(Received for publication, June 9, 1997)
From the Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068
The products of cleavage of bovine factor Va by activated protein C (APC) in the presence and absence of phospholipid (25% phosphatidylserine, 75% phosphatidylcholine, PCPS) were evaluated using sedimentation velocity/equilibrium methods in the analytical ultracentrifuge and by immunoprecipitation using an antibody directed against the light chain of the factor Va molecule. The molecular weight and sedimentation coefficient of the associated heavy and light chains of factor Va, 173,000 (7.9 S) is reduced to 132,000 (7.1 S) by APC cleavage at Arg505 and Arg662. Complete cleavage of the factor Va heavy chain (with APC-PCPS) at Arg505, Arg662 and Arg306 results in a drastic change in the molecular weight observed for the product. Two products are resolved with sedimentation coefficients of 3.3 and 6.3 S with estimated molecular weights of 48,000 and 114,000, respectively. Immunoprecipitation studies showed that the products of factor Va cleavage at Arg505 and Arg662 (A1A2N·A2C·LC) are mostly noncovalently associated and consequently immunoprecipitated with an antibody directed against the light chain of the factor Va molecule. In contrast, for factor Va cleaved at Arg505, Arg662, and Arg306 the precipitated complex consisted of the A1 domain (residues 1-306) and the light chain (residues 1537-2183) of factor Va (A1·LC). The fragments corresponding to residues 307-505 (A2N) and 506-662 (A2C) are found in the supernatant. The combined mass of these two products (48,000) is similar to the estimated mass of the 3.3 S fragment estimated from sedimentation velocity/equilibrium studies; while the combined mass of the 1-306 + 1537-2183 products corresponds to 114,000, the estimated mass of the 6.3 S fragment. These data lead to the conclusion that cleavages at Arg306, Arg505, and Arg662 of the factor Va molecule resulted in the dissociation of the entire A2 domain as two noncovalently associated fragments (A2N·A2C). Enzyme kinetic and light scattering data suggest that the complete inactivation of the factor Va molecule involves not only cleavage at Arg306 but also the dissociation of the A2 domain. These data also suggest that the complete APC inactivation of the factor Va molecule is analogous to the spontaneous inactivation of factor VIIIa, which occurs via the dissociation of the A2 domain.
Bovine factor V circulates in blood as a single chain 330,000 molecular weight protein (1), which is cleaved by thrombin to give rise to the active species, factor Va, which is composed of a heavy chain (A1A2, residues 1-713) and a light chain (A3C1C2, residues 1537-2183) (2, 3). The two subunits of the factor Va molecule are associated noncovalently (4) and contribute both the membrane receptor site for factor Xa, the serine protease component of the prothrombinase complex (5, 6) and an effector function which influences the kcat of the enzyme complex. As a consequence, this complex is 300,000-fold more efficient in the activation of plasma prothrombin than factor Xa at potential physiologic concentration. In addition to the proteolytic activation steps which result in a 400-fold (at a minimum) increase in activity (7) over factor V, the factor Va molecule is subject to proteolytic regulation by activated protein C (APC)1 generated as a consequence of thrombin-thrombomodulin cleavage of plasma protein C (8). APC cleaves the bovine factor Va heavy chain at Arg505, Arg306, and Arg662 to produce an inactive species (9, 10). The inactivation cleavages occur in sequential fashion with cleavage at Arg505 preceding cleavages at Arg306 and Arg662. Once cleaved the resulting product, factor Va, is no longer capable of binding factor Xa or prothrombin (11). While the cleavages at Arg505 and Arg306 are both influenced by phospholipid (1-palmitoyl-2-oleoyl phosphatidylserine and 1-palmitoyl-2-oleoyl phosphatidylcholine, PCPS), the cleavage at Arg306 is dependent upon the presence of anionic phospholipids (10). The factor Va molecule cleaved only at Arg505 and Arg662 is still capable of binding factor Xa and influencing thrombin activation, but the affinity for factor Xa and the efficiency of prothrombin activation are reduced (10, 12). The present study was undertaken to examine the alterations in factor Va molecular composition associated with cleavage by APC.
HEPES, Sepharose CL-4B, cyanogen bromide, bovine serum albumin,
PS, and PC were purchased from Sigma. Bovine factor Va, bovine factor
Xa, and bovine APC were purified as described previously (13, 14).
