From the Department of Biochemistry, University of Vermont, College
of Medicine, Burlington, Vermont 05405-0068, § Department of
Chemistry, College of St. Catherine, St. Paul, Minnesota 55105-1730, and Department of Medicine, Jagiellonian University
School of Medicine, Cracow 31-066, Poland
Received for publication, May 15, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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We report the effect of homocysteine on the
inactivation of factor Va by activated protein C (APC) using clotting
assays, immunoblotting, and radiolabeling experiments. Homocysteine,
cysteine, or homocysteine thiolactone have no effect on factor V
activation by Homocysteine is a nonprotein-forming sulfhydryl amino acid derived
from the metabolism of methionine. Homocysteine is metabolized in human
cells through two pathways of remethylation to methionine and one
pathway of transsulfuration to cysteine with cystathionine as an
intermediate (1). Congenital defects in the enzymes involved in
methionine metabolism, 5,10-methylenetetrahydrofolate reductase, N5-methyltetrahydrofolate
methyltransferase, and cystathionine The relationship(s) between elevated levels of homocysteine in the
blood and thrombosis has been a subject of considerable investigative
effort; however, the mechanisms by which hyperhomocysteinemia leads to
increased risk of thrombosis are not clear (14-17). In vitro studies provide evidence that supraphysiologic homocysteine levels have a direct toxic effect on the endothelium, probably through
free radical generation during oxidation of the homocysteine sulfhydryl
group (18, 19). Homocysteine has also been reported to impair the
binding of tissue-type plasminogen activator to its endothelial cell
receptor, annexin II (20), to induce tissue factor expression (21) and
to suppress heparan sulfate expression (22). Several studies suggest
that homocysteine may interfere with the protein C pathway (23-26).
The most important function of this pathway appears to be the
inactivation of factor Va, the essential cofactor for the
prothrombinase complex which converts prothrombin to thrombin.
Human factor V (Mr = 330,000) is activated by
The regulation of factor Va is a key process for maintaining
hemostasis, as evidenced by two congenital thrombotic disorders, i.e. the G1691A mutation (Arg-506 to Gln) in factor V
Leiden and protein C deficiency (33, 34). There is also evidence for impaired factor Va inactivation by APC in the absence of the factor V
Leiden mutation, which is associated with increased risk of venous
thrombosis (35).
The present study was undertaken to evaluate the ability of
homocysteine at concentrations of 1 mM or less to modulate
activation of human factor V and inactivation of human factor Va. We
examined the effect of homocysteine on these processes using clotting
assays and Western blot analyses along with radiolabeling of factor V by [35S]homocysteine. Our data suggest a potential
contributor to the prothrombotic tendency observed in hyperhomocysteinemia.
Materials
DL-Homocysteine, L-homocystine,
DL-homocysteine thiolactone, and L-cysteine as
well as HEPES were purchased from Sigma.
1,2-Dioleoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (PS) and
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (PC) were
purchased from Avanti (Alabaster, AL). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, and nitrocellulose were purchased from
Bio-Rad. Pooled normal plasma (number of donors >30) was purchased
from George King Bio-Medical (Overland Park, KS). Factor V was purified
from human plasma by immunoaffinity chromatography as previously
described (36). Factor V concentrations were estimated spectrophotometrically. Human APC and Methods
Homocysteine Modification and Functional Assays--
Factor V
was dialyzed against 20 mM HEPES, 0.15 M NaCl,
5 mM CaCl2, pH 7.4 (HBS-Ca2+) with
or without the addition of homocysteine or other thiols (solubilized in
0.15 M HCl) at varying concentrations at 4 °C for 2 h followed by dialysis against HBS-Ca2+ for 2 h.
Homocystine was solubilized in 0.15 M HCl. Factor V was
activated by treatment with 1 nM Gel Electrophoresis and Western Blotting--
Aliquots from the
activation/inactivation experiments were withdrawn and quenched in 2%
Activated Protein C Resistance Assay--
Factor V was dialyzed
against HBS-Ca2+ containing 1 mM
Plasma samples from individuals with elevated homocysteine levels were
a generous gift from Dr. Andrzej Szczeklik, Jagiellonian University
School of Medicine, Cracow, Poland. Plasmas were obtained from
four asymptomatic men with genetically determined hyperhomocysteinemia stemming from a mutation in the 5,10-methylenetetrahydrofolate reductase gene (C677T). The presence of factor V Leiden was excluded on
the basis of polymerase chain reaction analysis. Blood was drawn after
a 16-h overnight fast, and plasma homocysteine concentration was
determined according to Mansoor et al. (43).
Radiolabeling of Factor V with
[35S]Homocysteine--
L-[35S]Methionine
(201 mCi/mmol) was converted to
L-[35S]homocysteine thiolactone and
subsequently to L-[35S]homocysteine using the
method of Baernstein (44). The product was identical with authentic
homocysteine by thin layer and paper chromatography and contained a
small amount (<10%) of homocystine. Reported concentrations of
L-[35S]homocysteine were determined by liquid
scintillation counting. Radiolabeled L-homocysteine was
added to factor V that had been dialyzed against HBS-Ca2+
at room temperature for 2 h. Subsequently, factor V was activated with
Kinetic analyses of [35S]homocysteine modification of
factor V were performed in HBS-Ca2+ at room temperature in
which times of treatment were varied at a fixed
[35S]homocysteine concentration, or concentrations of
[35S]homocysteine were varied and the reaction allowed to
proceed for a fixed time. Reactions were quenched with iodoacetic acid (Sigma) that had been recrystallized from heptane. Aliquots from reacting mixtures of factor V and [35S]homocysteine were
combined with an equal volume of 100 mM iodoacetic acid,
0.5 M Tris, pH 8.7, before the addition of SDS-PAGE sample buffer. The radiolabeled factor V was resolved by electrophoresis on
4-12% linear gradient SDS-polyacrylamide gels under nonreducing conditions, and the gels were processed as described above.
