Mechanisms of Factor Xa-catalyzed Cleavage of the Factor VIIIa A1 Subunit Resulting in Cofactor Inactivation*

Keiji NogamiDagger , Hironao WakabayashiDagger , and Philip J. FayDagger §

From the Departments of Dagger  Biochemistry and Biophysics and § Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, December 20, 2002, and in revised form, February 4, 2003

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Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg336, which precedes the highly acidic C-terminal region, and a recently identified site at Lys36. By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A137-336, was shown to proceed via two pathways identified by the intermediates A11-336 and A137-372 and generated by initial cleavage at Arg336 and Lys36, respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an ~3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by ~2-fold. Although association of A1 or A137-372 with A3-C1-C2 enhanced the rate of cleavage at Arg336, inclusion of A3-C1-C2 did not affect the cleavage at Lys36 in A11-336. A synthetic peptide 337-372 blocked the cleavage at Lys36 (IC50 = 230 µM) while showing little if any effect on cleavage at Arg336. Proteolysis at Lys36, and to a lesser extent Arg336, was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys36 and Arg336 are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys36 appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.

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Factor VIII, a plasma protein deficient or defective in individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid surface-dependent conversion of factor X to Xa (1). Factor VIII is synthesized as an ~300-kDa precursor with domain structure A1-A2-B-A3-C1-C2 and is processed to a series of divalent metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1-A2-B domains and a light chain consisting of the A3-C1-C2 domains (2-4). Factor VIII is activated by thrombin or factor Xa by limited proteolysis at the A1-A2 domain junction (Arg372), A2-B junction (Arg740), and near the N terminus of the light chain (Arg1689) (5). The resultant factor VIIIa, a heterotrimer composed of A1, A2, and A3-C1-C2 subunits (6, 7), retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with weak affinity (Kd ~260 nM (8)) by primarily electrostatic interactions (7, 9). Thus at physiological concentrations (~1 nM), factor VIIIa is unstable, and loss of activity is attributed to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (7, 9, 10).

The association of factor VIIIa with factor IXa increases the kcat for factor Xa formation by several orders of magnitude compared with factor IXa alone (11). Recent studies have shown that modulation of factor IXa by A2 subunit enhances the kcat for factor Xa activation by ~100-fold (12) and that A1 subunit synergizes this effect (>15-fold) to alter the interaction of A2 subunit with factor IXa (13, 14). The C-terminal acidic region of the A1 subunit (residues 337-372) appears to represent an A2-interactive site and participates in the orientation of A2 yielding maximal stimulation of factor IXa (14). This region is also implicated in the binding of factor X (15), and recent evidence suggests that this contributes to the Km value for factor X interaction with factor Xase (16). Several proteases including activated protein C (5, 17), factor IXa (18, 19), and factor Xa (5) attack this site cleaving at Arg336 in the A1 subunit, and this event correlates with inactivation of the cofactor. Although the mechanism for inactivation following cleavage at this site is not fully understood, component effects include altered interaction of the A2 subunit with the truncated A1 and an increase in the Km value for substrate factor X. Recently, a second cleavage site specific for factor Xa, Lys36, has been identified in the A1 subunit (16). Attack at this site is also inactivating, and this effect results in part from altered conformation of A1 limiting productive interaction with the A2 subunit (16).

Although factor Xa functions as an activator of factor VIII, subsequent factor Xa-catalyzed cleavage(s) within the A1 subunit of factor VIIIa may contribute to the dampening of factor Xase activity. This potential role may be significant given that the protease is the product of the reaction catalyzed by factor Xase. However, the interactions of factor Xa with substrate factor VIIIa (A1 subunit) are poorly understood. In this study, we report on the catalytic mechanism during proteolytic inactivation due to cleavage within the A1 subunit by factor Xa. By using isolated subunits of the cofactor and by employing functional reconstitution assays and SDS-PAGE analyses, we show the initial reaction of the A1 subunit with the protease yields two distinct intermediates indicative of separate pathways leading to the terminal A137-336 product. Furthermore, specific cleavages at Lys36 and Arg336 are differentially modulated following interaction of the protease with the A3-C1-C2 subunit, as well as the acidic C-terminal region of the A1 subunit.

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Reagents-- Purified recombinant factor VIII preparations were generous gifts from Bayer Corp. (Berkeley, CA) and the Genetics Institute (Cambridge, MA). The monoclonal antibody 58.12 recognizing the N-terminal end of A1 (20) was a gift from Dr. James Brown, and monoclonal antibody C5 recognizing the C-terminal end of A1 (21) was a gift from Drs. Carol Fulcher and Zaverio Ruggeri. Phospholipid vesicles containing 20% phosphatidylserine, 40% phosphatidylcholine, and 40% phosphatidylethanolamine (Sigma) were prepared using N-octyl glucoside (22). TAP1 was a gift from Dr. Sriram Krishnaswamy. The reagents human alpha -thrombin, factor IXa, factor X, and factor Xa (Enzyme Research Laboratories, South Bend, IN) and the chromogenic Xa substrate S-2765 (N-alpha -benzyloxycarbonyl-D-arginyl-glycyl-L-arginyl-p-nitroanilide-dihydrochloride; DiaPharm Group, Westchester, OH) were purchased from the indicated vendors. The synthetic peptide corresponding to factor VIII residues Met337-Arg372 (designated as peptide337-372) was prepared by the Biotechnology Analytical and Synthesis Facility at Cornell University and has been described previously (23). Peptide337-359 and peptide360-372 were derived from a tryptic digest of peptide337-372 and purified as described previously (23). The peptide351-365 was a gift from Dr. Paul Foster.

