©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Model for the Factor VIIIa-dependent Decay of the Intrinsic Factor Xase
ROLE OF SUBUNIT DISSOCIATION AND FACTOR IXa-CATALYZED PROTEOLYSIS (*)

(Received for publication, July 13, 1995; and in revised form, October 17, 1995)

Philip J. Fay (1) (2)(§) Tammy L. Beattie (1) Lisa M. Regan (2)(¶) Lynn M. O'Brien (1)(**) Randal J. Kaufman (3)

From the  (1)Departments of Medicine and (2)Biochemistry, University of Rochester School of Medicine, Rochester, New York 14642 and the (3)Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and A1/A3-C1-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the A1 subunit. At low reactant concentrations (0.5 nM FVIIIa; 5 nM FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIII) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min) and paralleled the rate of proteolysis of A1 subunit. However, the rate of decay of FXase containing the FVIIIa was reduced (0.048 min) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.


INTRODUCTION

FVIII (^1)and FIX are essential plasma glycoproteins that when absent or defective, result in hemophilia A and B, respectively. The proteolytically activated forms of these proteins; FIXa, a serine protease, and FVIIIa, a protein cofactor, form a Ca- and surface-dependent complex referred to as the intrinsic FXase complex, that efficiently converts zymogen FX to FXa (see (1) for review). The role of FVIIIa in this complex is to increase the k for this reaction by several orders of magnitude(2) .

FVIIIa is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. (^2)The A1 and A3-C1-C2 subunits retain the Me ion linkage responsible for the association of the heavy and light chains in heterodimeric factor VIII and can be isolated as a stable dimer(3, 4) . The A2 subunit is weakly associated with the dimer (K = 260 nM; Refs. 5 and 6) primarily through electrostatic interaction (7) and at physiologic pH readily dissociates resulting in the loss of FVIIIa activity(5, 6, 7, 8) . Under the appropriate reaction conditions, FVIIIa activity can be reconstituted from the isolated A2 subunit and the A1/A3-C1-C2 dimer (6, 7, 8, 9, 10) .

Association of FVIIIa with FIXa in the presence of phospholipid and Ca stabilizes cofactor activity(11) . Furthermore, reconstitution of heterotrimeric FVIIIa from the isolated A2 subunit and A1/A3-C1-C2 dimer is enhanced severalfold in the presence of FIXa and phospholipid(9) . While prolonged interaction of FVIII(a) with FIXa results in a loss of FVIII(a) activity due to proteolytic cleavage within the A1 domain/subunit(12, 13) , stable enhancement of FVIIIa reconstitution can be achieved in the presence of active site-inhibited FIXa(9) . Thus FIXa exhibits the capacity to modulate FVIIIa activity, thereby affecting the catalytic efficiency of FXase.

FXase activity is labile, whereas FVIIIa-independent conversion of FX by FIXa is relatively stable. In this report we examine the FVIIIa-dependent lability of FXase, which has been referred to as a ``self-damping''(14) . Experiments assess the relative contributions of FVIIIa subunit dissociation and FIXa-catalyzed proteolysis of FVIIIa to the decay of FXase activity. This study was aided by use of a mutant FVIII molecule, FVIII, resistant to FIXa-catalyzed cleavage at the A1 site. Results of this analysis yield a model for the intrinsic instability of this enzyme complex.


MATERIALS AND METHODS

Reagents

Recombinant FVIII preparations were gifts from Dr. Jim Brown of Bayer Corp. and Debra Pittman of the Genetics Institute. TAP was a generous gift from Dr. S. Krishnaswamy (Emory University). The murine monoclonal antibody R8B12, which reacts with the C-terminal region of the FVIII A2 domain(4) , was prepared as described previously (7) . The anti-A1 subunit monoclonal antibody, specific for the NH(2)-terminal region, was provided by Dr. Jim Brown of Miles, Inc. The reagents alpha-thrombin, FIXa FX, and FXa (Enzyme Research Laboratories), hirudin (Sigma), inosithin (Associated Concentrates, Inc.), and the chromogenic substrate S-2765 (N-alpha-benzyloxycarbonyl-D-arginylL-glycyl-L-arginyl-p-nitroanilide-dihydrochloride; Pharmacia Biotech Inc.) were purchased from the indicated vendors.

