated with FXa (50 ng) followed by a mouse anti-human FX/Xa antibody (1 µg/ml) (American Diagnostica, Inc., Greenwich, CT) in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4, containing 0.05% Tween 20. After further incubation and washing, a rabbit anti-mouse antibody conjugated with alkaline phosphatase (Sigma number 2429 at 1/1000) was added. The color reaction was initiated by the addition of p-nitrophenyl phosphate disodium (1 mg/ml) in 0.05 M Na(2)CO(3), 1 mM MgCl, pH 9.8. An additional solid phase binding assay for complex determination was performed by linking FXa (50 ng) in 0.1 M Na(2)CO(3), pH 9.6, to the microtiter plate. After blocking the cuvette wells with bovine serum albumin, they successively were incubated with PN-2/AbetaPP (250 ng) and monoclonal antibody P2-1 to PN-2/AbetaPP in ascites fluid (Van Nostrand et al., 1989) followed by detection with a rabbit anti-mouse antibody conjugated with alkaline phosphatase (Sigma number 2429 at 1/1000 dilution).

Protein and Peptide Sequencing

Factor Xa was incubated with PN-2/AbetaPP (1 to 16 parts factor Xa to 1 part PN-2/AbetaPP, mol/mol) in 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4, containing 2 mM Ca for 1 h at room temperature. The reaction was stopped with sample buffer for SDS-PAGE. The PN-2/AbetaPP samples (1.0 µg/lane) were electrophoresed on a 6% SDS-PAGE and then electroblotted onto nitrocellulose. After blocking with Blotto, the protein was detected using monoclonal antibody P2-1 followed by a second antibody conjugated with horseradish peroxidase (Van Nostrand et al., 1990b) using the chemiluminescence system of Amersham. Factor Xa cleaved PN-2/AbetaPP in 4:1 (mol/mol) ratio of FXa to PN-2/AbetaPP was electrophoresed on 15-22 and 6% SDS-PAGEs, respectively, followed by electroblotting onto a Problot membrane (Applied Bioscience) and detected by staining. The NH(2)-terminal amino acid sequences of an 8.2-kDa peptide seen on the 15-22% SDS-PAGE and of the cleaved PN-2/AbetaPPs at 116, 97, and 90 kDa seen on the 6% SDS-PAGE were determined from the Problot membrane in the Protein and Carbohydrate Structure Facility at the University of Michigan, Ann Arbor, MI.

Statistics

Significance of difference in results between groups was measured by t test for groups of unpaired data.


RESULTS

PN-2/AbetaPP Inhibits Factor Xa

PN-2/AbetaPP was found to produce a K(i) of FIXa of 1.5 times 10M (a mean of two experiments) on chromogenic detection of a polylysine based-factor X activation assay versus a K(i) of FIXa of 6.9 times 10M when the same samples were studied on a factor X activation peptide assay. This 46-fold difference in inhibitory ability of the same samples on two different factor IXa enzymatic assays suggested that on the chromogenic assay, whose results are determined indirectly through factor X activation, additional inhibition must have occurred than that of FIXa alone. This result prompted the present investigation to determine if PN-2/AbetaPP and its KPI domain were direct inhibitors of FXa. In independent experiments, the K(m) of FXa for the chromogenic substrate N-tosyl-Gly-Pro-Arg-p-nitroanilide was found to be 0.26 ± 0.1 mM. Using conditions identical to the polylysine-based FX activation assay by FIXa, PN-2/AbetaPP inhibited FXa with a K(i) of 1.6 ± 0.4 times 10M (Table 1). If polylysine were excluded from the buffer of the assay, PN-2/AbetaPP inhibited FXa with a K(i) of 4.5 ± 2.3 times 10M (Table 1). These data indicated that polylysine itself potentiated the inhibition of FXa by PN-2/AbetaPP as measured on a chromogenic assay. AbetaPP, an isoform of AbetaPP which does not contain the KPI domain, did not inhibit FXa when used in up to 10-fold molar excess to enzyme. The discordance in inhibition of FIXa by PN-2/AbetaPP of the same samples between the chromogenic and FXa activation peptide assays also can be explained by the inhibitor blocking generated FXa. Additional investigations were performed to determine the degree of FXa inhibition by PN-2/AbetaPP and its KPI domain under various conditions. Similar to the results seen with isolated FXa and PN-2/AbetaPP, the presence of PSPC, FVIIIa, and/or FVa did not influence the degree of inhibition of FXa by PN-2/AbetaPP and its KPI domain (Table 1). These data indicated PN-2/AbetaPP and its KPI domain were equipotent inhibitors of FXa. Furthermore, the stoichiometry of FXa inhibition by PN-2/AbetaPP was 1:1. However, at 4 orders of magnitude molar excess KPI domain to FXa, FXa activity was not reduced to zero.