Densitometric data from resulting autoradiographs were normalized to
correct for variations in protein loading and then converted to the
percentage of maximum values using the highest densitometric value
within a data set. These transformed data were then fit to a double
exponential equation using software IGOR Pro Version 3.1, WaveMetrics Inc.
Activation of Factor V by Inactivation of Factor Va by APC in the Presence of
Phospholipids--
Preincubation of factor V with 1 mM
homocysteine for 2 h inhibited the APC anticoagulant effect toward
factor Va (Fig. 1). Factor V was treated
with homocysteine at 1 mM, 100 µM, or 30 µM at 4 °C for 2 h, dialyzed against
HBS-Ca2+ for an additional 2 h, and then activated by
To test whether the interference was related to a decrease in APC
activity after incubation with homocysteine, APC was preincubated with
homocysteine at 1 mM for 30, 60, and 120 min at 4 °C. No differences were observed in the rate of inactivation of 100 nM factor Va by 2.5 nM APC whether treated with
homocysteine or untreated (data not shown). Contrary to previous
reports (23, 25), we did not observe a direct effect of homocysteine on
APC activity per se.
Inactivation of Factor Va by APC in the Absence of
Phospholipids--
The cleavage at Arg-306 is
phospholipid-dependent, whereas the cleavage at Arg-506 is
enhanced in the presence of phospholipids (30). In the absence of
phospholipids, the rate of APC inactivation of factor Va derived from
factor V treated with 1 mM homocysteine (above) was
significantly delayed compared with controls (Fig. 2). The ~3-fold decrease in the rate
constant induced by homocysteine is similar to that observed in the
presence of phospholipids. Since the lipid-independent inactivation
proceeds only through cleavage at Arg-506, the significant impairment
of APC inactivation of factor Va without phospholipids indicates that
homocysteine must slow cleavage at Arg-506.
Immunoblot Analysis of Factor Va Inactivation by APC--
Factor V
(100 nM) was treated with either 1 mM
homocysteine, 1 mM homocystine, or HBS-Ca2+ at
4 °C for 2 h. After activation by Factor V Purified from Plasma Treated with Homocysteine--
Human
plasma contains a wide range of the sulfhydryl compounds, including
cysteine, cysteinylglycine, homocysteine, and glutathione, which are in
the reduced (<5%) or oxidized form or are protein-bound (43).
Total plasma concentration of cysteine, the most abundant aminothiol,
is around 250 µM, with levels of reduced cysteine amounting to 10 µM (43). Moreover, it is known that
homocysteine binds to numerous plasma proteins, mainly albumin (plasma
concentration 0.5-0.7 mM) at an equimolar ratio. To
determine whether the addition of homocysteine to normal human plasma
would alter the APC sensitivity of factor V subsequently purified from
such plasma, human plasma was incubated with homocysteine (10 mM) at 4 °C for 2 h, and then factor V was
purified, as described under "Methods." Homocysteine added to
plasma effectively retarded the inactivation of subsequently purified
and Radiolabeling of Human Factor V with
[35S]Homocysteine--
To determine whether homocysteine
can be incorporated into human factor V, factor V was incubated with
450 µM [35S]homocysteine at room
temperature for 2 h. It was then activated by
Samples obtained before and 10, 30, and 120 min after the addition of
APC were removed, mixed with 2%
To test whether factor V in plasma can incorporate homocysteine, human
plasma was incubated with 450 µM
[35S]homocysteine at room temperature for 2 h.
SDS-PAGE analysis of nonreduced samples was performed. The Coomassie
Blue-stained gel and the corresponding autoradiograph showed the
presence of a band with the same mobility as purified human factor V
(data not shown).
Time course analyses of the incorporation of homocysteine into factor V
were performed across a range of concentrations using [35S]homocysteine. At 450 µM
[35S]homocysteine, radiolabeling of factor V was complete
in less than 90 s (data not shown). At concentrations of
homocysteine between 10 and 450 µM, a 5-min reaction
period at room temperature was sufficient to achieve levels of
radiolabeling within 15% of those observed after 60 min. Fig.
6A illustrates the time course for the reaction with 300 nM factor V and 25 µM [35S]homocysteine. When iodoacetic
acid-quenched time points from reactions of factor V and
[35S]homocysteine were subjected to proteolysis by
To estimate the equilibrium constant describing the reaction of factor
V with homocysteine, factor V (300 nM) was incubated at
room temperature for 5 min with increasing concentrations of [35S]homocysteine, the radiolabeled product resolved by
SDS-PAGE, and the relative levels of factor V radiolabeling determined
by densitometry. The resulting binding curve is shown in Fig.
6B. Half-maximal modification of factor V was achieved at
~80 µM homocysteine.