Proteins

FVIII (15) and FVIIIa subunits (7) were prepared as described previously using recombinant FVIII as the starting material. FVIII activity was measured by a one stage clotting assay using plasma that had been chemically depleted of FVIII activity as described previously(16) . Construction of the FVIII cell line was described by Pittman and Kaufman(17) . Cell culture supernatants containing FVIII (<0.3 units/ml FVIII; 400 ml) were applied to a column of R8B12 antibody coupled to Affi-Gel 10. The bound FVIII was eluted in buffer containing ethylene glycol (7) and concentrated by dialysis against a polyethylene glycol (M(r) 20,000; 10%, w/v)-containing buffer. Yields were low and typically resulted in about 2 ml of 10 units/ml FVIII. FIXa-cleaved FVIII light chain was prepared by reacting the light chain (500 nM) with FIXa (30 nM) in 20 mM Hepes, pH 7.2, 100 mM NaCl, 5 mM CaCl(2), and 0.01% Tween 20 for 24 h at room temperature. These conditions resulted in >90% conversion as determined by SDS-polyacrylamide gel electrophoresis and silver staining. Reconstitution of the FVIII following reaction with FVIII light and heavy chains and subsequent chromatography of the heterodimer forms on Mono S were performed as described previously(16) . Protein concentrations were determined by the Coomassie Blue dye binding method of Bradford(18) . FIXa incorporated > 0.9-mol equivalents of dansyl-Glu-Gly-Arg chloromethyl ketone indicating a high specific activity for this preparation of enzyme.

FXa Generation Assays

The rate of conversion of FX to FXa was monitored in a purified system(19) . FVIII was activated with thrombin (20 nM, 30 s) in 20 mM Hepes, pH 7.2, 100 mM NaCl, 5 mM CaCl(2), and 0.01% Tween in the presence of 100 µg/ml phospholipid. Hirudin (0.65 units) was added to inactivate thrombin and time course reactions were initiated with the addition of FIXa (see figure legends for reactant concentrations). Aliquots were removed at the indicated times, reacted with FX (200 nM unless otherwise indicated) for 15, 30, 45, and 60 s at which time EDTA (50 mM final concentration) was added to stop the reaction. Initial rates of FXa generation were determined by addition of the chromogenic substrate, S-2765 (460 µM). Reactions were read at 405 nm using a V(max) microtiter plate reader (Molecular Devices).

Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using the method of Laemmli (20) with a Bio-Rad minigel electrophoresis system. Electrophoresis was carried out at 150 V for one hour. For Western blotting, unstained gels were transferred to polyvinylidene difluoride membranes (Bio-Rad, 0.2 µm) using a Bio-Rad minitransblot apparatus at 100 V for 1.5 h in buffer containing 192 mM glycine, 25 mM Tris, and 20% (v/v) methanol. Bands were visualized as described previously (21) using the anti-A1 subunit monoclonal antibody followed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody. Densitometric scans were performed using a Beckman DU 650 spectrophotometer equipped with Gel Scan software.

Kinetics of FXase Decay

The loss of FVIIIa-dependent FXase activity was determined using a multistep model to account for modes of cofactor inactivation by both subunit dissociation and proteolysis. The model comprises the following steps,

which represents the reversible dissociation of FVIIIa to A2 and A1/A3-C1-C2 subunits. In this and following steps, FVIIIa (or A1/A3-C1-C2) is bound to the PL surface. The rate constants, k(1) and k(2), are 0.32 min and 1.3 times 10^6M min, respectively(6) , and the K(d) is 260 nM(5) . The steady state concentration of FVIIIa was solved by quadratic equation as determined from,

where [A2] = [A1/A3-C1-C2] in the absence of exogenous A2 and [FVIIIa](0) is the initial concentration of FVIIIa following activation and equals the initial concentration of FVIII, [FVIII](0).