Phospholipid vesicles composed of 75% PC and 25% PS were prepared as
described previously (15). The concentration of the phospholipid
vesicles in each preparation was determined by phosphorous assay.
Dansylarginine 4-ethyl piperidine amide (DAPA) was a gift from
Haematologic Technologies, Essex Junction, VT. Factor Va protein
concentrations were estimated spectrophotometrically by absorbance at
280 nm, using 280nM1% = 17.4 (16). The compositions of the products of APC cleavage were
assessed via SDS-polyacrylamide gel electrophoresis using 5-15%
gradient gels under reducing conditions and stained with Coomassie
Brilliant Blue (17).
Bovine factor Va was extensively dialyzed against 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl2, pH 7.4 (HBS-Ca2+). Factor Va (2 µM) was incubated with bovine APC (40 nM) in the presence or absence of 20 µM PCPS vesicles. Following a 150-min incubation the reaction was terminated by addition of diisopropyl fluorophosphate to 1 mM.
Functionally defined apparent dissociation constants (Kd(app) for factor Xa binding to factor Va-PCPS were inferred from an assay that employs purified reagents and measures thrombin formation (18). In a typical experiment a mixture of prothrombin (1.4 µM) PCPS vesicles (20 µM) and DAPA (3 µM) were incubated in the dark to allow equilibration. At selected time intervals, an aliquot of the mixture was added to a cuvette containing factor Va product (0.5 nM) and the reaction initiated by various concentrations of factor Xa. Thrombin formation was measured using a Perkin Elmer MPF-44A spectrofluorometer using excitation and emission wavelengths of 280 and 550 nm (DAPA-thrombin fluorescence) (18). The initial rate of thrombin formation for each factor Xa concentration was calculated and plotted as a function of the factor Xa concentration using the software PRIZM (GraphPadTM, San Diego, CA), assuming that factor Xa has one binding site for factor Va.
Sedimentation studies were conducted using a Beckman Optima XL-I
analytical ultracentrifuge equipped with UV-visible absorbance scanning
and Rayleigh Interference Optics. Sedimentation velocity experiments
were performed at 50,000 rpm using an An50-Ti, 8-hole rotor in 12-mm
double-sector cells that housed sapphire windows. Data were collected
using absorbance optics ( = 280 nm). The dialysate (see above) was
used in the cell reference sector. All velocity analyses were performed
simultaneously. The measurements were made on 400-µl samples at
20 °C using a scan interval of 6 min. Weight average sedimentation
coefficients (s20,w) were determined by second
moment analysis (19). g*(s) analyses were
conducted as described previously (20). In both instances the
absorbance versus time scans were analyzed on an IBM PS350 using the software package Microcal Origin version 3.78 supplied by
Beckman with the X-LI. Estimations of the relative absorbance contributions for multiple species resolved by sedimentation (APC-PCPS cleavage) were made by graphical integration of the
g*(s) plots.
Sedimentation equilibrium studies were performed using the high speed
meniscus depletion method as described by Yphantis (21). Experiments
were performed at 15,500 rpm for 14 h at 19.9 °C. The
attainment of equilibrium was assessed empirically by the observation
of identical gradients over a 3-h period. Molecular weight average
estimates were evaluated using the Optima XL software package Microcal
Origin Version 3.78 from Beckman. The reaction products were visualized
on 5 to 15% SDS-polyacrylamide gels, prior to and after the analytical
ultracentrifuge runs. For all calculations a of 0.72 was used (22).
Estimates of the molecular weights of species present in heterogenous mixtures were accomplished by two approaches. One approximation made use of the relationship between sedimentation coefficient and molecular weight for nearly globular proteins.
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(Eq. 1) |
In the second approximation the sedimentation equilibrium profile derived from absorption optics was evaluated using a two-component ideal mixture model (XL-I software) in which the relative absorbance contributions of the two species known to be in the centrifuge cell were derived from integration of the g*(s) plots from the sedimentation velocity data. The relative absorbance area ratio for a:b was 4.35. For modeling, the mass of the species was varied from 114,000-120,000 and 40,000-48,000 to obtain the model fit. The model fit was subsequently evaluated using fits to plots of ln A versus r2 using Microsoft Excel (5.0a)
To conduct immunoabsorption experiments, an anti-factor Va light chain
antibody, HFV#1, was coupled to Sepharose CL-4B beads (23).
