Activated Protein C Resistance: Molecular

Transcription

Activated Protein C Resistance: Molecular
RAPID COMMUNICATION
Activated Protein C Resistance: Molecular Mechanisms Based on Studies
Using Purified G1n5O6-FactorV
By Mary J. Heeb, Yumi Kojima, Judith S. Greengard, and John H. Griffin
Gln=-factor V (W)
was purified from plasma
of an individual
homozygous for an Arg506Gln mutation
in FV that is associated with activatedproteinC
(APC)resistance.Purified
GlnW-FV, aswell as Glnm-FVa generated by either
thrombin
or FXa, conveyed APC resistance to FV-deficient plasma in
coagulation assays. Clotting assay studies also suggested
that APC resistance doesnot involve any abnormalityin FVAPC-cofactor activity. In purified reaction mixtures,GlnmFVa in comparison to normal N a showed reduced susceptibility to APC,because it was inactivated -10-fold slower
It was previously reported
that inacthan normal ArgW-FVa.
tivation of normal FVa by APC involves an initial cleavage
at Argm followed by phospholipid-dependent cleavage at
Argaos.lmmunoblotandaminoacid
sequenceanalyses
of Glnm-FVa was
showed that the 102-kDheavychain
cleaved at Argaos duringinactivation byAPC in a phospholipid-dependent reaction. This reducedbut measurable susceptibilityof Glnm-FVa to APC inactivation may help explain
why APC resistance is a mild risk factor for thrombosis becauseAPCcan
inactivatebothnormalFVa
andvariant
Glnm-FVa. In summary, this study shows that purified
Gln--FV can account for APC resistanceof plasma because
Gln=-FVa, whether generated by thrombin or M a , is relatively resistant to APC.
0 7995 by The AmericanSociety of Hematology.
T
with 2.6 for pooled nomal plasma using APTT assays as previously
described.' The father (age 63 years), mother (age 63 years), and
sister (age 26 years) of the individual were determined to be heterozygous for the mutation Arg506Gln in FV and to be A X resistant
(AP'IT ratios of 1.5, 1.7, and 1.65, respectively). A brother (age 29
years) was determined to have only normal FV and plasma that was
not APC resistant. The only notable thrombosis episode occurring
before the age of 60 years in this family was a documented incident
of thrombophlebitis and pulmonary embolism presented by the father
of the propositus at 49 years of age. The fraternal grandparents and
the fraternal uncle of the propositus were reported to have experienced deep vein thrombosis, stroke, and myocardial infarction after
60 years of age, respectively. Free protein S antigen (Asserachrom
free protein S ; Diagnostica Stago, Asnieres, France) and protein C
amidolytic activityx7were within the normal ranges for the propositus, his parents, and two siblings.
FVpurifcation. Blood (450 mL) from the individual homozygous for the mutation Arg506Gln in FV was collected after informed
consent into 50 mL of 0.11 m o a trisodium citrate. Plasma was
prepared by centrifugation at room temperature and made 50 pg/
mL in soybean trypsin inhibitor, 10 mmoIL in benzamidine, and
1 0 0 U/mL in aprotinin and frozen at -70°C. A second unit (450
mL) of blood was similarly obtained 8 weeks later, and the plasma
was treated with inhibitors and combined with thawed plasma from
HE PROTEIN C anticoagulant pathway provides a major physiologic mechanism that regulates thrombosis,
and defects in this pathway account for most currently
known genetic risk factors for venous thrombosis. Hereditary
heterozygous deficiency of protein C or of protein S is associated with increased risk for venous thrombosis in young
adults,'-3 whereas severe deficiency (< 1%) of protein C or
protein S is associated with purpura fulminans or massive
venous thrombosis in the neonatal peri0d.4.~Another hereditary abnormality of the protein C pathway involves a laboratory finding of a poor anticoagulant response to activated
protein C (APC), termed APC re~istance.~.'