Radiolabeling experiments carried out with
L-[35S]cysteine (124 mCi/mmol) indicate that
this thiol also readily forms disulfide adducts with factor V when
reacted under conditions and concentrations used with
[35S]homocysteine (data not shown). As observed in the
case of [35S]homocysteine, disulfide formation between
factor V and L-[35S]cysteine was apparent
after Effects of BME--
Since our data suggest that homocysteine is
incorporated into factor V through the formation of disulfide bonds, we
hypothesized that every plasma-derived factor V preparation would
reflect some level of derivatization with homocysteine and, thus, some
impairment with respect to APC inactivation. To test this hypothesis,
we dialyzed factor V against 1 mM APC Resistance Assay Performed in Plasmas Containing Various
Mixtures of Reduced (FVBME) and Homocysteinylated
(FVHCY) Factor V--
A series of reconstituted plasmas
were generated with varying proportions of partially reduced factor V
(FVBME) and homocysteinylated factor V (FVHCY)
(see "Methods") and tested in an APC resistance assay format.
Clotting times for each factor V mixture were measured after incubation
in the presence or absence of 1 nM APC. The clotting time
for each factor V mixture was then plotted against the percentage of
reduced factor V (FVBME) present in that mixture. Fig.
8 shows the results of such an
experiment. The composition of the factor V mixture had no effect on
the rate of clot formation in the absence of APC (closed
circles). However with APC, clotting times in those same mixtures
increased as a function of the percentage of FVBME present:
from 56 s with 0% FVBME,100% FVHCY to
89 s with 100% FVBME, 0% FVHCY
(open circles). A pooled plasma obtained from normal
individuals gave clotting times of 46 s in the absence of APC and
85 s in the presence of APC, corresponding to a
FVBME/FVHCY composition of 88%/12% (Fig.
8).
Plasmas from four individuals (see "Methods") with homocysteine
levels of 40, 44, 76, and 80 µM were then tested in this
assay format (Fig. 8). In the absence of APC, clotting times for
patient plasmas always fell between 40-45 s, the same range as
observed with the mixtures constructed in factor V-deficient plasma
(Fig. 8). In the presence of APC, clotting times for the pair with 40 µM levels of homocysteine averaged 79 s,
corresponding to a FVBME/FVHCY distribution of
68%/32%; clotting times for the plasmas with ~80 µM
homocysteine averaged 71 s, corresponding to a
FVBME/FVHCY composition of 44%/56%. Thus
plasmas with higher homocysteine levels behave in the APC resistance
assay as mixtures with a higher proportion of FVHCY.
We present evidence that in vitro homocysteine
treatment of factor V can protect The most probable mechanism by which homocysteine inhibits APC
inactivation of factor Va is by forming heterologous disulfide bonds.
The heavy chain of bovine factor Va contains four cysteine residues in
the A1 domain and six in the A2 domain (45). All cysteines in the A1
domain are associated with two disulfide bridges, whereas in the A2
domain, two free cysteines are located at positions 538 and 589 (45).
On the basis of homology, the A2 domain of the heavy chain of human
factor Va is likely to contain two free cysteines, Cys-539 and Cys-585
(Fig. 9). Since the light chain of bovine
factor Va contains two free cysteines (Cys-1953 and Cys-2100), two free
cysteine residues are anticipated in human factor Va light chain (46).
The autoradiograph depicted in Fig. 5B provides evidence
that both the heavy and light chains of factor Va are radiolabeled with
[35S]homocysteine. Of the three fragments resulting from
the APC cleavage of the heavy chain of factor Va, only the 28/26,000
doublet (residues 507-709), which contains Cys-539 and Cys-585,
incorporated [35S]homocysteine (Fig. 5B).
Under reducing conditions (2% -thrombin. Factor Va derived from homocysteine-treated
factor V was inactivated by APC at a reduced rate. The inactivation
impairment increased with increasing homocysteine concentration (pseudo
first order rate k = 1.2, 0.9, 0.7, 0.4 min
1 at 0, 0.03, 0.1, 1 mM
homocysteine, respectively). Neither cysteine nor homocysteine
thiolactone treatment of factor V affected APC inactivation of derived
factor Va. Western blot analyses of APC inactivation of
homocysteine-modified factor Va are consistent with the results of
clotting assays. Factor Va, derived from factor V treated with 1 mM
-mercaptoethanol was inactivated more rapidly than
the untreated protein sample. Factor V incubated with
[35S]homocysteine (10-450 µM) incorporated
label within 5 min, which was found only in those fragments that
contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113),
the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC
cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with
-mercaptoethanol removed all radiolabel. Plasma of patients assessed
to be hyperhomocysteinemic showed APC resistance in a clot-based assay.
Our results indicate that homocysteine rapidly incorporates into factor
V and that the prothrombotic tendency in hyperhomocysteinemia may be
related to impaired inactivation of factor Va by APC due to
homocysteinylation of the cofactor by modification of free cysteine(s).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synthase, along with
deficiencies in the essential cofactors folic acid and vitamins
B6 or B12 can result in elevated levels of
plasma homocysteine. In healthy individuals, plasma total homocysteine (the sum of homocysteine and the homocysteinyl derivatives homocystine and cysteine-homocysteine, whether free or bound to proteins) was
between 5 and 15 µM (2, 3). A number of
case-control and prospective studies have shown that
hyperhomocysteinemia, present in 5-7% of the population, is an
independent risk factor for atherosclerotic vascular disease in the
coronary (4-6), cerebrovascular (7), and peripheral vessels (8, 9) and
also for venous thromboembolism (10-13).