Approximately 30 µg of the factor Va treated with APC (±PCPS
vesicles) was incubated with 300 µl of
HFV-1 beads in 150 µl of
buffer. Incubation was allowed to proceed for 30 min with occasional
stirrings. Following 30 min of incubation the samples were centrifuged
at 10,000 × g for 5 min. The recovered supernatant was
saved for analyses of the unbound material. The antibody beads were
washed three times with 1 ml of 20 mM HEPES, 0.15 m
NaCl, 5 mM CaCl2. Elution of the beads was
performed with 2% SDS. The proteins were visualized on a 5 to 15%
polyacrylamide gel electrophoresis (17).
The APC enzyme concentration dependence of the rate of factor Va inactivation process was evaluated by mixing factor Va (1 µM) PCPS (20 µM) and HBS-Ca2+ at 37 °C with APC over a concentration range from 0 to 5 µM. Following a 3-min incubation period an aliquot of the reaction was removed for assessment of remaining activity in the prothrombin activation assay utilizing 10 nM factor Xa. The remaining sample was quenched into SDS buffer and analyzed by SDS-polyacrylamide gel electrophoresis (17).
Light scattering measurements were made in a photon counting Fluoromax II SPEX fluorimeter (Edison, NJ). Scattering intensity was measured at 320 nm (excitation and emission) with a band pass of 5 nm. Experiments were performed in degassed and filtered (0.2 µM) HBS-Ca2+ buffer in a fluorimeter cuvette with gentle stirring. Factor Va (100 nM) was incubated with 20 µM PCPS and the scattering intensity monitored. The decrease in scattering intensity upon addition of APC (20 nM) was continuously monitored over 30 min (24). A first order fit of the data was achieved using a single exponential fit. In control experiments, the addition of 10 nM APC to PCPS vesicles in the absence of factor Va resulted in no detectable change in light scattering intensity under the experimental conditions used.
Bovine factor Va was chosen for these studies because of its
relative abundance and stability which permits reasonably dependable physical analyses of the protein. The species studied included factor
Va, factor Va cleaved by APC, 5 mM Ca2+, in the
absence of phospholipid (designated factor VaAPC, cleavage at Arg505, Arg662, and
Arg1752/1753), and factor Va cleaved by APC, 5 mM Ca2+ in the presence of PCPS vesicles
(designated factor VaAPC/PCPS, cleavage at
Arg505, Arg306, Arg662, and
Arg1752/1753). Fig.
1 is a Coomassie Blue-stained SDS gel
that illustrates the nature of the reaction products in the present
study analyzed under denaturing conditions. Lanes 1 and
2 are bovine factor Va. Lane 2 illustrates the
lack of influence of PCPS. Lane 3 contains the products of
APC cleavage in the absence of PCPS. Lane 4 contains the
products of APC cleavage in the presence of PCPS vesicles. Unlike human
factor Va the light chain of bovine factor Va is cleaved at
Arg1752/1753 to give rise to products of
Mr(app) 46,000/48,000 and 30,000. This latter
cleavage has been shown to be irrelevant with respect to assembly or
the activity of the factor Va molecule (9, 10, 25-27). The titrations
of the activities of the various factor Va species as a function of
factor Xa concentration are illustrated in Fig.
2. The functional
Kd(app) of the factor Va species are
presented in Table I together with the
nomenclature of the relevant fragments which comprise the factor Va
products and their SDS apparent molecular weights.
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Sedimentation velocity and sedimentation equilibrium studies were performed on each of the products represented in Fig. 1 and these data are illustrated in Table II. Factor Va and the products derived from factor Va following APC cleavage in the absence of phospholipid are relatively homogenous species by sedimentation velocity analysis. A small but significant change (7.9 to 7.1 S) in sedimentation coefficient is observed upon cleavage of factor Va at Arg505, Arg662, and Arg1752/1753. In contrast, the APC-PCPS cleavage of factor Va at Arg306 (in addition to the other cleavage sites) shows a significant reduction in the weight average sedimentation coefficient (s20,w). g*(s) analyses indicated two components, a with s20,w 6.3 and b with s20,w 3.3. The molecular weight estimated for intact factor Va (173,000) is in reasonable agreement to the sum of the heavy and light chain masses estimated from SDS gel electrophoresis (168,000) (Table I) and consistent with the molecular weight reported by Laue and co-workers for bovine factor Va (28). The product of APC cleavage of factor Va (minus PCPS) displayed a heterogenous molecular weight distribution in sedimentation equilibrium with weight average molecular weight of 132,000. The light chain fragments are known to remain intact following cleavage at Arg1752/1753 (9), and the cleavage at Arg505 gives rise to fragments of 153,000 and 20,000. The velocity and equilibrium data combined with activity data (9, 10) suggest that APC cleavage results in a reversibly associated product A1 A2N·A2C·LC (Table I).