APC resistance
is detected in 20% to 50% of venous thrombosis patients,
depending on criteria for patient population selection.'.'' The
genetic and molecular basis for APC resistance involves a
mutation in the coagulation factor V (W)gene at nucleotide
1691 that causes the mutation, Arg506Gln.I3-l5Two reports
of functional activity studies suggested that the variant
GlnSM-FVais resistant to cleavage by APC.'3*'6One report,
using FV purified from two individuals heterozygous for the
ArgSO6Gln-W mutation, concluded that thrombin-activated
GlnsM-FVais APC resistantI6;however, another report, using
unpurified adsorbed plasma from an individual homozygous
for the Arg506Gln mutation, concluded that Gln5M-FVagenerated by Factor Xa ( m a ) activation is APC resistant,
whereas Gln5"-FVa generated by thrombin activation is not
APC resi~tant.'~ Todate, no studies comparing purified
Gln5"FV activated by FXa or by thrombin have been reported. To address this controversy and to assess the hypothesis that purified Glns"-FV indeed accounts for APC resistance in plasma, the present study based on purification of
GlnSM-FVfrom homozygous plasma was undertaken.
MATERIALS AND METHODS
Patient material. During testing of normal laboratory worker
volunteers for APC resistance, a 32-year-old man was identified as
homozygous for the DNA mutation predicted to give Gln5"-FV
using DNA analysis performed as previously described.14Clotting
assays of the proband's plasma showed it to be markedly APC
resistant with a ratio of (activated partial thromboplastin time
[AFTTI + APC)/(AFIT) of 1.1 to 1.3 on different dates, compared
Blood, Vol 85, No 12 (June 15). 1995: pp 3405-3411
From the Departments of Molecular and Experimental Medicine
and of Vascular Biology, The Scripps Research Institute, La Jolla,
CA.
Submitted February 16, 1995: accepted March 21, 1995.
Supported in part by National Institutes of Health Grants No.
ROI-HW2246, ROl-HL21544, and MOIRR00833 and by the Stein
Endowment Fund.
A preliminary report of this study was presented at the American
Heart Association Meeting, Dallas, l X , November, 1994, and at the
American Society of Hematology Meeiing, Nashville, TN, December,
1994.
Address reprint requests to SBRS, The Scripps Research Institute,
I0666 N Torrey Pines Rd. La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordnnce with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8512-0.00/0
3405
3406
the first collection. Diisopropylfluorophosphateand phenylmethylsulfonic acid were each added to a concentration of 1 mmoVL and
the plasma was barium adsorbed as described." The supernatant
solution was sequentially treated with 7% and then 13% polyethylene
glycol-6000 (PEG); the precipitate from 13%PEG containing FV
was further purified as described." FV was similarly prepared from
500 mL of plasma from an individual determined not to be APC
resistant by APTT and from 1.5 L of pooled plasma from 6 individuals of undetermined APC resistance status. In experiments described
below, normal FV from the single donor was used unless otherwise
stated.
FV characterization and activation. FV was characterized by
absorbance at 280 nm using Ala, = 8.9, by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), by enzyme-linked
immunosorbent assay (ELISA) for FV antigen, and by functional
assay. The sandwich ELISA was performed as described for protein
S antigen" using immobilized rabbit anti-FV IgG (Dako, Carpenteria, CA) at 7.5 pg/mL as the capturing antibody and monoclonal
anti-FVa light chain antibody at 5 pg/mL (Hematologic Technologies, Essex Jct, VT) as the detecting antibody. Dilutions of normal
plasma were simultaneously assayed and the data were used to construct a standard curve, assuming a plasma concentration of 30 nmol/
L FV antigen. FV activity was determined in a FXa-l-stage assay
in which 60 pL of FV-deficient plasma (George King Biomedical,
Overland Park, KS) was mixed with 20 pL of HEPES-buffered
saline, pH 7.4 (HBS), and 50 pL of cephalin (prepared as recommended by the manufacturer, Sigma, St Louis, MO). After 2 minutes
of preincubation of this reaction mixture at 37"C, IO pL containing
20 nmoVL FXa (Enzyme Research Laboratories, South Bend, IN)
and IO pL of a dilution of normal plasma or of a test sample as a
source ofFV or FVa were added. Finally, 50 pL of 33 mmol/L
CaCI,inHBS
containing 1% bovine serum albumin (BSA) was
added and clot time was measured at 37°C using an ST4 coagulometer (American Bioproducts, Parsippany, NJ). FV activity in the
test samples was calculated by reference to a standard curve constructed with dilutions of normal plasma. Activity was expressed in
units, in which 1 U equals the FV coagulant activity in I mL of
normal plasma. FV (1 00 nmoUL) was activated at room temperature
in HBS containing 0.5% BSA and 5 mmoUL CaCI2 using either I
nmol/L thrombin (Enzyme Research Laboratories) or IO nmol/L
FXa and 25 MmoVL phospholipid vesicles (20% phosphatidylserine
and 80% phosphatidylcholine). Maximal activation time for FV was
about 1 hour in each case, after which thrombin was neutralized2'
and the sample was diluted in HBS-0.5% BSA for immediate further
use.