The interaction of FIXa with FVIIIa is given by,

where the K(d) = 23 nM(22) is equivalent to the interaction of FIXa with A1/A3-C1-C2 dimer (11 nM; (23) ) and is assumed to be equivalent for the interaction with the mutant FVIIIa form. The concentration of FIXa-FVIIIa-PL is calculated using a (single site) ligand binding curve,

where [FIXa-FVIIIa-PL](max) = B(max) = [FVIIIa-PL](0) and [FIXa] [FVIIIa]. The reversible dissociation of FVIIIa subunits in the presence of FIXa is given by,

At low FVIIIa concentrations, the inter-FVIIIa subunit interaction is stabilized by the presence of FIXa and phospholipid(11, 12) . Furthermore, preliminary data suggest the association rate constants in the absence (k(2)) and presence (k(6)) of FIXa are similar. (^3)Thus we assume that k(1) > k(5). Because reactions use low (sub nM) concentrations of FVIIIa, the contribution of the association rate constants (k(2) and k(6)) for the dimer/A2 subunit interaction is negligible (e.g. at 1 nM FVIIIa, k(2)= 0.0013 min) so only the dissociation rate constants need to be considered in assessing rates of FXase decay. Therefore, under these conditions,

where k(1)` is the experimentally observed dissociation rate constant for FVIIIa at a given concentration of FIXa. The denominator of the equation represents 1 + a stabilization factor. This factor, which we define as the ratio of dissociation rate constants in the absence (k(1)) and presence (k(5)) of FIXa multiplied by the mole fraction of FVIIIa in complex with FIXa, retards the dissociation rate constant for FVIIIa (k(1)) and thus would reduce the overall rate of FXase decay. As [FIXa] approaches 0, the quantity [FIXa-FVIIIa-PL]/[FVIIIa-PL](0), which is calculated from , approaches 0 and k(1)` equals k(1). From the above equation the calculated value for k(5) = 0.039 ± 0.003 min (n = 4) was determined over a range of FIXa levels (5-50 nM) using the mutant FVIIIa so that FXase decay was solely attributed to subunit dissociation.

A final consideration is FXase decay resulting from proteolysis. Cleavage of FVIIIa at Arg by FIXa (12) inactivates the cofactor by markedly reducing the affinity of A2 for the truncated A1/A3-C1-C2 dimer(4) . Therefore, proteolytic inactivation can be represented by,

where k(7) is the rate constant for proteolysis at a given concentration of FIXa.

Thus, the observed rate of FVIIIa-dependent decay of FXase for a given concentration of FIXa can be represented as a sum of the contributing rate constants as follows,

Data Analysis

Nonlinear curve fitting using the Marquardt algorithm was performed with UltraFit software (Biosoft). Rate of loss of FXase activity was fit to a single exponential decay with offset (activity = initial activity bullet e + offset) or double exponential decay. The initial activity level was determined from the 30-s time point. The offset was employed to account for the stable, residual FXase activity observed following prolonged incubations. Rates of proteolysis of FVIIIa A1 subunit were determined from the Western blot as a ratio of scanning densities of A1 fragment to intact A1 for each time point so as to eliminate lane to lane variability in loading/staining. Equivalent binding affinities of the anti-A1 monoclonal antibody for the A1 subunit and A1 fragment were assumed.


RESULTS

Lability of FXase

The above model for FXase lability was tested under conditions of relatively low reactant concentrations. For these experiments, FVIII (0.5 nM; 0.3 units/ml) was rapidly activated with an excess of thrombin, which was subsequently inhibited with hirudin as described under ``Materials and Methods.'' Reactions were initiated with the addition of FIXa (5 nM), and time points were removed and assayed for residual FXase activity. In Fig. 1A, residual FXase activity is plotted as nM FXa generated/min as a function of incubation time and the data fitted to a single exponential decay curve. The fitted curve (circles) yields a decay rate of 0.106 ± 0.017 min. This value is similar to that predicted for k(1)`, suggesting that decay resulted primarily from subunit dissociation. Since the curve approaches the x axis asymptotically, an offset value (6.4 nM/min for this decay curve) represents the value for the rate of FXa generated at prolonged incubation time.