Isolation of Factor VIIIa Subunits-- Factor VIII (1.5 µM) was treated overnight at 4 °C in buffer containing 10 mM MES, pH 6.0, 0.25 M NaCl, 50 mM EDTA, and 0.01% Tween 20, and the light and heavy chains were isolated following chromatography on SP-Sepharose and Q-Sepharose columns (Amersham Biosciences) as described previously (24). The purified heavy chain was cleaved by thrombin, and the A2 and A1 subunits were purified by fast protein liquid chromatography using a Hi-Trap heparin column and a Mono-Q column as reported previously (14). The A3-C1-C2 subunit was prepared as described previously (15). The A1/A3-C1-C2 dimer was isolated from thrombin-treated factor VIIIa using a Mono-S column chromatography, and the residual A2 subunit was removed using an anti-A2 subunit monoclonal antibody coupled to Affi-Gel 10 (20). The A3-C1-C2 subunit was further purified following dissociation of the dimer by EDTA and chromatography on Mono-S (15). A truncated A11-336 subunit was prepared as described earlier (14). SDS-PAGE of the isolated subunits showed >95% purity. Protein concentrations were determined by the method of Bradford (25).

Cleavage of A1 or A1/A3-C1-C2 by Factor Xa-- The A1/A3-C1-C2 dimer was reconstituted from isolated A1 and A3-C1-C2 subunits (2.4 µM each) overnight at 4 °C in 20 mM HEPES, pH 7.2, 0.3 M NaCl, 25 mM CaCl2, and 0.01% Tween 20. Human factor Xa was added to the isolated A1 subunit or A1/A3-C1-C2 dimer in a 1:10 ratio (mol/mol) in a buffer containing 20 mM HEPES, pH 7.2, 0.1 M NaCl, 5 mM CaCl2, and 0.01% Tween 20, and the reactions were run at 22 °C. Samples were taken at the indicated times, and the reactions were immediately terminated and prepared for SDS-PAGE by adding SDS and boiling for 3 min.

Factor Xa Generation Assays-- The rate of conversion of factor X to factor Xa was monitored in a purified system (26). To prepare A1/A3-C1-C2 dimers containing the cleaved A1 subunit, the cleavage reaction was quenched using TAP at an ~5-fold molar excess relative to factor Xa. This mixture was adjusted to 500 nM and was reconstituted with 500 nM A3-C1-C2 subunit. Factor VIIIa was reconstituted from the A1/A3-C1-C2 dimer form(s) (5 nM) plus A2 subunit (20 nM), and reacted with 20 nM factor IXa and 10 µM phosphatidylserine-phosphatidylcholine-phosphatidylethanolamine vesicle for 30 s in 20 mM HEPES, pH 7.2, 0.05 M NaCl, 5 mM CaCl2, 100 µg/ml bovine serum albumin, and 0.01% Tween 20. Reactions were initiated with the addition of 500 nM factor X. Aliquots were removed at appropriate times to assess initial rates of product formation and added to tubes containing EDTA (final concentration; 50 mM) to stop the reaction. Rates of factor Xa generation were determined by the addition of the chromogenic substrate, S-2765 (final concentration; 0.46 mM). Reactions were read at 405 nm using a Vmax microtiter plate reader (Molecular Devices, Sunnyvale, CA). All reactions were run at 22 °C.

Electrophoresis and Western Blotting-- SDS-PAGE was performed with 8% gels using the procedure of Laemmli (27). Electrophoresis was carried out using a Bio-Rad minigel apparatus at 150 V for 1 h. Bands were visualized following staining with GelCode Blue Stain Reagent (Pierce). For Western blotting, the proteins were transferred to a polyvinylidene difluoride membrane using a Bio-Rad mini-transblot apparatus at 50 V for 2 h in buffer containing 10 mM CAPS, pH 11, and 10% (v/v) methanol. Proteins were probed using the 58.12 and C5 monoclonal antibodies followed by goat anti-mouse alkaline phosphatase-linked secondary antibody. The signal was detected using the ECF system (Amersham Biosciences), and the blots were scanned at 570 nm using a Storm 860 instrument (Molecular Devices). Densitometric scans were quantitated using ImageQuant software (Molecular Devices).