-thrombin cleavages at Arg-709, Arg-1018, and Arg-1545 (27-28). The
product, factor Va, is composed of a heavy chain, residues 1-709
(Mr = 105,000), derived from the NH2
terminus of factor V (A1-A2 domains) and a noncovalently associated
light chain, residues 1546-2196 (Mr = 74,000),
derived from the COOH terminus (A3-C1-C2 domains) (27-28). Factor Va
is proteolytically inactivated by activated protein C
(APC)1 by three cleavages of
the heavy chain at Arg-506, Arg-306, and Arg-679 (29, 30). The cleavage
of factor Va at Arg-506 is enhanced in the presence of a phospholipid
surface, whereas cleavage at Arg-306 is lipid-dependent
(30). After cleavage at Arg-306, the A2 domain (residues 307-709)
dissociates as two fragments (residues 307-506 and 507-709) from the
factor Va molecule, leading to the complete inactivation of factor Va
(31, 32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin were gifts from Hematologic Technologies Inc. (Essex Junction, VT). Hirudin was obtained from Genentech (South San Francisco, CA). Phospholipid vesicles (PCPS) composed of 75% PC and 25% PS were prepared according to Barenholz et al. (37), with the concentrations determined by phosphorous assay. Monoclonal antibodies against the heavy chain of
human factor Va (
FVaHC 17) were from the Biochemistry Antibody Core Laboratory (University of Vermont). Goat anti-mouse IgG
peroxidase was purchased from Southern Biotech (Birmingham, AL).
Molecular weight standards were purchased from Life Technologies, Inc.
The chemiluminescent substrate, luminol, was purchased from PerkinElmer
Life Sciences as was the fluorography reagent, Entensify. L-[35S]Methionine and
L-[35S]cysteine were purchased from Amersham
Pharmacia Biotech.
-thrombin for 10-13
min at 37 °C.
-Thrombin was inhibited by the addition of hirudin at a 4-5-fold molar excess. APC at various concentrations (see legends
to figures) was added to factor Va in the presence or absence of
phospholipids. At selected time intervals, samples were removed and
diluted into HBS-Ca2+ for assay. Factor Va activity was
immediately measured in a clotting assay at 37 °C using factor
V-deficient plasma (38). In a typical assay, 50 µl of factor V or Va
diluted in HBS-Ca2+ was added to an equal volume of factor
V-deficient human plasma followed by the addition of 100 µl of PT
(prothrombin time) reagent (Simplastin, Organon Teknika, Durham,
NC). All experiments were repeated three times on separate days with
comparable results. Time course data for the APC inactivation of factor
Va were fit to a single exponential equation using software IGOR Pro
Version 3.1, WaveMetrics Inc. (Lake Oswego, OR). Initial slopes from
the fit curves were used to calculate the pseudo first order rate constants. Pseudo first order rate constants are all normalized to
constant APC concentration (2.5 nM).
-mercaptoethanol, 2% SDS for electrophoretic analysis. Samples were
separated on a 5 to 15% linear gradient gel (SDS-PAGE) under reducing
conditions according to Laemmli (39). Transfer from the gel to
nitrocellulose (Bio-Rad) was performed as previously described (40).
Immunoreactive fragments were detected using the monoclonal antibody
FVaHC 17, which recognizes an epitope located between
residues 307 and 506 in the heavy chain of factor Va (41). Blots were
developed using the Renaissance chemiluminescent reagent (Dupont). Film
was developed in a Kodak X-Omat on Reflection film (PerkinElmer Life
Sciences). Densitometry of immunoblots was performed on a
Hewlett-Packard ScanJet 4C/T equipped with a transparency adapter for
backlighting the x-ray film (Hewlett-Packard, Palo Alto, CA) and
analyzed using NIH Image 1.6 (Bethesda, MD).
-mercaptoethanol (BME) or 2 mM homocysteine (HCY) for
2 h at 4 °C and then for 2 h at 4 °C against
HBS-Ca2+. Dialysis was performed in filled, capped bottles
in buffer that had been boiled in a reflux apparatus and purged
extensively with nitrogen. The resulting stocks, FVBME (2.2 µM) and FVHCY (2.0 µM) were
used immediately. FVHCY could be flash-frozen in
methanol/dry ice and successfully recovered, whereas FVBME
proved sensitive to freezing, showing a decrease in APC sensitivity
after thawing. Mixtures of FVBME and FVHCY with
a total factor V concentration of 400 nM in which the
amount of FVHCY varied from 0 to 100% were constructed by
appropriate dilution in HBS-Ca2+, and their relative
susceptibility to APC proteolysis assessed by a modification of the
method of Dahlback (42). Five µl of each factor V mixture was added
to 95 µl of factor V-deficient (immunodepleted) plasma followed by
the addition of 100 µl of aPTT (activated partial thromboplastin
time) reagent (Organon Teknika). This reformulated plasma
([factor V] = 20 nM) was incubated for 5 min at 37 °C.
One hundred µl of 3 nM APC in HBS-30 mM
Ca2+ or 100 µl of HBS-30 mM Ca2+
was then added, the mixture rocked at 37 °C, and clotting time was
recorded. Clotting times were plotted against the percentage of
FVBME present. Best-fit lines were determined by linear
regression analysis. The slopes of these lines from 3 separate sets of
FVBME/FVHCY mixtures varied by less than 10%.
To assay plasma, 100 µl of patient or normal plasma was combined
directly with 100 µl of aPTT reagent and then processed as above.
-thrombin, and then, upon addition of hirudin and PCPS
vesicles, factor Va was inactivated by APC. At designated time points,
aliquots were removed from the mixture and subjected to SDS-PAGE.