Figure 1: Time-dependent decay of FXase activity. A, reactions contained 5 nM FIXa, 100 µg/ml phospholipid, and 0.3 units/ml FVIIIa in the absence (circles) or presence (squares) of 100 nM A2 subunit. Reactions were initiated with addition of FIXa and aliquots were removed at the indicated times and assayed for FXa generating activity. Data were fitted to a single exponential decay with offset as described under ``Materials and Methods.'' Decay rates and offset values were 0.106 ± 0.017 min and 6.4 nM FXa/min and 0.084 ± 0.005 min and 19.5 nM FXa/min for reactions run in the absence and presence of exogenous A2, respectively. B shows a plot of offset value (circles) and calculated FVIIIa concentration (squares, from ) determined for the indicated exogenous A2 concentrations.



In order to further assess the role of FVIIIa subunit dissociation in the decay of FXase, a similar reaction was performed with a high level (100 nM) of exogenous A2 subunit added at the initiation of the reaction (Fig. 1A, squares). This condition was predicted to reduce the rate and/or extent of FVIIIa dissociation, thereby increasing cofactor stability. While the presence of the added A2 subunit resulted in a similar rate of decay (0.084 ± 0.014 min), the offset value was significantly increased (20 nM/min). This suggested that while the dissociation rate constant of FVIIIa still remained much faster than the association rate constant, as the time course progressed, a new equilibrium was established reflecting the higher offset in response to the exogenous A2. Fig. 1B shows that the offset, as measured from FXase decay curves in the absence and presence of several levels of exogenous A2 (primary data for intermediate A2 concentrations not shown), is linearly related to the concentration of A2 over the concentrations used. This result would be expected for all A2 subunit levels below the K(d) for the A2-dimer interaction (260 nM; (5) and (6) ) if subunit dissociation were a primary reason for the decay of FXase. Furthermore, the offset values correlated well with the calculated concentrations of FVIIIa (from ). These results suggested that at the above FVIIIa and FIXa concentrations employed, proteolysis was not a factor in loss of FXase activity.

Analysis Using a FVIII Mutant

While the above results suggest that FVIIIa dissociation is a primary reason for FXase decay, the potential role of FIXa-catalyzed cleavage at Arg was not directly considered. To include this aspect in our analysis, a series of experiments was performed using a mutant FVIII molecule, FVIII, in which Arg is replaced with Ile. This FVIIIa form is resistant to FIXa-catalyzed cleavage in the A1 subunit, as verified by Western blot analysis (Fig. 2). In this figure, wild type and mutant FVIIIa forms were reacted with a high concentration of FIXa over a 30-min time course. Resultant A1 subunits were visualized using a monoclonal antibody specific for its NH(2) terminus; thus, both full-length A1 subunit and the A1 fragment truncated at Arg appear on the blot. The failure of FIXa to cleave the A1 subunit of the mutant FVIII permitted us to dissect the roles of dissociation and proteolysis in the FVIIIa-dependent decay of FXase.


Figure 2: Western blot of FVIIIa forms following reaction with FIXa. FVIIIa was prepared from native FVIII (15 units/ml) and the FVIII mutant (5 units/ml) following reaction with thrombin (3 nM) for 2 min and subsequent inhibition of the thrombin with hirudin. FIXa (100 nM) and phospholipid (50 µg/ml) were added to each reaction and aliquots were removed at 2 and 30 min, subjected to SDS-polyacrylamide gel electrophoresis, transferred, and probed with an anti-A1 subunit monoclonal antibody as described under ``Materials and Methods.'' Lanes 1-3 and 4-6 represent reactions before and at 2 and 30 min after addition of FIXa to FVIIIa and wild type FVIIIa, respectively. The high molecular weight band likely represents residual heavy chain (contiguous A1-A2) not cleaved by thrombin. Note that this band also persists in the mutant FVIIIa preparation, whereas it disappears with time in the wild type FVIIIa preparation.