Data Analysis-- The concentrations of remaining intact A1 and factor Xa-cleaved A1 products were calculated from band densities using the formula A1 = (density of A1 at the indicated time/density of initial A1) × initial A1 concentration, or A1 fragment = (density of A1 fragment at the indicated time/density of initial A1) × (mol mass A1/mol mass fragment) × initial A1 concentration, respectively. In order to evaluate the catalytic efficiency of factor Xa, we calculated the cleavage rate constants based on the densitometric values. Assuming the cleavage event and release of products are rapid, the concentration of free factor Xa should be constant. Therefore, the rate constant correlates with the concentration of substrate. The apparent rate constant (k) for A1 subunit cleavage by factor Xa was determined by employing Equation 1,


[A]<SUB>t</SUB>=[A]<SUB>0</SUB>·e<SUP>−kt</SUP> (Eq. 1)
where [A]t is the intact A1 subunit concentration at time point t.

The apparent rate constant values (k11, k12, k21, and k22) in the following Scheme 1 are based on parallel and series reactions for intact A1 subunit cleavage by factor Xa and were estimated by nonlinear least squares regression using multiple equations as described in Equations 2-5.
[A]<SUB>t</SUB>=[A]<SUB>0</SUB>·e<SUP>−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)t</SUP> (Eq. 2)

[B1]<SUB>t</SUB>=<FR><NU>[A]<SUB>0</SUB>·k<SUB>11</SUB>·(e<SUP>−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)t</SUP>−e<SUP>−(k<SUB>12</SUB>+k<SUB>4</SUB>)t</SUP>)</NU><DE>(k<SUB>12</SUB>+k<SUB>4</SUB>)−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)</DE></FR> (Eq. 3)

[B2]<SUB>t</SUB>=<FR><NU>[A]<SUB>0</SUB>·k<SUB>21</SUB>·(e<SUP>−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)t</SUP>−e<SUP>−(k<SUB>22</SUB>+k<SUB>4</SUB>)t</SUP>)</NU><DE>(k<SUB>22</SUB>+k<SUB>4</SUB>)−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)</DE></FR> (Eq. 4)

[C]<SUB>t</SUB>=<FR><NU>(K<SUB>1</SUB>+K<SUB>2</SUB>)·(e<SUP>−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)·t</SUP>−e<SUP>−k<SUB>4</SUB>·t</SUP>)</NU><DE>k<SUB>4</SUB>−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)</DE></FR>

−<FR><NU>K<SUB>1</SUB></NU><DE>k<SUB>11</SUB></DE></FR>·(e<SUP>−(k<SUB>12</SUB>+k<SUB>4</SUB>)·t</SUP>−e<SUP>−k<SUB>4</SUB>·t</SUP>)−<FR><NU>K<SUB>2</SUB></NU><DE>k<SUB>22</SUB></DE></FR>·(e<SUP>−(k<SUB>22</SUB>+k<SUB>4</SUB>)·t</SUP>−e<SUP>−k<SUB>4</SUB>·t</SUP>) (Eq. 5)

<FENCE>K<SUB>1</SUB>=<FR><NU>[A]<SUB>0</SUB>·k<SUB>11</SUB>·k<SUB>12</SUB></NU><DE>(k<SUB>12</SUB>+k<SUB>4</SUB>)−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)</DE></FR>, K<SUB>2</SUB>=<FR><NU>[A]<SUB>0</SUB>·k<SUB>21</SUB>·k<SUB>22</SUB></NU><DE>(k<SUB>22</SUB>+k<SUB>4</SUB>)−(k<SUB>11</SUB>+k<SUB>21</SUB>+k<SUB>3</SUB>)</DE></FR></FENCE>
where [A]t, [B1]t, [B2]t, and [C]t are the concentrations at time point (t) of intact A1 and A1 fragments consisting of residues 37-372 (A137-372), residues 1-336 (A11-336), and residues 37-336 (A137-336). We observed a reduction in total staining intensity for these fragments as the time course progressed, in parallel with the appearance of diffuse, low molecular mass staining material. For this reason it was necessary to incorporate expressions for rate values for these small degradation products (designated D1-D4). Because the initial loss of mass was greatest at the initial time points, we assume k3 > k4. Furthermore, rate values for k4 could not be differentiated for species designated B1, B2, and C.


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Scheme 1.  


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Proteolysis of A1 Subunit by Factor Xa-- We recently demonstrated that factor Xa cleaves the A1 subunit (residues 1-372) of factor VIIIa at Lys36 (16) in addition to Arg336 (5). Identification of reaction products was determined following reaction of the isolated A1 subunit (2.4 µM) with factor Xa (0.24 µM). Fig. 1A shows the stained gel following electrophoresis of intact A1 and the products of A1 subunit cleavage by factor Xa after reaction for 4 h at 22 °C. Three fragments were observed in addition to the intact subunit. These fragments were further characterized by Western blot analysis employing anti-A1 domain monoclonal antibodies specific for the N- or C-terminal sequences. Although the intact A1 (fragment 1) and fragment 3 were reactive with monoclonal antibody 58.12 (an antibody directed to residues 1-12 of the A1 sequence), fragments 2 and 4 were not identified (Fig. 1B, a). Use of the C5 antibody (specific for residues contained within the C-terminal acidic region of A1) showed reactivity with the intact A1 (fragment 1) and fragment 2 but did not react with either fragments 3 or 4 (Fig. 1B, b). These results, taken together with identified factor Xa cleavage sites for intact A1 (A11-372) at Lys36 and Arg336, identify fragment 2 as A1 residues 37-372 (A137-372), fragment 3 as A1 residues 1-336 (A11-336), and fragment 4 as A1 residues 37-336 (A137-336). Fig. 1C schematically illustrates the products of A1 digestion by factor Xa. Pretreatment of factor Xa with a molar excess of TAP failed to yield any proteolysis of A1 subunit, indicating that both cleavages were catalyzed by factor Xa (data not shown).