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The weight average molecular weight of the APC-PCPS cleaved product is significantly reduced corresponding to a loss in mass of >50,000. Since the cleavage products of the light chain (LCN and LCC) are known to remain self associated, the change in mass can only be accounted for either by the dissociation of the A1 domain (residues 1-306) or the fragments obtained from the A2 domain, A2N (residues 307-505) and A2C (residues 506-662). Estimation of the mass of the sedimenting products resolved using the g*(s) approach suggest that the molecular weights of these fragments are approximately 120,000 and 47,000, assuming that their frictional coefficients are similar to those of factor Va and factor Va HC respectively (28).
The relative absorbance contributions of a and b to the concentration profiles in sedimentation equilibrium experiments were inferred from integration of the areas from the g*(s) plot. The values of 48,000 and 114,000 provided the best fit to the sedimentation data assuming an "ideal" noninteracting two component model. These collected data suggest that following cleavage at Arg306 the entire A2 domain dissociates from the remainder of the molecule (A1·LC) and exists primarily as the noncovalently associated product A2N·A2C.
The identical materials used in the sedimentation experiments (Fig. 1)
were subjected to immunoprecipitation analyses using an anti-factor Va
light chain antibody coupled to Sepharose. The results of this
experiment are shown in Fig. 3. The
factor Va molecule cleaved at Arg505, Arg662,
and Arg1752/1753 (lane 2) is quantitatively
immunoprecipitated by the Sepharose coupled anti-light chain antibody
(lane 4). No products are found in the supernatant
(lane 3). These results are consistent with the conclusion
of reversible association of the products of APC cleavage at
Arg505, Arg662, and Arg1752/1753. A
similar immunoprecipitation experiment conducted with the material
cleaved using APC + PCPS (lane 5) reveals that while the
light chain fragments (LCC and LCN) and the A1
domain are bound to the immunoprecipitate (lane 7) the
A2N and A2C fragments are found in the
supernatant (lane 6).
The issues then arise as to whether ultimate inactivation is associated
with cleavage at position Arg306 or whether inactivation is
associated with cleavage or the dissociation of the components
A2N + A2C which together comprise the entire A2
domain of the factor Va heavy chain or both the cleavage and dissociation process. Fig. 4 presents an
analysis of the inactivation of factor Va as a function of APC
concentration. It can been seen in this plot that the reaction is
decidedly nonlinear with respect to enzyme concentration, ultimately
becoming independent of enzyme at the highest concentrations evaluated
(~1.5-5 µM). These inactivation analyses are
fundamentally more complicated than the analysis of the spontaneous
inactivation factor VIIIa since the inactivating reaction is lipid
dependent and because the identification of the factor Va inactivation
associated with cleavage at Arg306 must be conducted by
quantitative analysis of thrombin generation (at high factor Xa
concentrations) rather than simply by a clotting assay. The inset in
Fig. 4 illustrates plots of the activity loss, post heavy chain
cleavage for two APC concentrations in the experiment described in Fig.
4. These points correspond to the activity losses in the intervals
following complete cleavage of the heavy chain, at 2.5 and 1 min
following the addition of 1.7 µM (triangle)
and 5 µM (squares) APC, respectively. In both
cases activity remained after the factor Va heavy chain was entirely
cleaved as assessed by gel analyses. However, the activity still
remaining (~20%) decays at a rate (k = 0.4 min1) independent of the enzyme concentration. These data
suggest an enzyme independent process, consistent with dissociation of the A2 domain fragments, is associated with complete activity loss.
However since the rate of the cleavage-independent activity loss
overlaps the Arg306 cleavage rate it is not possible to
assign a totally independent value to the residual activity present in
the factor Va cleaved at Arg505 and Arg306
product prior to dissociation.