APC resistance assays. APTT assays were performed as follows. FV-deficient plasma (45 pL) wasmixed with 5 pL of test
sample (consisting of plasma, 1 nmoUL final FV, or 22 pmol/L final
FVa) and 50 pL of Platelin LS (Organon Teknika, Durham, NC).
After 200 seconds of preincubation at 37°C the mixture was recalcified with 30 mmol/L CaCI, in HBS, 0.5% BSA at 37°C in the
presence or absence of 16 nmoVLAPC in this solution. The ratio
of ( A m + APC)/(A€TT) was calculated. An FXa-l-stage assay
was performed as indicated above, except that CaCl' was added in
the presence or absence of 48 nmol/L final concentration of APC.
The ratio of (clotting time + APC)/(clotting time) was calculated.
APC inactivation of FVa. FVa or Gln5"-FVa was reacted with
APC at various concentrations at room temperature in the presence
of 25 pmol/L phospholipid vesicles, HBS, 0.5% BSA, and 5 mmol/
L CaC1,. Aliquots were removed at suitable times and immediately
assayed for residual FVa activity in prothrombinase assays or boiled
for 1 minute in SDS-PAGE sample buffer for immunoblotting analysis.
Ofher analyses. Prothrombinase assays were as described else-
HEEB ET AL
Table 1. APC Resistance of W-Deficient Plasma Containing
Aliquots of Plasma, F
V
,or FVa
Normal ArgWB-FV
Test Sample
Plasma
Purified FV
1.9
Thrombin-activated3.0
FVa
FXa-activated FVa 2.8
Xa-l- APT
Stage Assay
2.1
3.1
4.6
3.2
Variant Gln50B-FV
APTT
Stage Assay
1.8
1.2
1.4
1.3
1.2
1.6
1.5
Xa-l-
1.4
1.4
where2' and used 20 pmoVL FVa or GlnSLm-FVa,
1 nmoVL FXa, 25
p m o K phospholipid vesicles, 5 mmoK CaCI,, 0.1 mmol/L " l , ,
and 0.3 pmollL prothrombin in HBS-0.5% BSA. The rate of thrombin formation was assessed using the chromogenic substrate S-2238
in a Biotek (Winooski, VT) microplate reader using Kineticalc software. SDS-PAGE was performed with 4% to 15% gradient gels in
a mini-gel apparatus (Hoeffer, San Francisco, CA). For immunoblotting, gels were transferred to Immobilon membranes (Millipore, Bedford, MA), blocked, and reacted as described:' except that detection
used rabbit anti-FVa heavy chain antiserum (1:500), followed by
biotinylated antirabbit IgG diluted 1:1,000 (Pierce Chemical CO,
Rockford, IL), streptavidin-alkaline phosphatase 1 :500 (Pierce), and
phosphatase substrate kit (Bio-Rad, Hercules, CA). Antiserum to FV
heavy chain was provided by Drs Jan Rosing, Guido Tans, and
Harry Bakker (University of Limburg, Maastricht, The Netherlands).
NH,-terminal amino acid sequences were obtained from bands on
an Immobilon membrane using an Applied Biosystems (Foster City,
CA) model 470A gas phase sequencer under the supervision of Dr
Lisa Bibbs in The Scripps Research Institute Core Laboratory.
Each experiment presented in Table 1 or in Figs I through 3 was
performed on at least three separate occasions, with good agreement
of results.