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Fig. 1.   SDS-PAGE and Western blot of native and factor Xa-cleaved A1 forms. A, purified A1 subunit (2.4 µM) was reacted with factor Xa at 22 °C for 4 h as described under "Materials and Methods." Samples were run on an 8% gel and stained with GelCode Blue. Lane a, intact A1 subunit; lane b, A1 following reaction with factor Xa. B, Western blot of the factor Xa-treated A1 using anti-A1 monoclonal antibodies 58.12 specific for the N-terminal region (lane a) and C5 specific for the C-terminal region (lane b). C, schematic of the cleaved A1 fragments generated following reaction with factor Xa.

Factor Xa Cleavage of A1 Subunit Occurs by Two Pathways-- Time course assays were used to correlate rates of cleavages in the A1 subunit and residual cofactor activity following the reconstitution of the factor Xa-treated A1 with other factor VIIIa subunits. Fig. 2, A and B, shows the results of SDS-PAGE of the cleavage reactions. Densitometry scanning of the stained gel was employed to quantitate band density of the substrate A1 and each cleavage product. The A11-372 subunit gradually diminished following addition of factor Xa, and degradation was essentially complete by 8 h. Appearance of the A137-372 and A11-336 fragments correlated with loss of A11-372. However, rates of generation and degradation of these two forms were not identical. Generation of A137-372 appeared somewhat faster compared with the A11-336 based upon the initial slope of the curve, and the former was obtained in greater yield based upon total band density. Subsequently, complete degradation of A137-372 occurred within 10 h of peak level, while the A11-336 persisted at ~30% of peak level for 18 h, suggesting a markedly reduced rate of cleavage of the latter compared with the former. The A137-336, truncated at both termini, appeared after a lag of ~2 h, and its generation increased over the remainder of the time course (24 h). This band showed no loss in density over an extended (24 h) reaction time suggesting the fragment represented a terminal digestion product.


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Fig. 2.   Time course of proteolysis and cofactor activity of A1 subunit following reaction with factor Xa. A, SDS-PAGE of A1 subunit (2.4 µM) was reacted with factor Xa (0.24 µM) as described under "Materials and Methods." At the indicated times, the reaction was terminated, and samples were run on an 8% gel and stained with GelCode Blue. The cleaved A1 sample (adjusted at 500 nM) was reconstituted with the A3-C1-C2 (500 nM), followed by reaction with A2 subunit (20 nM). Reconstituted factor VIIIa activity was measured using a factor Xa generation assay as described under "Materials and Methods." B, quantitative evaluation by densitometry. The concentration of each A1 form was determined from scanning densitometry of the stained gel shown in A. The symbols used are as follows: open circles, A11-372; closed circles, A137-372; open squares, A11-336; closed squares, A137-336; and open triangles, total concentration of A1 and A1-derived fragments based upon summing the staining density. Data were fitted to the model described under "Materials and Methods." The inset shows factor VIIIa activity (% of control) (open circles). C, schematic of proposed pathways for A1 cleavage by factor Xa. The arrows show the site of factor Xa cleavage.

The simultaneous generation of A11-336 and A137-372 indicated the presence of two pathways for A1 cleavage of factor Xa to yield the terminal product, and these alternatives are schematically illustrated in Fig. 2C. Estimation of apparent rate constants for each cleavage reaction was derived from fitting the data shown in Fig. 2B to equations by a parallel series reaction as described under "Materials and Methods." Based upon staining density, the total concentration of intact A1 plus the fragments A11-336, A137-372, and A137-336 (Fig. 2B, triangles) decreased during the reaction time course. This result suggested additional, minor cleavage pathways and was supported by the appearance of diffuse staining, low molecular mass fragments in the gels (data not shown). To account for these auxiliary fragments, the model incorporated conversion of the initial substrate, intermediates, and terminal fragment to degradation products designated D1-D4 as described under "Materials and Methods." Because the greatest rate of loss occurred within the first 2 h, this result suggested the rate constant for D1 derived from degradation of intact A1 subunit was greater than those constants reflecting generation of D2-D4. We assume these latter rates (k4) are equivalent to fairly compare the rate constants (k11, k12, k21, and k22) for the primary cleavage pathways. The data are summarized in Table I. By using the isolated A1 subunit, there appeared to be a modest (2-fold) preference for factor Xa to cleave Lys36 compared with Arg336. However, the apparent cleavage rate at Lys36 in the intact A1 subunit (k11) was ~8-fold greater than that observed for the A11-336 substrate (k22). This result suggested that the C-terminal region (residues 337-372) represented a critical region for factor Xa-dependent cleavage at this site. Conversely, the N-terminal region of A1 subunit (residues 1-36) showed little influence on cleavage at the Arg336 site (k12 ~2-fold greater than k21).