The rate of mass reduction following APC inactivation of factor Va was
studied using right angle light scattering. The data presented in the
Fig. 5 illustrate the light scattering
change associated with addition of APC (20 nM) to a factor
Va (100 nM), PCPS (20 µM) reaction mixture. A
dramatic decrease in light scattering intensity occurs upon addition of
APC to the factor Va-phospholipid vesicle complex. At these
concentrations the cleavage reactions would have been complete within
approximately three minutes an interval accounting for about 70% of
the total scattering intensity reduction. Thus cleavage and
dissociation are occurring on roughly the same time scale. A 1st order
exponential approximation to the data would suggest a dissociation rate
k = 0.5 min1 similar to the rate of the
nonenzymatic factor Va decay rate (Fig. 4, inset).
The collected data lead to the conclusion depicted in Fig.
6. The complete inactivation of the
factor Va molecule which occurs with cleavage of the heavy chain at
Arg306, Arg505, and Arg662 results
in the dissociation of the A2 domain fragments from the remainder of
the factor Va molecule. The larger product at 6.3 S is a noncovalently
associated trimeric complex composed of A1·LC (Mr(app) 114,000). This product is incapable of
binding factor Xa or prothrombin or serving in the
prothrombinase complex (Fig. 2). The dissociated product (s)
corresponds to the entire A2 domain, has a
s20,w of 3.3 and is composed of two
peptides A2N and A2C with a combined
molecular weight of 48,000. It is likely, based upon the molecular
weight estimated from the s20,w of
the component b and the best fit of sedimentation
equilibrium data (Table II) that the two fragments, are
noncovalently associated, most likely in a reversible interaction.
The observation that the inactivating cleavage at Arg306
results in the dissociation of the A2 domain suggest that the
inactivated product, factor Vai, is equivalent in gross structure to
the inactive factor VIIIa product which results following the thrombin
activation cleavage of factor VIII at Arg372 (29). This
required activation cleavage in factor VIII (at Arg372)
results in the subsequent dissociation of the A2 domain and the
spontaneous inactivation of factor VIIIa (30, 31). The factor Va APC
cleavage at Arg306 produces a result similar to the
Arg372 cleavage in factor VIII resulting in dissociation of
the A2 domain and loss of factor Va activity. The process for factor Va
occurs at a faster rate (>0.5 minl) than the process for
factor VIIIa A2 dissociation (0.32 min
l)
(32).
The mechanism of complete inactivation of factor Va by APC-PCPS has
been controversial. The present data are consistent with the mechanism
illustrated in Fig. 6 in which activity is totally lost following
cleavage at Arg306 and the largely irreversible
dissociation of the A2 domain fragments. Under the experimental
conditions described in the present study (6 µM Va) it
is clear that any affinities of the ultimate cleavage products for each
other (if present at all) are relatively weak. However, since the
dissociation of the A2 fragment is most likely the direct cause of the
total factor Va inactivation, it is entirely possible that the
rate-limiting step of factor Va inactivation can be shifted from
peptide bond (Arg306) cleavage to A2 domain fragment
dissociation by other constituents of the reaction mixture. In addition
the products of the cleavage at Arg505 also appear to
engage in a reversible binding phenomena which may also be influenced
by third component binding. For example, it has been reported and
confirmed by numerous studies that factor Xa stabilizes the factor Va
molecule from inactivation by APC (33-36). Similarly it has been shown
that factor IXa stabilizes the thrombin activated factor VIIIa molecule
presumably by preventing dissociation of the A2 subunit (37, 38). The
stabilization of factor Va by factor Xa may be, in part, a consequence
of an altered dissociation of the products following APC cleavage. If this is indeed the case the presence of factor Xa or prothrombin during
APC inactivation may be major confounding elements in kinetic evaluations of factor Va inactivation but elements that are
biologically relevant.
The ultimate deconvolution of the relative activity losses associated with factor Va cleavage at position 306 and the activity loss associated with the dissociation of the fragments of the A2 domain is a matter that will require further extensive experimental analysis. However the present results clearly indicate that the kinetics of APC inactivation of factor Va are associated with complex processes which influence both the binding of the enzyme factor Xa and the effector function of factor Va in the prothrombinase complex and include both the proteolysis and the dissociation of fragments of the A2 domain ultimately leading to a totally inactivated factor Va molecule.