Reactions similar to those shown in Fig. 1A were performed using equivalent activity levels (based upon the one-stage clotting assay) of the FVIII mutant (Fig. 3). Results showed that the decay rates in the two reactions (0.120 ± 0.044 min and 0.126 ± 0.046 min for the absence and presence of 100 nM exogenous A2 subunit, respectively) were equivalent and similar to the values observed with the wild type FVIII. Furthermore, the offset value in the presence of A2 was about twice that observed in its absence. Since the A1 subunit of this material is not cleavable by FIXa, the above results, taken together, indicated that at low FVIIIa/FIXa concentrations, loss of FXase activity is primarily caused by dissociation of FVIIIa subunits with little if any contribution of proteolysis to FXase decay.


Figure 3: Decay of FXase containing FVIIIa. Reactions were as described in the legend to Fig. 1and run in the absence (circles) and presence (squares) of 100 nM A2 except that the mutant FVIIIa replaced the native form. Decay rates (single exponential) and offset values were 0.120 ± 0.044 min and 7.1 nM FXa/min and 0.126 ± 0.046 min and 12.1 nM FXa/min for reactions run in the absence and presence of exogenous A2, respectively.



An additional series of experiments, using the same level of FVIIIa as above but with a 10-fold increase in the concentration of FIXa (50 nM), was evaluated, since the potential for high ratios of protease to cofactor are physiologically possible based upon their relative plasma concentrations. Results are presented in Fig. 4and show decay of FXase composed of either wild type FVIIIa (circles) or FVIIIa (squares). The rate of decay of FVIII is actually reduced by a factor of 3-fold (0.048 ± 0.022 min) compared with the low FIXa situation (Fig. 3). This reduction in FXase decay rate suggests the high FIXa level promotes FVIIIa subunit association and verifies the concept of the stabilization factor described in . On the other hand, the rate of decay of FXase containing wild type FVIIIa was evaluated using a double exponential decay curve to extract rate constants for proteolysis (k(7)) and subunit dissociation (k(1)`) for that concentration of FIXa. Values of 0.819 and 0.046 min were obtained. The latter was equivalent to the above value obtained with the mutant molecule suggesting this represented the decay rate attributed to subunit dissociation; whereas the former value was increased by 8-fold compared with that observed with the lower FIXa level (Fig. 1A). These results suggest that at high stoichiometries of FIXa relative to FVIIIa, proteolysis contributes significantly to the decay of FXase, outweighing any inter-FVIIIa subunit stabilizing activity.


Figure 4: Decay of FXase containing wild type and mutant FVIIIa in the presence of high FIXa. Reactions contained 0.3 units/ml wild type (circles) or FVIII mutant (squares) FVIIIa, 100 µg/ml phospholipid, and 50 nM FIXa and were run as described under ``Materials and Methods.'' Decay rates for the wild type FVIIIa used a double exponential curve fit to account for contributions of proteolytic inactivation and subunit dissociation. The fitted constants were 0.819 ± 0.181 min and 0.046 ± 0.007 min. Decay rate for the mutant FVIIIa was fitted to a single exponential and yielded a value of 0.048 ± 0.022 min.



FIXa-catalyzed Cleavage at Arg Does Not Inactivate FVIIIa

In addition to the site at Arg, FIXa cleaves (slowly) at Arg in the A3 domain (light chain) of FVIII(a)(12, 13) . To ensure that cleavage at this site was not relevant to our model for FXase decay, an analysis of its effect on FVIII activity was performed. Isolated FVIII light chain or FIXa-cleaved light chain was recombined with FVIII heavy chain in the presence of Mn as described under ``Materials and Methods.'' The resultant FVIII heterodimer forms were isolated by Mono S chromatography and assessed for activity. The specific activity of the FVIII form composed of the FIXa-cleaved light chain was higher (3-fold) compared with the authentic FVIII, suggesting that cleavage of the light chain actually contributes to activation. Both FVIII forms could be activated to similar extents with thrombin (Fig. 5). These results indicate that proteolysis at Arg is not inactivating and thus does not contribute to FXase decay.


Figure 5: Thrombin activation of reconstituted FVIII forms. FVIII heterodimers (25 nM) isolated from the Mono S column were reacted with thrombin (1 nM). Aliquots were removed at the indicated times and assayed for activity using a one stage assay. Circles represent the authentic FVIII, and squares represent FVIII reconstituted from native heavy chain and FIXa-cleaved light chain.