                              
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Table I
Relative rate constants estimated for A1 subunit cleavage by factor Xa
Parameter values were calculated from the fitted data shown in Figs. 2 and 3 using the formula presented under "Materials and Methods." All data are represented as rate constant values (k) × 1000.

The analysis of A1 cleavage was complemented with assessment of the change of cofactor activity for factor VIIIa reconstituted from the cleaved A1. For this analysis, cleavage of A1 was quenched at the indicated time points by addition of TAP, and the A1 forms were reconstituted with an equimolar concentration of A3-C1-C2 as described under "Materials and Methods." The resultant A1/A3-C1-C2 dimer forms were diluted and reacted with A2 (20 nM) to generate factor VIIIa heterotrimers, and cofactor activity was measured in a factor Xa generation assay. The amount of exogenous factor Xa resulting from the cleavage of A1 subunit was measured in a control assay, and the data were corrected prior to plotting. Loss of cofactor activity as a result of A1 cleavage was correlated with proteolysis of the A11-372 and the appearance of the A137-336 via the intermediates A137-372 and A11-336 (Fig. 2, A and B). However, this inactivation was incomplete, representing ~40% of the activity for cofactor reconstituted with intact A1. This level of residual activity was consistent with the value obtained for factor VIIIa reconstituted with purified A137-336 (16).

Effect of A3-C1-C2 Subunit on A1 Cleavages by Factor Xa-- The above results showed slow rates of cleavage of the isolated A1 subunit at either terminus. Earlier studies have demonstrated optimal reaction rates for factor Xa activity directed against factor VIII substrates require a phospholipid surface (5). Furthermore, a factor Xa interactive site was recently mapped to the C2 domain of the cofactor (28), which also contains residues required for the interaction of cofactor with anionic phospholipid surfaces (29). Thus, these factors likely contribute to catalytic efficiency of factor Xa-catalyzed cleavage of A1. In order to determine the roles for C2 and phospholipid, isolated A1 subunit was reconstituted with isolated A3-C1-C2, and the resultant A1/A3-C1-C2 dimer was employed as substrate for factor Xa in reactions performed in the presence of phospholipid. Comparison of factor Xa cleavage of isolated A1 with that for A1 reconstituted in the dimer is shown in Fig. 3. Time course experiments were run using 2.4 µM of either substrate and 0.24 µM factor Xa at 22 °C as described under "Materials and Methods." Data obtained from the stained gel (Fig. 3A) were subjected to densitometry scans (Fig. 3B) and are summarized in Table I. The cleavage of A1 in the dimer was rapid compared with the isolated subunit. Generation of significant levels of the A11-336 fragment from this substrate appeared within 2 min and reflected an ~6-fold increase in constant k21(+A3C1C2) compared with constant k21(-A3C1C2). Subsequent conversion of this fragment to the terminal digestion product showed little if any influence of the A3-C1-C2 based upon near equivalence of the estimated rate constants (k22(+A3C1C2)/k22(-A3C1C2) ~1). In contrast to the rapid appearance of the A11-336 fragment following cleavage of the dimer, generation of the A137-372 was not significantly enhanced by the presence of the A3-C1-C2 subunit (k11(+A3C1C2)/k11(-A3C1C2) ~1.8). However, subsequent conversion to the A137-336 terminal fragment was markedly enhanced by this association (k12(+A3C1C2)/k12(-A3C1C2) ~6). These results indicate that the cleavage at Arg336 rather than at Lys36 is markedly affected by inclusion of the A3-C1-C2 subunit and the surface, suggesting a selective role for interaction of protease via binding to the C2 domain in catalyzing cleavage at the C-terminal site. Moreover, the cleavage at Arg1721 at the A3-C1-C2 subunit by factor Xa appeared within 2 min and was almost complete by 120 min as described previously (5).


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Fig. 3.   Effect of A3-C1-C2 subunit on cleavage of A1 by factor Xa. A, SDS-PAGE of A1 subunit (2.4 µM) either reconstituted with equimolar A3-C1-C2 subunit or alone reacted with factor Xa (0.24 µM) in the absence or presence of phospholipid vesicles (10 µM). Reactions were performed as described under "Materials and Methods." At the indicated times, the reactions were terminated, and samples were run on an 8% gel followed by staining with GelCode Blue. B, quantitative evaluation by densitometry. The concentration of each A1 form was determined from scanning densitometry of the stained gel shown in A. The symbols used are as follows: open circles, intact A11-372; closed circles, A137-372; open squares, A11-336; closed squares, A137-336. Data were fitted to the model described under "Materials and Methods."