PN-2/AbetaPP and Factor Xa Interactions

Investigations next were performed to determine if FXa and PN-2/AbetaPP formed a complex as determine by solid phase binding assay (Fig. 1). When PN-2/AbetaPP was coupled to microtiter plate wells, FXa specifically bound to the PN-2/AbetaPP as detected by an antibody to FXa followed by a second antibody conjugated with alkaline phosphatase (Fig. 1, top). Likewise, when FXa was linked to microtiter plate wells, PN-2/AbetaPP specifically bound to the FXa as detected by an antibody to PN-2/AbetaPP followed by a secondary antibody conjugated with alkaline phosphatase (Fig. 1, bottom). These studies indicated that FXa and PN-2/AbetaPP formed a complex characteristic of Kunitz-type inhibitors.


Figure 1: Solid phase binding assay between PN-2/AbetaPP and FXa. Top, purified PN-2/AbetaPP (250 ng) was linked to microtiter plate cuvette wells (see ``Experimental Procedures''). After blocking with bovine serine albumin, purified FXa (50 ng), antibody to Factor X, and a second antibody to detect the antibody to Factor X was added in sequential order. In various wells, one of the components of the complex assay was excluded. No APP represents wells where no PN-2/AbetaPP were linked to the cuvette wells; No FXa indicates wells where no FXa was added; No AntiFXa represents wells were no primary antibody to Factor X was added; No 2nd Ab indicates wells where no alkaline phosphatase-conjugated second antibody were added; and ALL represents cuvette wells were all components were added. The data presented represents the mean ± S.E. of nine experiments. Bottom, purified FXa (50 ng) was linked to microtiter plate cuvette wells (see ``Experimental Procedures''). After blocking with bovine serine albumin, purified PN-2/AbetaPP (250 ng), antibody to PN-2/AbetaPP, and a second antibody to detect the antibody to PN-2/AbetaPP was added in sequential order. In various wells, one of the components of the complex assay was excluded. NoFXa represents wells where no Factor Xa was linked to the cuvette wells; No APP indicates wells where no PN-2/AbetaPP was added; and No AntiAPP represents wells were no primary antibody to PN-2/AbetaPP was added. The data presented represents the mean ± S.E. of nine experiments.



Additional investigations showed that PN-2/AbetaPP was a substrate of FXa (Fig. 2). When PN-2/AbetaPP was incubated with increasing concentrations of FXa (1-16-fold molar excess), there was a decrease in the large, dark 124-kDa band of the starting material on an immunoblot of a nonreduced 6% SDS-PAGE and the appearance of 3 new bands at 116, 97, and 90 kDa, respectively, that migrated further into the gel (Fig. 2A). At a presumed 1:1 molar ratio of FXa to PN-2/AbetaPP some cleavage in PN-2/AbetaPP occurred (Fig. 2A). As the concentration of FXa to PN-2/AbetaPP increased from 4:1 to 16:1, all of the 124-kDa starting material was converted into lower molecular mass bands at 116, 97, and 90 kDa. Four-fold molar excess FXa to PN-2/AbetaPP liberated a single 8.2-kDa peptide which was detected on a reduced 18% SDS-PAGE (data not shown). The amino terminus sequence of this peptide was LEVPTDGNAG . . . which is the known amino-terminal sequence of PN-2/AbetaPP after cleavage of its signal peptide at alanine 17 (Fig. 3). The liberated peptide was a single peptide because on multiple gel electrophoreses using different percentage acrylamide gels (15-22%), only this single amino-terminal sequence was obtained. These data indicated that FXa liberated an amino-terminal peptide from PN-2/AbetaPP.