RESULTS
Variant GlnS"-FV waspurified from plasma obtained
from an APC-resistant subject who was homozygous for the
DNA mutation of G to A at nucleotide 1691 that predicts
the mutation Arg506Gln. Normal FV and GlnSo6-FV were
2 5 0 % pure as judged byfinal antigedprotein ratio and
270% pure as judged by SDS-PAGE (data not shown). A
purification of greater than 2,880-fold was achieved based
on initial versus final antigedprotein ratio, with a recovery
of 10% antigen.
To define the APC resistance of the variant Gln5"-FV in
comparison to normal F V , APTT assays were performed
using FV-deficient plasma reconstituted with samples containing normal or variant forms of FV or FVa. Test samples
including normal plasma, homozygous Gldo6-FV plasma,
purified normal FV or FVa, or purified GlnSoh-FVor Gin'"'FVa were analyzed (Table 1). The APTT values for each
test sample were determined in duplicate in the presence and
in the absence of APC. The ratio of APTT in the presence
ofAPC to the APTT valuein the absence of APCwas
calculated in each case and the mean value for this APC
resistance ratio is given in Table 1. When purified normal
FV was used to reconstitute FV-deficient plasma, the APC
resistance ratio was 3.1, similar to that seen for normal
plasma, whereas the APC resistance ratio for the addition of
APC RESISTANCE AND PURIFIED GLN~-FACTORV
purified Gln'"-FV was 1.2, similar to that for whole plasma
from the same subject homozygous for the Arg506Gln mutation. This finding shows that purified Glns"-FV indeed conveysAPC resistance to plasma, as predi~ted.'~"~
Subsequently, experiments were performed to determine whether
normal and variant FVa generated by either thrombin or FXa
activation conveyed APC resistance in AP?T assays. FVdeficient plasma was reconstituted with the various FVa species, as described under experimental procedures, and the
APC resistance ratios were determined (Table 1). Normal
FVa, whether generated by thrombin or by FXa activation,
was quite susceptible to APC as indicated by APC resistance
ratios of 4.6 and 3.2, respectively (Table 1). The data in
Table 1 indicate that the variant Glns"-FVa, whether activated by thrombin or by FXa, was relatively resistant to
APC, giving ratios of 1.6 and 1.5, respectively. These results
indicate that, in comparison to normal FVa, the purified
variant Glns"-FVa is relatively resistant to APC independent
of whether it is activated by thrombin or FXa.
If the variant Glns"-FVa were insusceptible, ie, completely resistant, to AFT, one would predict that the APC
resistance ratio should be 1.00. However, values for APC
resistance ratios of Glns"-FVa in APTT assays were 1.05 to
1.6 (Table 1 and data not shown). Ratios greater than 1.0
could conceivably reflect inactivation of FVIIIa byAPC
during coagulation as monitored in APTT assays. To exclude
any contribution of inactivation of FVIIIa by APC, FXa-1stage assays were also used for APC resistance tests because
these are independent of FVIIIa. The APC resistance ratios
from the ma-l-stage assays were also greater than 1.0, ie,
1.2 to 1.4 for Gln5"-FV and Gln5"-FVa (Table l), suggesting
that Gln5"-FV and/or G1n'"-FVa are not completely insusceptible to APC.
Interestingly, in the FXa-l-stage assays, the APC resistance ratio of normal FV was 1.9, whereas for normal FVa
activated by FXa or thrombin the ratios were 2.8 and 3.0,
respectively (Table 1). Similarly, when the APC resistance
ratios were determined using APTT assays, the ratios for
normal and variant FVa were greater than those for normal
or variant FV (Table 1).
Studies were made to define the influence on APC resistance ratios of the concentration of purified normal and variant FV that was added to FV-deficient plasma. When [FV]
was varied from 0.9 to 4.5 nmol/L, the APC resistance ratios
in FXa-l-stage assays for either normal FV or Gln'"-FV
were essentially constant (1.90 2 0.05 or 1.15 -I- 0.05, respectively). Similarly, the APC resistance ratios in APTT
assays were constant, ie, independent of [FV] between 0.9
and 4.5 nmoVL (3.35 +. 0.35 or 1.03 ? 0.05; data not shown).