Effects of FX

Although in the above reactions FXase decay is measured in the absence of FX, a final consideration is the effects of substrate on the FVIIIa-dependent decay of activity. Two parameters were examined: that of influencing the inter-FVIIIa subunit affinity and that of modulating proteolytic cleavage of the FVIIIa A1 subunit. Factor VIIIa reconstitution experiments, performed using a one stage clotting assay, showed that the presence of FX neither enhanced nor inhibited the regain in activity following mixing of the isolated A2 subunit and A1/A3-C1-C2 dimer (data not shown). This result suggested that substrate FX did not affect the association or dissociation rate constants for the inter-FVIIIa subunit interaction.

On the other hand, inclusion of FX had a marked affect on the cleavage of FVIIIa (Fig. 6). In these experiments, FVIIIa (50 nM), FIXa (70 nM), and phospholipid vesicles (100 µg/ml) were incubated in the presence and absence of FX. Aliquots were removed at the indicated times, run on a gel, and Western blotted with the anti-A1 subunit monoclonal antibody. In the presence of FX (A), the A1 subunit was rapidly cleaved to a fragment of size consistent with cleavage at Arg. However, after a few minutes, both bands disappeared suggesting that the FXa generated by FXase further cleaved the A1 to small fragments and/or within the epitope such that no products were detected. To eliminate the effect of generated FXa, a similar reaction was performed using the specific FXa inhibitor, TAP(24) . Thus any FXa generated would be sequestered from FVIIIa. Sufficient TAP was included to result in a t of inhibitor-substrate formation of less than 1 s(25) . This concentration of TAP did not effect the rate of FIXa-catalyzed proteolysis of FVIIIa in the absence of FX (0.519 min) as shown in B. The bottom panel (C) shows the reaction performed in the presence of FX plus TAP. Scans of this blot show that the presence of FX reduced the rate at which FVIIIa A1 subunit was cleaved by about 10-fold (0.050 min). This result suggested that the (FXa-independent) damping of FXase via proteolysis is dependent upon the substrate availability such that FIXa-catalyzed proteolysis of A1 is reduced when FX is present.


Figure 6: Effect of FX on FIXa-catalyzed proteolysis of FVIIIa. All reactions contained FVIIIa (50 nM), FIXa (70 nM), and phospholipid (100 µg/ml). Additional components were: A, FX (400 nM); B, TAP (1 µM), and C, FX plus TAP. Reactions were run and blotted as described under ``Materials and Methods.'' Lanes 1-9 represent time points at 0, 1, 2, 4, 7, 10, 15, 25, and 40 min after FIXa addition. Data from densitometric scans were analyzed using a single exponent decay. Rates of conversion of the A1 subunit to the A1 fragment were 0.519 ± 0.046 min and 0.051 ± 0.025 min, for reactions shown in B and C, respectively.




DISCUSSION

In this report, we present a model to evaluate the instability of the intrinsic FXase complex. A scheme summarizing the reaction pathways is shown is Fig. 7. The FVIIIa-dependent decay of FXase arises from both the weak affinity interaction between the A1/A3-C1-C2 dimer and A2 subunit and FIXa-catalyzed proteolysis of the A1 subunit. The relative contribution of either mechanism is dependent upon the level of FIXa present. Rate constants for dissociation of FVIIIa into subunits (k(1), k(5)) predominate at low reactant concentrations. At high relative FIXa concentrations, while dissociation of subunits is minimized by a FIXa-dependent stabilizing activity, FVIIIa inactivation by proteolysis of A1 subunit (k(7)) predominates. Thus, the presence of FIXa has profound effects on FVIIIa stability/activity. These effects have been controversial and can now be related to the above model.


Figure 7: Reaction pathways in the formation and degradation of FXase. Association of all FVIIIa and A1/A3-C1-C2 dimer forms with PL is implicit. Rate constants for the FIXa-dimer interaction (k(3)`, k(4)`) are assumed to be equivalent to those for the FIXa-FVIIIa interaction (k(3), k(4)). The constant k(7) may be severalfold greater than k(7)` from comparison of cleavage of the two substrates(12) .