Figure 2: PN-2/AbetaPP is a substrate of FXa. Panel A, PN-2/AbetaPP (1.0 µg or 8.3 pmol) were incubated with equal to 16-fold molar excess FXa in 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4, in the presence of 2 mM Ca for 1 h at room temperature. The reactions were stopped with sample buffer and applied nonreduced to a 6% SDS-PAGE. The samples were electroblotted onto nitrocellulose and an immunoblot was performed using monoclonal antibody P2-1. The immunoblot was detected by chemiluminescence. The figure is a photograph of an autoradiogram. PN-2 represents the immunoblot of 8.3 pmol of PN-2/AbetaPP being applied to the SDS-PAGE. Ratio 1:1 to 1:16 represent the ratio of PN-2/AbetaPP to FXa (mol/mol) in the incubation mixture. Panel B, the figure represents a Coomassie-stained 6% SDS-PAGE of reduced 83 pmol of PN-2/AbetaPP alone (PN2) or 332 pmol of FXa and 83 pmol of PN-2/AbetaPP, ratio 4:1 of FXa to PN-2/AbetaPP (Xa PN2). The numbers and stained bands to the right of the figure represent molecular mass standards (M(r) standards) in kilodaltons. Panel C, the figure represents a photograph of a Coomassie-stained reduced 6% SDS-PAGE of 83 pmol of AbetaPP alone (695) or 332 pmol of FXa and 83 pmol of AbetaPP, ratio of 4:1 of FXa to AbetaPP (Xa 695). The numbers and stained bands to the right of the figure represent molecular mass standards (M(r) standards) in kilodaltons.




Figure 3: The FXa cleavage site in PN-2/AbetaPP. The figure is the amino-terminal sequence of PN-2/AbetaPP using the single letter code for each amino acid. The arrow after alanine 17 represents the cleavage site for the single peptide for PN-2/AbetaPP (Ponte et al., 1988). The arrow after arginine 102 represents the FXa cleavage site in PN-2/AbetaPP.



Further investigations sought the FXa cleavage site on the amino-terminal side of PN-2/AbetaPP. When PN-2/AbetaPP (a major band at 124 kDa and two minor bands at 105 and 98 kDa) was cleaved by 4-fold molar excess FXa, three new corresponding lower molar mass bands were detected (a major one at 115 kDa and two minor bands at 97 and 90 kDa) when the sample was reduced and electrophoresed on a 6% SDS-PAGE, as detected by a Coomassie Blue staining (Fig. 2B). The amino-terminals of each of these three bands were sequenced and a single amino acid sequence (KQCKTHPHFV . . . ) was found for all three of these bands (Fig. 3). These data indicated that molar excess FXa to PN-2/AbetaPP cleaved PN-2/AbetaPP at a single site after arginine 102. Additional investigations showed that AbetaPP, a form of AbetaPP which does not contain the KPI domain and does not inhibit FXa, also was a substrate of the enzyme (Fig. 2C). These data indicated that inhibition of FXa was independent of the inhibitor being a FXa substrate. Further isolated KPI domain was not cleaved by FXa (data not shown).