Protein bands were visualized by staining the gel with Coomassie Blue and destaining by diffusion. Gels were then soaked in the fluorography reagent as per the manufacturer's instructions and dried, and radiolabeled protein was detected using Fuji Medical x-ray film (Fuji
Medical Systems USA, Inc., Stamford CT). Densitometry was performed on
the Coomassie-stained gel before drying and on the resulting
autoradiographs as described for immunoblots. Details are given in the
legends to figures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Thrombin--
There was no
significant alteration in
-thrombin activation of factor V after
dialysis against 1 mM homocysteine in HBS-Ca2+
for 2 h. Homocystine, homocysteine thiolactone, and cysteine incubated with factor V under the same conditions did not affect activation of factor V by
-thrombin (data not shown).
-thrombin. A progressive decrease in the initial rates of APC
inactivation of factor Va, generated from the homocysteine-treated
factor V, is associated with increasing homocysteine concentrations
(Fig. 1). The rate constant for the APC-catalyzed inactivation of
factor Va, generated from factor V treated with 1 mM
homocysteine, is about one-third the rate constant for control
experiments (Fig. 1).
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Fig. 1.
Effect of increasing concentrations of
homocysteine. Factor V was dialyzed against HBS-Ca2+
containing homocysteine at the following concentrations: 0 ( ), 30 µM (
), 100 µM (
), 1 mM
(
) as described under "Methods." Subsequently, factor V (100 nM) was activated by
-thrombin (1 nM) at
37 °C for 10 min followed by the addition of hirudin (5 nM). Upon the addition of PCPS vesicles (20 µM) and then APC (2.5 nM), the activity of
factor Va was evaluated at 1, 3, 5, and 10 min using a one-stage
clotting assay (see "Methods"). The results were expressed as the
percentage of initial activity of factor Va as a function of time. The
inset presents the rate constants for APC inactivation. Data
represent one of three experiments.
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Fig. 2.
Effect of homocysteine in the absence of
phospholipids. Factor V was dialyzed against HBS-Ca2+
with 1 mM homocysteine ( ) or without (
), as described
under "Methods." Subsequently, factor V (100 nM) was
activated by
-thrombin (1 nM) at 37 °C for 10 min
followed by the addition of hirudin (5 nM). After the
addition of APC (10 nM), the activity of factor Va was
evaluated at 1, 3, 5, and 10 min using a one-stage clotting assay (see
"Methods"). The results were expressed as the percentage of initial
activity of factor Va as a function of time. The inset
presents the rate constants for APC inactivation. Data represent one of
three experiments.
-thrombin (see
"Methods"), the factor Va preparations were cleaved by 2.5 nM APC at 37 °C, and the time course of each reaction
was followed by Western blot analysis using the monoclonal antibody
FVaHC 17, which recognizes an epitope in the fragment
307-506 (36), the ultimate APC cleavage product of factor Va heavy
chain. Data for this experiment over 60 min are presented in Fig.
3. Lanes 1-6 correspond to
the control; lanes 13-18 correspond to the
homocystine-treated factor V. The samples from treatment with
homocysteine are presented in lanes 7-12. In both the
control and in the homocystine-treated samples, the 60-min reaction
sample (lane 6 and lane 18) show almost complete cleavage of the heavy chain and the appearance of fragments
corresponding to the final cleavage product of factor Va. In contrast,
some heavy chain remains intact in the homocysteine-treated samples at
60 min (lane 12). The data show that homocysteine inhibits cleavage of factor Va heavy chain by APC and that the reaction requires
the reduced form of the amino acid. This effect on inactivation is
homocysteine-specific, since for factor V preincubated with 1 mM L-cysteine for 2 h, the time course of
the inactivation of factor Va by APC was identical to those in control
and homocystine experiments (data not shown).
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Fig. 3.
Immunoblots of factor Va after
treatment. Human factor V was dialyzed in HBS-Ca2+
alone, with homocysteine (1 mM), or with homocystine (1 mM), as described under "Methods." Factor V (100 nM) was then incubated with -thrombin (0.5 nM) at 37 °C for 13 min, followed by the addition of
hirudin (2 nM). In the presence of PCPS vesicles (40 µM), factor Va was inactivated by APC (2.5 nM) at 37 °C. At six time points (see below), aliquots
taken from the mixture were added to 2%
-mercaptoethanol, 2% SDS
(see "Methods") After transfer to nitrocellulose, immunoreactive
fragments were detected with the monoclonal antibody
FVaHC 17. Lanes 1-18 depict aliquots
withdrawn immediately before (0 min) and at 1, 5, 10, 20, and 60 min
after the addition of APC to factor Va untreated (1-6),
dialyzed against 1 mM homocysteine (7-12), and
against 1 mM homocystine (13-18). Approximately
100 ng of protein was applied per lane. The position of the
molecular weight markers is indicated on the left.
HC, heavy chain.
-thrombin-activated factor V by APC in the presence of PCPS
vesicles (Fig. 4). The ~3-fold decrease
in the rate constant is the same as observed when purified factor V was
treated with 1 mM homocysteine (Fig. 1). Slower
inactivation of factor Va, derived from factor V purified from plasma
treated with homocysteine, was also observed in the absence of
phospholipids (data not shown).
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Fig. 4.
Effect of homocysteine on factor V in normal
plasma. Homocysteine (10 mM) ( ) or buffer control
(
) was added to plasma for 2 h (see "Methods"). Factor V
was purified using immunoaffinity chromatography according to Katzmann
et al. (36). Factor V (100 nM) was treated with
1 nM
-thrombin, after which hirudin (5 nM),
PCPS vesicles (40 µM), and finally APC (2.5 nM) were added to the mixture. At 1, 3, 5, 7, and 10 min
after the addition of APC, samples taken from the mixture were assayed
for factor Va activity. The inset presents the rate
constants for APC inactivation. Data represent one of three
experiments.