Several years ago, Jesty (14) observed that the rate and yield of FXa formed by the intrinsic FXase decreased with increasing FIXa concentration and a constant level of FVIIIa. He suggested that, while the most probable cause for the damping of FXase activity was the spontaneous decay of the labile FVIIIa, this was likely not the only consideration since the rate constant for FXase decay varied with FIXa concentration. At lower FIXa concentrations there was a tendency toward a minimum decay rate of 0.2 min, whereas this value was as high as 0.94 min at high levels of FIXa. That high concentrations of FIXa result in an acceleration of the decay of FXase (14, 26) is compatible with FIXa-catalyzed proteolysis of FVIIIa indeed contributing to the instability of this complex. Results presented in this study show similar rates of proteolysis of A1 subunit (0.51 min; Fig. 6B) and the contribution to FXase decay by proteolysis (0.82 min, Fig. 4). Furthermore, it has been argued that because of the proteolytic activity of FIXa toward FVIIIa, FIXa does not contribute to FVIIIa stabilization(13) . However, this conclusion was based upon examination of only very high reactant concentrations (100-200 nM).

Lollar et al.(11) determined that at low FVIIIa (<1 unit/ml) and FIXa (5 nM) levels and in the presence of a phospholipid surface, porcine FVIIIa was stabilized from spontaneous decay. This effect was also observed with an active site-modified FIXa (27) . Thus, these investigators concluded that association of FVIIIa with the other components of FXase markedly reduced the lability of the cofactor. Furthermore, the observed decay of porcine FVIIIa in that study was slower than that of the human protein (14, 22, present study). The enhanced stability of porcine VIIIa compared with human FVIIIa is consistent with a 3-fold greater dissociation rate constant for the latter(6) .

The effect of FIXa in the enhancement of FVIIIa reconstitution from isolated subunits clearly supports the role for a stabilizing activity. Reconstitution analyses typically use higher FVIIIa and FIXa concentrations (10-40 nM) compared with assays where FVIIIa is generated from in situ activation of FVIII by thrombin. For this reason, the FIXa-dependent enhancement of FVIIIa reassociation is transient in the presence of native enzyme, with decay correlating with proteolysis of A1 subunit(9) , while active site-modified FIXa yields a greater -fold enhancement of FVIIIa reconstitution that is relatively stable.

Use of a mutant form of FVIII, FVIII, allowed us to assess the degree to which FIXa modulates the stability of the labile FVIIIa trimer, since FXase containing this FVIIIa molecule decays via a nonproteolytic mechanism. Comparison of this decay with the predicted first order dissociation rate constant for FVIIIa (k(1)) allowed us to derive an expression for the extent of added stability conferred by association with FIXa on the PL surface, based upon the product of mole fraction of FVIIIa complexed and the ratio of k(1)/k(5). Saturating FIXa would maximally inhibit this decay by 9-fold (k(1)/k(5) = 8.2). Recently, it was observed that the first order decay of human FVIIIa (2 nM) was reduced 8-fold with saturating FIXa (10 nM)(22) . This -fold reduction would be predicted from our model as a result of maximal reduction of FVIIIa dissociation and little or no contribution of proteolysis to decay as a consequence of low reactant concentration. Furthermore, this value is similar to the maximal enhancement (8-10-fold) of FVIIIa reconstitution we observe in the presence of active site-modified FIXa(9, 28) .

The mechanism by which FIXa stabilizes the labile FVIIIa heterotrimer is not known. A high affinity site for FIXa has been localized to the FVIII light chain, possibly within the A3 domain(28) . Furthermore, a peptide comprised of A2 residues 558-565 inhibits the FIXa-dependent enhancement of FVIIIa reconstitution (29) as well as the A2-dependent contribution to the FVIIIa-dependent fluorescence anisotropy of fluorescein-Phe-Phe-Arg-labeled FIXa(23) . Thus FIXa likely tethers sites within the A1/A3-C1-C2 dimer and the A2 subunit.