In the absence of APC, the FXa-l-stage clotting times for
the mixture of FV-deficient plasma with normal FV varied
from 55 to 35 seconds, and the clotting time for the FVdeficient plasma containing Glns"-FV varied from 51 to 34
seconds depending on the [FV] (0.9 to 4.5 nmol/L). The
preparations of normal and variant FV used for these studies
were mostly single-chain FV. Thus, the APC resistance ratio
in FXa-l-stage and in APTT assays was independent of FV
final concentration between 0.9 and 4.5 nmol/L, equivalent
to an initial plasma FV level of 9% to 45%.
3407
Studies were performed to compare the sensitivity of purified Gln5"-FVa with that of normal FVa to inactivation by
APC and to determine if these sensitivities were markedly
different for thrombin-activated versus FXa-activated FVa
species. For these experiments, normal and variant purified
FV were separately activated either by FXa or by thrombin.
The time courses for maximal activation were similar in each
case (data not shown). Each of the various species of FVa
was mixed withvarying amounts of APC (0 to 1.33 nmol/L).
Inactivation of normal and Gln5%-FVa thatoccurred during
3 minutes of incubation was quantitated by measuring the
residual FVa activity using a purified prothrombinase assay
(Fig 1). Under the conditions used, thrombin-activated normal FVa was inactivated by APC with the same dose response as FXa-activated normal FVa (Fig 1, lower curves).
In comparison to normal ArgS"-FVa,both thrombin-activated and FXa-activated Gln5"-FVa were relatively resistant
to APC, with the apparent sensitivity to APC of the variant
FVa species approximately 10-fold less than the normal FVa
species (Fig 1). Moreover, Gln5%-FVaactivated by thrombin
was indistinguishable from Gln'"-FVa activated by FXa in
its relative resistance to APC.To compare further the relative
sensitivities of each FVa to APC, the time course of APC
inactivation was examined at two doses of APC (Fig 2). At
100 pmol/L APC, 20% to 25% inactivation of Gln5"-FVa
was observed within 30 minutes of incubation, whereas a
similar extent of inactivation of normal FVa was observed
within approximately 2 to 3 minutes (Fig 2, left panel). The
time for 50% inactivation of normal FVa by 100 pmoVL
APC was 4 to 7 minutes, whereas for 50% inactivation of
Gln'"-FVa it was 8 minutes for 800 pmol/L APC (Fig 2,
right panel). These comparisons suggest that, under the conditions of these experiments, GlnSo6-FVais approximately
8- to 10-fold less susceptible than normal FVato APC inactivation.
At 800 p m o m APC, 80% inactivation of Gln5%-FVa was
observed within 30 minutes, with a similar extent of inactivation of normalFVa within about 5 minutes (Fig 2, right
panel). These results were independent of whether the Gln5"FVa or normal FVa had been generated by thrombin or by
FXa. In other experiments with longer times and higher APC
concentrations, greater than 90% inactivation of Glns"-FVa
was observed.
To compare the proteolytic fragments of normal and variant FVa produced by APC during inactivation, cleavage of
the heavy chains of Glns"-FVa and normal FVa was monitored by immunoblotting using a polyclonal antibody against
the human FVa heavy chain (Fig 3). The first heavy chain
fragments observed during incubation of normal FVa with
APC were of apparent molecular mass 76 and 30 kD (Fig 3
and data not shown), suggesting initial cleavage at Arg'" as
reported by Kalafatis et al.23 Subsequent appearance of a
fragment of approximately 44 k D , the size of a polypeptide
containing residues 1-306, was consistent with a secondary
cleavage at Arg3%in normal FVa, in agreement with previous work.23Generation of the fragment containing residues
1-306 would also be associated with release of a 30-kD
fragment corresponding to residues 307-506, and indeed a
HEEB ET AL
3408
110 -
.?
I'
Gln50B-FVa(FIla-activated)
\
-0
\
\
"Q..
. ..
....
.
\
90
~
..