-thrombin, and
the resulting factor Va was treated with 6 nM APC in the
presence of phospholipids. At selected time points aliquots were
removed and subjected to analysis by SDS-PAGE. Coomassie Blue staining
of the gel with nonreduced samples (Fig.
5A) displays [35S]homocysteine-treated factor V (lane 1),
the derived factor Va (lane 2), and the time course of APC
degradation of factor Va (lanes 3-8). Degradation of the
heavy chain of factor Va, derived from
[35S]homocysteine-treated factor V, is not complete at
180 min (lane 8). In contrast, APC degradation of factor Va,
derived from untreated factor V, is complete under these conditions in
60 min (30). The APC cleavage of factor Va, derived from
[35S]homocysteine-treated factor V, yielded products of
Mr = 45,000 (), Mr = 30,000 (), and Mr = 28/26,000
(). Bands corresponding to the light chain of factor Va are
stable throughout the 180-min incubation with APC. This pattern is
identical to that seen with untreated factor V (30). Comparing the
Coomassie Blue-stained (Fig. 5A) gel with the corresponding
autoradiograph (Fig. 5B) revealed that only the protein
fragments that contain free sulfhydryl groups (45, 46) (intact factor V
(lane 1), the heavy and light chains of factor Va
(lane 2), the B region (lanes 2-8), and the doublet of Mr = 28/26,000 (lanes
3-8)) incorporate [35S]homocysteine. Consistent
with the pattern observed by Coomassie Blue staining, the intensity of
the radiolabeled heavy chain band decreased with time, whereas the
intensity of the light chain bands remained unchanged. The fragments of
Mr = 45,000 () and 30,000 (),
which do not contain any free cysteines, are not visualized on the
autoradiograph (Fig. 5B).
View larger version (89K):
[in a new window]
Fig. 5.
Modification of human factor V with
[35S]homocysteine. Factor V (600 nM) was
incubated with 450 µM [35S]homocysteine in
HBS-Ca2+ at 25 °C for 2 h (lane 1) and
then treated with -thrombin (12 nM) for 10 min
(lane 2). After the addition of hirudin (60 nM)
followed by PCPS vesicles (40 µM), factor Va was
subjected to APC (6 nM) proteolysis. At selected time
intervals (5, 10, 30, 60, 120, and 180 min in lanes 3-8,
respectively), 110-µl aliquots of the mixture were withdrawn and
analyzed by a 5-15% linear gradient SDS-PAGE under nonreducing
conditions. Duplicate aliquots were also removed before and 10, 30, and
120 min after the addition of APC, mixed with 2%
-mercaptoethanol,
2% SDS, and analyzed by a 5-15% linear gradient SDS-PAGE. Coomassie
Blue-stained gel run under nonreducing conditions (panel A)
and its autoradiograph (panel B) are shown. Coomassie
Blue-stained gel run under reducing conditions (panel C) and
the corresponding autoradiograph (panel D) are shown. The
positions of the molecular weight markers are indicated. HC,
heavy chain; LC, light chain.
-mercaptoethanol/SDS, and analyzed
by SDS-PAGE. When samples were reduced, none of the bands seen on the
gel stained with Coomassie Blue (Fig. 5C) could be detected
on the autoradiograph of this gel (Fig. 5D) even with periods of autoradiography 20 times longer than those required to
detect bands on the gels with the nonreduced samples. Thus the
radiolabel in factor V/Va was totally removed by the disulfide-reducing agent.
-thrombin and analyzed by SDS-PAGE, no differences in the rates of
homocysteine incorporation into the heavy and light chains of factor Va
were observed (data not shown). At elevated homocysteine
concentrations, e.g. 450 µM, periods of
incubation with factor V greater than 60 min resulted in an additional,
slower increase in the level of factor V radiolabeling.
-Thrombin
digestion indicated that this radiolabeling reflected adduct formation
in the B region of factor V, presumably with Cys-1085 (data not
shown).
View larger version (14K):
[in a new window]
Fig. 6.
Kinetics of factor V modification by
[35S]homocysteine. Factor V (300 nM) was
incubated with [35S]homocysteine at concentrations
ranging from 10 to 450 µM in HBS-Ca2+ at
25 °C for times ranging from 15 s to 120 min. Reactions were
quenched with iodoacetic acid, and the radiolabeled factor V was
resolved by SDS-PAGE under nonreducing conditions. Relative levels of
radiolabeled factor V were determined by densitometric analysis of the
resulting autoradiographs. Panel A shows a time course
analysis of the reaction of 25 µM
[35S]homocysteine with factor V. The curve fit results in
a percentage of maximum density value at infinite time of 93.5%.
Panel B shows the result of a 5-min treatment of factor V at
the following [35S]homocysteine concentrations: 10, 20, 40, 80, 100, 200, and 450 µM. The curve fit results in a
percentage of maximum density at infinite time of 105.1%.
-thrombin proteolysis in the heavy chain, the light chain, and
the B region; proteolysis by APC located the heavy chain radiolabeling
only in the 28/26,000 doublet (residues 507-709).
-mercaptoethanol for
2 h. Factor Va, derived from the thiol-reduced factor V, was
inactivated by APC about 50% more rapidly than the control (Fig.
7).
View larger version (22K):
[in a new window]
Fig. 7.