PN-2/AbetaPP Inhibits Factor Xa in the Prothrombinase Complex

The possible importance of PN-2/AbetaPP and its KPI domain to inhibit FXa is dependent upon whether these proteins produce inhibition in biologic assays. Studies were performed to determine if PN-2/AbetaPP and its KPI domain could inhibit FXa in the prothrombinase complex. Initial experiments determined the K(m) and k of prothrombin activation on phospholipid vesicles in the presence of FVa. By double reciprocal plot, FXa activation of FII in the presence of PSPC and FVa was shown to have a mean K(m) of 0.62 µM (range 0.23-1.6 µM) with a V(max) of 7 nM alpha-thrombin formed min (range 3.6-19.1 nM min) (Fig. 4). These values calculated to a k of 7 min and k/K(m) of 10.2 µM min. Both PN-2/AbetaPP and its KPI domain blocked FXa activity in the prothrombinase complex (Table 2). When FXa and PN-2/AbetaPP or its KPI domain were preincubated for 5 min with PSPC and FVa prior to the addition of FII and chromogenic substrate, the K(i) were 1.9 ± 1.5 times 10M and 1.3 ± 0.8 times 10M, respectively (Table 2). These results only were 2.8- and 4.9-fold more inhibition for PN-2/AbetaPP and its KPI domain, respectively, than the K(i) determined (5.3 ± 2.5 times 10M and 6.4 ± 2.3 times 10M, respectively) when the incubation mixture included the inhibitor, PSPC, FVa, and FII and the reaction was started by the addition of FXa (Table 2). Under these latter conditions, 100-fold molar excess of substrate (i.e. 1 µM FII) had little influence on PN-2/AbetaPP (2-10 nM) blocking 1 nM FXa. Regardless of the order of addition of reactants, both PN-2/AbetaPP and its KPI domain inhibited FXa to the same degree, indicating that the inhibitory domain of PN-2/AbetaPP for FXa was confined to the KPI region and not due to being a competing substrate. In the prothrombinase complex, 4 orders of magnitude molar excess KPI domain did not bring the level of FXa activity to zero. When samples were prepared for measurement of FXa enzymatic activity by a chromogenic assay and measurement of prothrombin fragment F, the degree of inhibition seen was the same (Table 3). These data indicated that PN-2/AbetaPP and its KPI domain did not inhibit alpha-thrombin, consistent with other reports (Van Nostrand et al., 1990a; Smith et al., 1990; Schmaier et al., 1993).


Figure 4: Substrate/velocity plot (A) and double reciprocal plot (B) of FII activation by FXa. End point rates of FII activation (mean ± S.E.) were determined as described under ``Experimental Procedures'' at each of the various concentrations of added FX as indicated in the graph. The graph is the result of FII activation by FXa (1.0 nM) in the presence of PSPC (25 µM), FVa (5 nM), and 2 mM Ca. The plotted results at each point are the mean ± S.E. of four independent experiments.







Investigations were next performed to determine the comparative inhibitory abilities of PN-2/AbetaPP versus antithrombin III and heparin on isolated FXa and FXa in the prothrombinase complex (Table 4). When PN-2/AbetaPP was incubated for 5 min with isolated FXa in the presence of polylysine, its second-order association rate constant (k) (1.4 ± 0.4 times 10^7M min) for FXa inhibition was 5-fold higher than that of antithrombin III and heparin (k = 3.0 ± 3.3 times 10^6M min). Interestingly if heparin were present in the reaction mixture with PN-2/AbetaPP, no inhibition of FXa was detected (data not shown). Alternatively, if polylysine was absent from the reaction mixture, the inhibitory abilities of PN-2/AbetaPP and antithrombin III were reversed. PN-2/AbetaPP inhibited FXa with a k = 3.0 ± 2.0 times 10^5M min; antithrombin III and heparin blocked FXa with a k = 1.3 ± 0.3 times 10^7M min. The data indicated that polylysine had opposite effects on both PN-2/AbetaPP and antithrombin III. In the prothrombinase complex, the biologically relevant assay, the k = 1.8 ± 0.7 times 10^6M min for PN-2/AbetaPP was essentially the same as the k = 3.0 ± 0.2 times 10^6M min seen with antithrombin III and heparin. These data indicated that in the prothrombinase complex, PN-2/AbetaPP in the absence of heparin and antithrombin III and heparin were equipotent inhibitors of FXa.