Kinetics of A1 Proteolysis by Factor Xa-- A series of experiments was performed to quantitate the kinetics of A1 cleavage (initial event) and the contributions made by other factor VIIIa subunits and the phospholipid surface. Fig. 4 shows the rate of loss of intact A1 subunit under the different experimental conditions. Results were derived from scanning densitometry of the stained gels, and data points were fitted using Equation 1 as described under "Materials and Methods." The rate of cleavage of the isolated A1 subunit (2.4 µM) by factor Xa (0.24 µM) was independent of phospholipid (8.12 ± 0.39 (×10-3) min-1 and 7.59 ± 0.25 (×10-3) min-1, in the presence and absence of phospholipid, respectively). Association of A1 subunit with the A3-C1-C2 yielded an ~3-fold increased rate of A1 cleavage (19.2 ± 1.09 (×10-3) min-1), and this rate was further increased ~2-fold (40.7 ± 3.04 (×10-3) min-1) in the presence of phospholipid. Reconstitution of the factor VIIIa heterotrimer by subsequent addition of the A2 subunit resulted in no further increase in the rate of A1 subunit cleavage (42.6 ± 4.23 (×10-3) min-1). Overall, the incorporation of A1 subunit in the A1/A3-C1-C2 dimer bound to the phospholipid resulted in an ~6-fold increase in cleavage rate constant compared with isolated A1 subunit alone.


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Fig. 4.   Effect of other factor VIIIa subunits on the cleavage of A1 subunit by factor Xa. A1 subunit (2.4 µM) in the presence (circles) or absence (squares) of A3-C1-C2 subunit (2.4 µM) was reacted with factor Xa (0.24 µM) in the presence (open symbols) or absence (closed symbols) of phospholipid vesicles (10 µM) for 2 h as described under "Materials and Methods." In addition, A1/A3-C1-C2 dimer reconstituted with the A2 subunit (2.4 µM) was reacted with factor Xa (triangles). Samples were run on an 8% gel followed by staining with GelCode Blue, and the concentration of remaining intact A1 was calculated using the formula described under "Materials and Methods." Data were plotted and fitted using Equation 1 described under "Materials and Methods."

Cleavage at Lys36 in A11-336 by Factor Xa-- Results presented above indicate a slow rate of proteolysis of the A11-336 to the A137-336 terminal fragment. In order to further study the mechanism for this cleavage, a purified A11-336 fragment was used as substrate for factor Xa. A11-336 was isolated from intact A1 following cleavage by activated protein C using Mono-S-Sepharose as described previously (14). Cleavage of this intermediate (2.4 µM) by factor Xa (0.24 µM) was evaluated by SDS-PAGE and densitometry (Fig. 5). Surprisingly, the purified A11-336 subunit persisted throughout an extended time course with ~80% of material uncleaved following a 15-h reaction with factor Xa. This observation is consistent with results presented in Fig. 2 showing the slow rate of loss of the A11-336 intermediate when derived from initial cleavage from A1. Furthermore, the apparent rate of this cleavage was not affected by the presence of the A3-C1-C2 subunit. These results support the hypotheses that the acidic C-terminal region of the A1 subunit (residues 337-372) is necessary for the effective cleavage at Lys36, and this cleavage is independent of the A3-C1-C2 subunit. Furthermore, these results suggest that this C-terminal region may represent an interactive site for factor Xa.


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Fig. 5.   Cleavage of purified A11-336 subunit by factor Xa. The isolated A11-336 (2.4 µM) or A11-336/A3-C1-C2 dimer (2.4 µM) was reacted with factor Xa (0.24 µM). Samples were removed at the indicated times and run on an 8% gel followed by staining with GelCode Blue.

Synthetic Peptide 337-372 Inhibits the Cleavage of the A1 Subunit by Factor Xa-- To examine further the possibility that this C-terminal region of A1 contained an interactive site for factor Xa, a synthetic peptide to this region (residues 337-372) was evaluated as a potential inhibitor of A1 proteolysis by factor Xa. In this experiment, the A1 subunit (2 µM) was reacted for an extended period with factor Xa (0.2 µM) in the absence and presence of increasing concentrations of the peptide. Reaction products were visualized by SDS-PAGE and quantitated by scanning densitometry (Fig. 6). In the absence of peptide, A1 subunit was essentially fully converted to the A137-336 terminal product. Increasing concentrations of peptide resulted in a diminution of the terminal product and concomitant increase in the concentration of the A11-336 intermediate, and this effect was dose-dependent (Fig. 6A). Fig. 6B illustrates the relative concentrations of the terminal product and intermediate. At 1 mM peptide, >80% of the intermediate persisted indicating significant inhibition of factor Xa-catalyzed cleavage at Lys36. From these data, an IC50 value for this inhibition was calculated to be 230 µM. Low levels of intact A1 subunit and A137-372 also persisted at the highest levels of peptide employed (~10% of the total protein), suggesting that the peptide had a marginal effect in inhibiting the cleavage at Arg336. Interestingly, neither the three overlapping peptides derived from the 337-372 sequence and representing residues 337-359, 351-365, and 360-372 nor the C5 antibody which binds residues 351-365 (21) inhibited the cleavage of the A1 subunit by factor Xa using the above conditions (results not shown). Taken together, these results suggest that the C-terminal sequence of A1 comprises an extended interactive surface for binding factor Xa, and this association is required for the selective proteolysis of A1 subunit at Lys36.