Effects of BME. Factor V was dialyzed
against HBS-Ca2+with 1 mM BME ( ) or without
(
), as described under "Methods." Factor V (100 nM)
was activated with
-thrombin (0.5 nM), followed by the
addition of hirudin (2 nM). In the presence of PCPS
vesicles (40 µM), factor Va was inactivated by APC (2.5 nM) at 37 °C. The activity of factor Va was evaluated at
1, 3, 5, and 10 min after the addition of APC (see "Methods"). The
results were expressed as the percentage of initial activity of factor
Va versus time. The inset presents the rate
constants for APC inactivation of factor Va.
View larger version (13K):
[in a new window]
Fig. 8.
APC resistance in plasmas containing various
mixtures of reduced (FVBME) and homocysteinylated
(FVHCY) factor V. Mixtures of FVBME and
FVHCY with a total factor V concentration of 400 nM were constructed, diluted 20-fold in factor V-deficient
plasma, and tested for reactivity with APC. Each factor V mixture was
assessed in the presence ( ) or absence (
) of 1 nM
added APC. Clotting time is plotted against the percentage of
FVBME present in each mixture. Data are from a
representative experiment. Best-fit lines were calculated by linear
regression analysis. Clotting times measured in the presence of 1 nM APC for plasmas from patients (see "Methods") with
defined levels of plasma homocysteine (40, 44, 76, and 80 µM) are indicated with arrows as is the
clotting time from pooled plasma obtained from normal donors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin-derived factor Va from
inactivation by APC. This effect is observed at concentrations of
homocysteine as low as 30 µM, similar to those found in
the blood of patients with "moderate hyperhomocysteinemia." As the
homocysteine concentration used to treat factor V increases, impairment
of APC-mediated proteolysis of human factor Va is more evident (Fig.
1). Our data indicate that the rate of APC cleavage at Arg-506 of
homocysteinylated factor Va is diminished about 2.5-fold. (Fig. 2). The
magnitude of this inactivation impairment is similar to that observed
when APC inactivation of factor V Leiden is assessed in a
function-based assay (47).
-mercaptoethanol) [35S]homocysteine was released from factor V, since none
of the bands seen on the Coomassie Blue-stained gel (Fig.
5C) were visualized on the corresponding autoradiograph
(Fig. 5D). These results are consistent with the conclusion
that incubation of human factor V with [35S]homocysteine
results in the formation of protein-S-[35S]homocysteine
disulfide adducts in a pattern consistent with the involvement of the
five free cysteines of factor V.
View larger version (21K):
[in a new window]
Fig. 9.
Factor V and Va with cleavage sites by
thrombin and APC. Positions of all free cysteines and disulfide
bridges are depicted.
It has been reported that reduction of disulfide bonds by homocysteine
or other reducing agents can result in the impaired function of such
proteins as protein C or thrombomodulin (25). No evidence of disruption
of intrachain disulfides of factor V was seen in our study either by
direct or functional criteria. The APC-generated heavy chain fragments
containing only disulfides (Mr = 30,000 and
Mr = 45,000, see Fig. 9) were not radiolabeled when factor V were treated for time periods up to 2 h either with 450 µM [35S]homocysteine (Fig.
5B) or 450 µM
L-[35S]cysteine (data not shown). Factor V
subjected to our treatment regimens with 1 mM thiol,
whether DL-homocysteine, L-cysteine, or
-mercaptoethanol, was indistinguishable from untreated factor V in
the kinetics of its activation by
-thrombin and in the activity of
its product, factor Va. In addition, 1 mM
L-cysteine exerted no effect on APC-mediated proteolysis of
factor Va, despite incorporation of
L-[35S]cysteine into the heavy and light
chains of the protein. Taken together, these observations do not
support a mechanism in which homocysteine efficacy derives from
disulfide exchange reactions between homocysteine and the structural
disulfides of the factor V molecule.
Our data offer some grounds for speculation concerning the mechanism of adduct formation between factor V and aminothiol. Blood constitutes a dynamic system where the thermodynamic and kinetic constants defining the chemistries of disulfide formation through oxidation and subsequent disulfide exchange reactions compete with the biological processes maintaining the specific circulating half-lives of the individual proteins, oxidized aminothiols, and reduced aminothiols involved in these reactions. In normal individuals, the total concentration of aminothiols (including homocysteine, cysteine, cysteinylglycine, and glutathione) is in the range of 300 µM, with cysteine levels constituting about 80% of the total (43, 48). Aminothiols in the reduced form constitute about 7% of the total. Greater than 50% of the oxidized pool for each major aminothiol occurs as a protein-S-S-X adduct (43). The ratio of reduced to total aminothiol for each thiol appears specific for that thiol: homocysteine, 1% (48); cysteine, 5% (43); cysteinylglycine, 10% (43); and glutathione, 60% (43). Our data detailing the modification of factor V by homocysteine combined with our preliminary observations indicating a similar reactivity with cysteine can be extrapolated to predict that factor V derived from plasma with 10-20 µM reduced aminothiol will have about 20% of its free cysteines in the form of heterologous disulfides. Thus in our reactions between homocysteine or cysteine and purified factor V, the majority of the initial bond-forming events would be oxidation reactions, presumably involving molecular oxygen as the electron acceptor. Factor V shows significant sequence homology with the copper-binding protein ceruloplasmin (49) and has been demonstrated to contain 1 mol of copper/mol of factor V (50). The spectral properties of the factor V copper complex are consistent with those of other copper-binding proteins expressing oxidase activity (50). We have observed that factor V fully homocysteinylated with unlabeled homocysteine and then incubated with [35S]homocysteine rapidly radiolabels in all regions of factor V containing free cysteines (data not shown). This indicates that disulfide exchange between free aminothiol and factor V homocysteine disulfide adducts will also be an ongoing process.