DISCUSSION

The present investigations expand the coagulation protease inhibitory spectrum of PN-2/AbetaPP. Although first recognized as a hemostatic inhibitor to factor XIa (Smith et al., 1990; Van Nostrand et al., 1990b), recent investigations have shown that PN-2/AbetaPP also is a potent inhibitor of factor IXa (Schmaier et al., 1993, 1995) and its KPI domain has some inhibitory activity to tissue factor-factor VIIa (Dennis and Lazarus, 1994a, 1994b). Initial reports suggested that PN-2/AbetaPP was not an inhibitor of FXa to any great extent (Smith et al., 1990; Van Nostrand et al., 1990b); however, other reports, consistent with coagulant assays, suggest otherwise (Kitaguchi et al., 1990; Petersen et al., 1994; Schmaier et al., 1993). Our investigations indicate that PN-2/AbetaPP is a direct inhibitor of FXa both as an isolated protein and in the prothrombinase complex. The inhibitory activity of PN-2/AbetaPP resides completely in its KPI domain because both the parent protein and its isolated KPI domain inhibit FXa to the same degree. Although the stoichiometry of inhibition appears to be 1:1, inhibition by the KPI domain does not appear to be active site-directed. Four orders of magnitude for the KPI domain to FXa does not reduce the FXa activity to zero, both in assays of isolated FXa and FXa in the prothrombinase complex. These data are different from those found with FIXa. Infinite concentrations of KPI domain abolished FIXa activity in the tenase complex (Schmaier et al., 1995). Nonactive site-directed FXa inhibitors also recently have been described for the hookworm-derived inhibitor of human FXa (Cappello et al., 1995).

We found that when using a polylysine-based FX activation assay, some of the measured inhibition of FIXa by PN-2/AbetaPP can be accounted for by PN-2/AbetaPP inhibiting generating FXa. Since the degree of FXa generated in this assay is small (1-2 nM), the concentration of PN-2/AbetaPP or KPI domain present in the assays would have been sufficient to inhibit both FIXa and the generated FXa (Schmaier et al., 1993, 1995). It also was of interest to learn that polylysine itself potentiated the degree of inhibition of FXa by PN-2/AbetaPP and reduced that of antithrombin III/heparin. The mechanism for this independent activity of polylysine is not known. Since polylysine itself can be an independent variable contributing to PN-2/AbetaPP's inhibitory ability, it should probably be avoided in assays of FIXa.

In addition to inhibition of enzymatic activity, we were able to demonstrate a physical interaction between PN-2/AbetaPP and FXa. On a solid phase binding assay, specific complex formation was detected between PN-2/AbetaPP and FXa. This information suggests that PN-2/AbetaPP is an inhibitor of FXa of the slow, tight class characteristic of Kunitz type inhibitors. PN-2/AbetaPP was also a substrate of FXa when the enzyme was in molar excess to inhibitor. It appears that FXa proteolyzes the major band of PN-2/AbetaPP at 124 kDa and the two minor bands (105 and 98 kDa) into corresponding lower molecular mass species (116, 97, and 90 kDa, respectively), each with the same new amino terminus as seen on immunoblot and Coomassie-stained gels. It is possible that FXa also cleaves PN-2/AbetaPP at a single point on the carboxyl-terminal side of the protein liberating an approximate 30-34-kDa protein. This result would explain the intensification of the post-cleaved 90-kDa band of PN-2/AbetaPP seen in Fig. 2, A and B. However, we never have found any evidence of such a band on our Coomassie-stained gels since it would be migrating with one of the subunits of FXa and thus be hidden. The fact that PN-2/AbetaPP is a substrate to molar excess FXa does not indicate that its mechanism of inhibition of the enzyme is substrate inhibition. First, AbetaPP is a substrate of FXa but it is not a FXa inhibitor. Second, the isolated KPI domain of PN-2/AbetaPP which does not contain the FXa cleavage site inhibits FXa to the same degree as its parent protein. Third, the isolated KPI domain is not cleaved by FXa. It is of interest that FXa cleaves PN-2/AbetaPP through its heparin binding domain. Since heparin neutralizes PN-2/AbetaPP's inhibitory activity on FXa, cleavage through this domain may preserve the inhibitory function of PN-2/AbetaPP for FXa.