70
""
Gln50B-FVa(FXa-activated)
50
FVa(Flla-activated)
30
FVa( FXa-act ivat ed)
10
0
0.33
0.67
APC, nM
1 .o
1.33
Fig 1. Inactivation of thrombin-activated and FXa-activated
FVa and Glnm-Na as a function
of APC concentration. Various
APC concentrations as indicated
were incubated with phospholipids and normal or variant N a
(200 pmollLl for 3 minutes. The
mixtures were then assayed
for residual FVa activity using
prothrombinase assays as described in Materials and Methods.
tion was shown to cosegregate withriskof thrombosis in
30-kD doublet was observed on this and other blots (data
familial APC re~istance.'""'.'~ Toshow that this DNA defect
not shown). Because the 30-kD banddid not increase in
is indeed the direct biochemical cause ofAPC resistance,
intensity after 3 minutes, it is possible that the original 30we purified FV from plasma of a subject homozygous for
kD fragment probably corresponding to residues 507-709
was further cleaved by APC at residue 679, as reported.'.'
Gln'"-FV. Coagulation studies (Table I ) show that purified
However, no fragments smaller than 30 kD were observed
GldDh-FVimparts to plasma resistance to APC.
We previously reported experiments suggesting that
with the particular anti-heavy chain antibodies used in this
thrombin-activated Glns"-FVa
is
relatively
resistant
to
study.
APC";in
contrast. others suggested that FXa-activated
In contrast to the results for normal FVa, the fragments
Gld"-FVa, but not thrombin-activated Glns"-FVa, is resisthat appeared upon incubation of Gln"'"-FVa with APC were
tant to APC." Studies described here show that Gln'"-FVa
at approximately 44 and 58 kD, consistent with a single
cleavage at Arg"" (Fig 3). To identify the site of phosphoconveys APC resistance to plasma and that Glns'K-FVa is
relatively resistant toAPC
independent of whetherthe
lipid-dependent cleavage of Gld"-FVa by APC, the heavy
chain fragments of 44 kD and 58 kD were sequenced. The
Glns"-FVa is generated by thrombin or by FXa, thusclarifying this important conceptual conflict.
44-kD fragment had the expected NH,-terminal sequence of
residues 1-9 of FV, Lys-Gln-Leu-Thr-Gln-Phe-Tyr-Val- The molecular defect responsible for APC resistance was
Ala-. The 58-kD fragment had the sequence X-Leu-Lysinitially hypothesized to involve a new APC cofactor I1 defiLys-He-X-Arg-, corresponding to the expected sequence of
cit"; subsequently, it was suggested to involvedefective APC
residues 307-3 13, Asn-Leu-Lys-Lys-Ile-Thr-Arg-.
Because
cofactor activity of FV in APC-resistant patients.2sThe incleavage at Arg'" of normal FVa is known to be phosphofluence of the concentration of normal or variant FV added to
lipid dependent? Gln"-FVa was treated with 700 pmol/L
FV-deficient plasma onthe response to APC wasdetermined
APC in the absence of phospholipid for 90 minutes.No
using APTT and FXa-I -stage assays, and the results showed
inactivation (S10%) was observed and no heavy chain fragthat the APC resistance ratios are insensitive to FV concenmentation was observed on immunoblots. In contrast, in the
tration. Because increasing the amounts of FV does not imabsence of phospholipids, -25% inactivation of normal FVa
prove the APC resistance ratio, ie, apparently does not enwas observed in 3 minutes, with no further loss of activity
hance APC activity, these findings do not support the
observed in 90 minutes (data not shown). These observations
hypothesis that single-chain normal FV or GlnsM-FV can act
are consistent with inactivation of Glns"-FVa via phosphoas a significant cofactor for APC anticoagulant activity in
lipid-dependent cleavage at Arg'".
plasma under typical conditions of the clotting assays used.
Studies in which purified Gln'"-FVa was added to FVDISCUSSION
deficient plasma consistently gave APC resistance ratios
suggesting that GlnsOh-FVawas not insusgreater than 1 .W,
APC resistance has been associated with a DNA mutation
ceptible toAPC. In studies usingpurified proteins, dosecausing replacement of Arg'"" by Gln in F V , and this muta-
APCRESISTANCE
AND PURIFIED GLN~-FACTOR
V
3409
[APC] =800pM
[APC] =l OOpM
100
I
...................................................
,
A
*
3
A\
Ita-activated Arg-FVa
.;. ................ .9.t!a-activated GIMV~ . . . . .
80
*Xa-activated Arg-FVa
\\
'p\
Q:\
A-Xa-activated Gln-FVa
......,\ ...........................................