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Fig. 6.   Inhibition of factor Xa-catalyzed cleavage of A1 subunit by factor VIII peptide337-372. A mixture of A1 subunit (2 µM) and serial dilutions of peptide337-372 was reacted with factor Xa (0.2 µM) at 22 °C overnight. Samples were run on an 8% gel followed by staining with GelCode Blue. A, SDS-PAGE; lane 1 shows intact A1. Lanes 2-7 show A1 subunit plus factor Xa in the presence of peptide (0, 62.5, 125, 250, 500, and 1000 µM, respectively). B, densitometry scan of the stained gel shown in A. Density of the A137-336 band in lane 2 of the stained gel was used to represent the 100% level for the terminal product. The symbols used are as follows: open circles, A137-336; and closed circles, A11-336.

Heparin Blocks A1 Cleavage by Factor Xa-- A hallmark of the C-terminal region of A1 subunit is the high content of acidic amino acids (13 Glu + Asp/36). Thus, this region could define an interactive region for an anion-binding site on the protease. Indeed, a heparin-binding exosite on factor Xa was described recently (30). In order to test whether this exosite contributed to the cleavage of the A1 subunit at Lys36 and/or Arg336, an experiment was performed to evaluate proteolysis of A1 in the presence of heparin (100 and 500 units/ml). Results shown in Fig. 7 indicated a dose-dependent inhibition of factor Xa-catalyzed cleavage of A1 by heparin. Although both cleavages appeared to be inhibited by heparin, it appeared that cleavage of Lys36 was somewhat more reduced than cleavage at Arg336, based upon relative concentrations of the A11-336 intermediate and A137-336 terminal product. These observations support a role for the heparin-binding exosite of factor Xa in the proteolytic inactivation of factor VIIIa as mediated through cleavages at Arg336 and Lys36.


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Fig. 7.   Effect of heparin on A1 subunit proteolysis by factor Xa. The A1 subunit (2.5 µM) was preincubated with heparin for 15 min followed by reaction with factor Xa (0.25 µM) and CaCl2 (5 mM) at 22 °C overnight. Samples were run on an 8% gel followed by staining with GelCode Blue. Lane 1 shows intact A1. Lanes 2-4 show the cleavage of A1 by factor Xa in the presence of heparin (0, 100, 500 units/ml, respectively).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of human factor VIII by thrombin or factor Xa results from limited proteolysis at identical sites. However, the latter protease attacks additional sites in the factor VIII/VIIIa substrate. Cleavage within the A3 domain at Arg1721 (5) appears benign based upon factor VIIIa reconstitution studies using factor VIII light chain cleaved at this site in place of the thrombin-cleaved chain (31). Three additional sites have been mapped within the A1 domain/subunit of factor VIII/VIIIa. Porcine factor VIII is cleaved at Arg219 as a part of the activation process, yielding a factor VIIIa form possessing two subunits derived from the original A1 domain (32). This residue is Gln in other factor VIII molecules and thus is not attacked. Interestingly, porcine factor Xase composed of factor Xa-activated factor VIIIa shows a severalfold lower catalytic efficiency than factor Xase composed of the thrombin-activated cofactor. Factor Xa also cleaves at Arg336 (5), preceding the anionic C-terminal region and Lys36 (16). Proteolysis at these two sites correlates with inactivation of cofactor function. Furthermore, these effects appear additive with factor VIIIa composed of A1336 and A137-336 possessing ~60 and ~30% of native factor VIIIa activity, respectively (16). Cleavage at Arg336 alters the interaction of A1 relative to A2 subunit reflecting reduced kcat values (14), as well as resulting in a severalfold increase in Km of factor Xase for substrate factor X (16). Loss of activity resulting from cleavage of A1 at Lys36 is less well understood but appears to alter its conformation markedly affecting the affinity of this subunit for A2 subunit (16).

In this study we examine the mechanisms of interaction of factor Xa resulting in the limited proteolysis of the A1 subunit, thus reflecting proteolytic pathways leading to inactivation of the cofactor. The identification of two intermediates, A137-372 and A11-336, indicated alternative pathways, designated I and II (Fig. 2C), respectively, leading to the formation of the terminal product, A137-336. Facile determination of the intermediates was achieved following Western blotting with monoclonal antibodies specific for the N and C termini. By using isolated A1 subunit as substrate, pathway I resulting in initial cleavage at Lys36 (k11) demonstrated an ~2-fold faster relative rate than initial cleavage at Arg336 (k21). However, whereas cleavage of A137-372 (k12) occurred at an appreciable rate, cleavage of the A11-336 intermediate (k22) was markedly reduced. This result suggested a role for the C-terminal acidic region (residues 337-372) in this catalytic step. This conclusion was supported by several observations including a slow rate of cleavage of Lys36 using a purified A11-336 subunit and selective inhibition of cleavage at this site by the 337-372 peptide.