PN-2/AbetaPP is a potent anticoagulant of FXa in the prothrombinase complex. In our laboratory the K(m) and k/K(m) ratio of prothrombin activation by FXa is 0.62 µM and 10.2 µM min, respectively, results which are comparable to the findings of other investigators (Krishnaswamy et al., 1987) using a fluorescent marker instead of a chromogenic substrate to monitor prothrombin activation. The degree of inhibition of FXa by PN-2/AbetaPP and its KPI domain is to the same order of magnitude in the prothrombinase complex as with the isolated pure enzyme. Regardless of the order of addition of reactants, PN-2/AbetaPP and its KPI domain inhibit FXa on PSPC. PSPC in the presence of FVa must have oriented FXa such that it was susceptible to inhibition by PN-2/AbetaPP or its KPI domain even though the inhibitors were competing with 5 orders of magnitude more substrate. The finding that PN-2/AbetaPP and its KPI domain inhibit FXa in the prothrombinase complex make this class of inhibitors more important than what would be appreciated by just examining isolated FXa inhibition. Since PN-2/AbetaPP is not a plasma protein but rather a cell surface-associated protease inhibitor, influencing FXa activity in the prothrombinase complex suggests that this class of Kunitz-type protease inhibitors may be important regulators of various hemostatic enzymes. In fact, PN-2/AbetaPP and its homologue, amyloid beta-protein precursor-like protein-2, may constitute a new class of serine protease inhibitors modulating hemostasis (Sprecher et al., 1993).

Although our investigations show that artificial agents can influence the degree of FXa inhibition by PN-2/AbetaPP or antithrombin III and heparin, in the prothrombinase complex assembly, PN-2/AbetaPP and antithrombin and heparin were comparable inhibitors. In the absence of added heparin, the degree of antithrombin III inhibition of FXa was orders of magnitude less potent than that seen with PN-2/AbetaPP. In plasma, antithrombin III would be the predominant inhibitor because its plasma concentration is 4 µMversus the 30 nM level of PN-2/AbetaPP which may be achievable in plasma when platelets are activated (Van Nostrand, et al., 1991). However, it is not known which may be the predominant inhibitor on cell membranes. The second-order rate constants for antithrombin III and heparin inhibition of FXa that we obtained were 1-2 orders of magnitude higher than that reported by other investigators (Olson et al., 1992; Ellis et al., 1982). Differences in heparin preparations and concentrations and ionic strengths in the buffers may account for these variations. Alternatively, in the presence of heparin, PN-2/AbetaPP did not inhibit FXa. PN-2/AbetaPP is known to have a heparin binding domain which allows heparin to potentiate its inhibition of factor XIa but not factor IXa (Smith et al., 1990; Van Nostrand et al., 1990b; Schmaier et al., 1993). It is of interest that the FXa cleavage site on PN-2/AbetaPP is at arginine 102 which is in the heparin binding region of the protein (Small et al., 1994). Excess FXa could be preventing PN-2/AbetaPP from associating with heparin on cell membranes. These investigations show that PN-2/AbetaPP in the absence of heparin and antithrombin III in the presence of heparin are the naturally occurring inhibitors of FXa. Isolated KPI domain of PN-2/AbetaPP is a fragment from a naturally occurring human protein which may have potential use as an anticoagulant since its inhibitor activity is equal to the tick anticoagulant peptide (Waxman et al., 1990).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL49566 (to A. H. S. and W. E. V. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Hematology and Oncology, Dept. of Internal Medicine, University of Michigan, 102 Observatory St., Ann Arbor, MI 48109-0724. Tel.: 313-747-3124; Fax: 313-764-2566: aschmaier{at}uv1.im.med.umich.edu.

(^1)
The abbreviations used are: AbetaPP, amyloid beta-protein precursor; PN-2, protease nexin-2, AbetaPP, or AbetaPP; KPI, Kunitz protease inhibitor; FIXa, human factor IXa; FXa, activated human coagulation factor X; FVa, thrombin-activated coagulation factor V; FII, human coagulation factor II (prothrombin); PSPC, phosphatidylserine/phosphatidylcholine vesicles; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Kenneth A. Bauer for performing the factor X activation peptide release assays. We appreciate the technical assistance of Linda D. Dahl.


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