Results of the experiments with factor V treated with 1 mM
-mercaptoethanol suggest that reduction of heterologous disulfide bonds between factor V free cysteines and homocysteine leads to a
markedly faster APC inactivation of factor Va. Since plasma concentrations of homocysteine differ among even healthy subjects, the
degree of impairment of APC inactivation of factor Va could vary
significantly. Our observations may offer an explanation for the
reported differences in APC activity toward factor Va derived from
different preparations of factor V.
Kinetic studies using [35S]homocysteine indicate that the
equilibrium between free homocysteine and homocysteinylated factor V is
reached in less than 5 min. This equilibrium is characterized by an
apparent Kd of 80 µM. Since the
circulating half-life of factor V is about 720 min, it can be estimated
that a fairly complete spectrum of factor V modification (10-85%)
could occur within the physiologically relevant range of elevated
homocysteine concentrations (10-450 µM). No difference
in the rates of modification between the free cysteines in the heavy
chain and those of the light chain was observed, suggesting an
equivalence in the reactivity of these sites. Combined with the
observation that modification of factor V by homocysteine does not
alter its rate of activation by -thrombin, we hypothesized that a
two component model for factor V populations might be sufficient to
characterize the relationship between homocysteine level and the APC
sensitivity of derived factor Va. One component is unmodified, virgin
factor V, produced in vitro by treating purified factor V
from pooled normal plasma with
-mercaptoethanol (FVBME)
and representing the source of factor Va most rapidly inactivated by
APC (Fig. 7). The other component is homocysteine-modified factor V,
generated in vitro by treating factor V with 2 mM homocysteine (FVHCY), representing the
source of "impaired" factor Va. The rate constant for the latter
inactivation by APC is ~25% that for unmodified factor Va. Plasma
factor V may then be conceived of as a mixture of these species, the
amounts of each being a function of plasma homocysteine and factor V
concentrations and the equilibrium constant. When such mixed
populations were systematically constructed from FVBME and
FVHCY stocks and tested in an APC resistance assay format, clotting times increased in a linear manner with increasing
representation by FVBME in the factor V mixture (Fig. 8).
This curve was used as a standard curve for translating plasma
homocysteine levels into estimates of their factor Va sensitivity to
APC degradation. Preliminary results with plasmas from four individuals
with elevated homocysteine levels support the potential for this
approach. Plasma homocysteine levels of 40, 44, 76, and 80 µM yield calculated values for percent unmodified factor
V of 67, 65, 51, and 50%, respectively, and predict clotting times of
79, 78, 73.4, and 73 s using the standard curve shown in Fig. 8.
The measured clotting times for these plasmas were 80, 78, 72, and
70 s. The clotting time of 85 s observed with the normal pool
plasma translates into a plasma homocysteine concentration of 10.9 µM, a value within the range of normal homocysteine
levels (2, 3, 43).
In vivo, increased thrombin formation would be the consequence of the reduced rate of APC-mediated proteolysis of factor Va, because this process leads to accumulation of active cofactor for the prothrombinase complex (30). Decreased efficiency of the APC-mediated factor Va inactivation may thus contribute to the prothrombotic activity of homocysteine. Our study may explain why the coexistence of factor V Leiden and hyperhomocysteinemia has a synergistic effect and confers a markedly higher risk of thrombotic disorder, as reported in a large epidemiological study by Ridker et al. (51).
Since platelets contain factor V that is secreted upon platelet
activation, the procoagulant activity of homocysteine through modification of platelet factor V may be important in regulating coagulation reactions at the site of vascular injury. If this is the
case, the property of homocysteine described here could also contribute
to an increased risk of arterial thrombosis reported in patients with
hyperhomocysteinemia. In summary, our results indicate that
homocysteine markedly impairs the APC-mediated inactivation of factor
Va. This effect is likely to be the consequence of modification of
factor V through attachment of homocysteine to free cysteine(s). Our
study strongly suggests that at the least the action of homocysteine on
factor Va inactivation is specific, involves the entire molecule, and
takes place at submillimolar homocysteine concentrations. The novel
pathophysiological mechanism reported here may partly explain the
thrombotic tendency observed in patients with hyperhomocysteinemia. Although the relationship between the pathology of hyperhomocysteinemia and APC resistance is by no means established by the present work, our
data do indicate that homocysteine modification of factor V is a
potential confounder of current APC resistance assays.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the excellent
technical assistance of Jed Pauls and thank Ted Foss for writing the
slope-finding software. We thank Dr. Andrzej Szczeklik and Dr. Teresa
B. Domagala for providing plasmas from individuals with a genetically
determined hyperhomocysteinemia and Dr. Richard Jenny for providing
human APC and -thrombin. We thank the United States Information
Agency Fulbright Scholar Program for its support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Merit Award HL-34575 (to K. G. M.) and the United States Information Agency Fulbright Scholar Program (to A. U.).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, Given Bldg., Health Science Complex, University of Vermont, College of Medicine, Burlington, VT 05405-0068. Tel.: 802-656-0335; Fax: 802-862-8229; E-mail: kmann@protein.med.uvm.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M004124200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
APC, activated
protein C;
BME, -mercaptoethanol;
HCY, homocysteine;
TEMED, N,N,N',N'-tetramethylethylenediamine;
PS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]
(sodium salt);
PC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine;
PCPS, phospholipid vesicles;
PAGE, polyacrylamide gel
electrophoresis.
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