Prolonged reaction with FIXa results in cleavage of FVIIIa. Results from this study suggest that relatively high concentrations of FIXa are required for efficient proteolysis. The reason for this is not clear but may suggest that the scissile bonds within the cofactor are not readily accessible to the bound enzyme and/or that cleavage results from FIXa molecules other than the one complexed with FVIIIa. The site cleaved by FIXa that correlated with FVIIIa inactivation was identified as Arg in A1 domain following N-terminal sequence analysis of a FIXa-generated FVIII fragment(12) . Previously, we proposed a mechanism for inactivation as a result of cleavage at this site(4, 7) . This cleavage would liberate the acidic C-terminal region of the A1 subunit (residues 337-372), which is involved in A2 subunit retention (30) following thrombin cleavage at Arg(31) in the FVIII heavy chain.

In a recent study, Neuenschwander and Jesty (32) measured decay of thrombin-activated FVIII in the presence of FIXa using a continuous FXa generation assay which contained acetylated-FX, a modified zymogen that is activated to a form of FXa that does not react with FVIII(a) substrates(33) . It was shown that a plot of 1/kversus [FIXa] was linear with a positive slope. Thus high levels of FIXa reduced the apparent decay of cofactor activity from 0.3 to 0.06 min. This result is consistent with the results presented in the current study, since we also find that the presence of substrate (plus an inhibitor of the generated product) reduced the contribution of k(7) to FXase decay by 10-fold, while the high levels of FIXa would modulate (reduce) the apparent dissociation rate constant (k(1)` parameter).

At high FVIIIa/FIXa concentrations and in the presence of the generated FXa, the FVIIIa A1 subunit was cleaved at Arg. This site represents a proposed FXa cleavage site (30) as well as a FIXa site (12) and thus would be inactivating. Additional proteolysis of A1 was observed to be FXa-dependent and suggested multiple sites in this subunit. This further proteolysis was observed following several minutes in the FXa generation time course. Thus, in vivo, FXa could potentially modulate FXase activity by inactivating the cofactor. Alternatively, the FXa generated could be sequestered/channeled from FXase to FVa (K(d) 1 nM; (34) ) in forming the prothrombinase complex. It is of interest to note that the FVIII mutant exhibits a 3-fold greater specific activity than wild type FVIII based upon a Coatest (FXa generation) assay(17) , whereas similar specific activities are obtained with the one-stage clotting assay. (^4)In the former assay, FVIII is preincubated with FIXa, FX, phospholipid, and Ca for several minutes prior to the activity determination. Thus, the higher activity of the mutant may reflect its resistance to cleavage by FIXa and/or generated FXa.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 30616 and HL 38199 (to P. J. F.) and HL 52173 and HL 53777 (to R. J. K.). 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: Hematology Unit, P. O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-6576; Fax: 716-473-4314.

Present address: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

**
Postdoctoral trainee supported by National Institutes of Health Grant HL 07152.

(^1)
The abbreviations used are: FVIII(a), factor VIII(a); FIX(a), factor IX(a); FX(a), factor X(a); PL, phospholipid; FXase, the PL-bound complex of FVIIIa and FIXa; TAP, tick anticoagulant peptide; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

(^2)
FVIIIa subunits are designated relative to the domainal sequence A1-A2-B-A3-C1-C2 (35) and are as follows: A1, residues 1-372; A2, residues 373-740; A3-C1-C2, residues 1690-2332. Noncovalent subunit associations are denoted by a shill (/) and covalent associations are denoted by a hyphen(-).

(^3)
L. M. O'Brien and P. J. Fay, unpublished observation.

(^4)
D. A. Michnick and R. J. Kaufman, unpublished observation.


ACKNOWLEDGEMENTS

We thank Micheline Mousalli for excellent technical assistance, Dr. Jim Brown and Bayer Corp. for the anti-factor VIII monoclonal antibody as well as recombinant factor VIII, Debra Pittman and The Genetics Institute for recombinant factor VIII, Dr. Sriram Krishnaswamy for the generous gift of tick anticoagulant peptide, and Dr. Harold Scheraga for helpful discussions. We also thank the anonymous reviewer of this manuscript for significant suggestions concerning the presentation of the kinetic model.


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