Reconstitution of A1 subunit with the A3-C1-C2 subunit to form the A1/A3-C1-C2 dimer and inclusion of a phospholipid surface resulted in disparate effects on the cleavages catalyzed by factor Xa. Although the rate for initial cleavage at Lys36 was marginally increased in the dimer (k11(+A3-C1-C2)/k11(-A3-C1-C2) ~1.8), the rate for initial cleavage at Arg336 was increased ~8-fold. This result suggested a differential regulation in the two cleavage reactions and suggested that cleavage of Arg336 was facilitated by interaction of the protease with the surface-bound substrate. Recently, a factor Xa interactive site was identified in the C2 domain of factor VIII (28). Results presented in that study showed the C2 domain-specific antibody NMC-VIII/5 did not block factor Xa-catalyzed activation of factor VIII (cleavage at Arg372). Interestingly, examination of the gel presented in that report shows no subsequent cleavage of the A1 subunit in the presence of the antibody. This result suggested that (i) interaction of factor Xa with C2 may facilitate cleavage of A1 subunit, and (ii) factor Xa may interact differentially with this domain in catalyzing cleavages that result in activation compared with those resulting in inactivation.

Although cleavage at Arg336 appears to be selectively enhanced by contribution of protease binding C2, cleavage at Lys36 appears to be modulated by the acidic C-terminal region of the A1 subunit. Earlier studies have indicated that this region constitutes a factor X interactive site. Solid phase binding analyses indicated an affinity of factor X for A1 subunit of ~1-3 µM (15) and showed that cleavage of A1 at Arg336 by activated protein C abrogated this interaction (33). Zero-length cross-linking studies identified a salt bridge(s) between residues in this region of A1 and a portion of the protease domain of the zymogen exclusive of the activation peptide (34). Based upon these observations, and the results of the present study showing markedly reduced rates of cleavage at Lys36 using the A11-336 substrate compared with intact A1 and specific inhibition of attack at this site by the 337-372 peptide, we suggest that this C-terminal region of A1 constitutes an interactive site for factor Xa as well as factor X.

Previous studies (30) have identified a series of basic residues in the heparin-binding exosite of factor Xa. In addition to binding heparin, some of these residues contribute to factor Xa interaction with cofactor and/or substrate recognition by prothrombinase. Results from the current study show selective inhibition by heparin in factor Xa-catalyzed cleavage at the Lys36 and Arg336 sites. Although high concentrations of heparin inhibited proteolysis at both sites, a lower level of heparin selectively blocked cleavage at the Lys36 site. These results suggest involvement of the heparin-binding exosite of the protease in the catalysis of A1 cleavage. Furthermore, given the highly anionic nature of the C-terminal region of A1, we suggest that this sequence interacts with the protease via residues that form its heparin-binding exosite.

Factor VIII is homologous to factor V (35), the activated form of which serves as cofactor for factor Xa in the prothrombinase complex. Although factor V shows no acid-rich region separating the A1 and A2 domains, a recent report indicates an acidic residue-rich sequence localized to the N-terminal region of the A2 domain of factor Va (residues 323-331, net charge = -3) represents a binding site for factor Xa (36). This conclusion was based upon peptide-dependent inhibition of prothrombinase activity as well as direct inhibition of the factor Va-factor Xa interaction determined by fluorescence techniques. Although this enzyme-cofactor binding interaction in prothrombinase clearly differs functionally from the association of enzyme and substrate described in this study, the similarities in these interactions relative to location of sites and sequence characteristics of the cofactor proteins are noteworthy and suggest homologous sites of interaction.

The physiological significance of factor Xa-catalyzed cleavage of A1 subunit resulting in inactivation of cofactor function remains to be determined. Although the catalytic rates for these reactions are slow, the concentrating effect of the thrombogenic surface in restricting reactants to a two-dimensional space and the generation of high local concentrations of protease by the factor Xase complex (kcat ~200 min-1 (12)) support the hypothesis that factor Xa, the product of factor Xase, may contribute to the dampening of this enzyme complex.

    ACKNOWLEDGEMENTS

We thank Lisa Regan of Bayer Corp. and Debra Pittman of Genetics Institute for the gifts of recombinant factor VIII. We also thank Jan Freas, Julie Dill, and Kyla Schmidt for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL 38199 and HL 30616. A preliminary account of this work was presented at the 44th annual meeting of the American Society of Hematology, December 7, 2002, Philadelphia, PA.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 correspondence should be addressed: Dept. of Biochemistry and Biophysics, P. O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-6576; Fax: 585-473-4314; E-mail: Philip_Fay@urmc.rochester.edu.

Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M213044200

    ABBREVIATIONS

The abbreviations used are: TAP, tick anticoagulant peptide; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)1-propanesulfonic acid.

    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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