60
*.
40
20
0
0
10
20
30
0
10
20
30
TIME, min
TIME, min
Fig 2. Time course of inactivation of thrombin-activated andFXa-activated normal FVa and Gin*-FVa. Normal or variantFVa (200 pmol/
L) was incubatedwith APC and phospholipids. At various timesas indicated, aliquots were removed and
assayed for residual FVa prothrombinase activity as described in Materials and Methods.
For the left panel, APC concentration was100 pmol/L. For the rightpanel, APC concentration was800 pmol/L. ( 0 )Thrombin-activated normalFVa, [A)FXa-activated normal FVa, ( 0 )thrombin-activated Glnm-FVa, (A) FXa-activated
GlnmFVa.
response studies and time course studies at different concentrations of APC (Figs 1 and 2 ) confirmed that purified Gln'"FVa is not completely resistant to APC and suggested that
the variant Glr?"-FVa is approximately IO-fold less sensitive to APC than is normal FVa. These findings imply that
the inactivation of Glns"-FVa by APC
can occur in vivo,
albeit at a reduced rate compared withnormalFVa. This
implication may help explain why APC resistance appears
tobe a rather mildrisk factor for venous thrombosis and
why a combination of genetic risk factors for venous thrombosis is found in a significant fraction of symptomatic patients.7.~n.?4.?~.~9
The APC-dependent cleavage patterns for the FVa heavy
chains of normal FVa and GlnS"-FVa were compared using
immunoblotting. The observed time-dependent cleavage patterns for normal FVa were consistent with a report showing
that inactivation of normal FVa involves an initial cleavage
at Arg'" followed by a phospholipid-dependent cleavage at
FVa
Gln506=FVa
I
Std.
I1
t = 60 3 60 126 '60 3 30 60
Fig 3. lmmunoblot of FVa and Gln=-FVa heavy
chains after APC inactivation. Normal RI or GlnSOBFV were each activated with thrombin
and then incubated at 5 nmol/L with APC (350 pmol/L) and phospholipidslCa". Atthetimes indicated, aliquots containing 60 n g of FVa wereremovedand
subjected
t o boiling, SDS-PAGE, and immunoblotting for FVa
heavy chain epitopes as indicated in Materials and
Methods. FVa activity assays performed in parallel
indicated thatGln5w-FVawas greater than 80% inactivated at 120 minutes and that normal FVa was
greater than 90% inactivated
at
60 minutes.
min.
-200
Mr x 103
765844-
30APC-
I
+ + +
-
+ + +
HEEB ET AL
3410
Arg306.23 Incontrast to the pattern for normal FVa, inactivation of Gln506-FVaby AFT was associated with appearance
of polypeptide fragments of approximately 44 and 58 kD
(Fig 3 ) because of cleavage at Arg306,as confirmed by NH2terminal amino acid sequencing. These results show that
cleavage by APC at Arg306in FVa is sufficient to inactivate
FVa, but that this cleavage is inefficient, approximately 10
times slower, unless prior cleavage at kgso6occurs.
Baboon studies suggest that FVa rather than FV is the
physiologic substrate for APC. APC at antithrombotic levels
of 6 and 30 nmoYL circulating in vivo for up to 1 hour in
baboons did not significantly decrease FV or FVIII levels
even though the AFTT was very markedly prolonged, eg,
36 to 169 seconds at 30 nmoYL
In contrast, when
thrombin was infused at 2 U/kg/min into baboons for 1
hour, levels of FV and FVIII decreased significantly (40%
to 60%).31Infused thrombin activates FV and FVIII to FVa
and FVIIIa, increases the level of circulating APC to -7
nmol/L, and prolongs the APlT (eg, 32 to 158 seconds at
2 U thr~mbin/kg/h).~~
Compared with normal FVa, GlnSMFVa has a relatively reduced rate of inactivation by APC
in the presence of phospholipids. The fact that Arg506Gln
mutation is clearly associated with increased risk of thrombosis6-12.24 and that cleavage of GlnSM-FVby APC is similar
to that of normal F V 3 ’ (Heeb, Kojima, and Griffin, unpublished data) further supports the concept that FVa rather than
FV is the physiologic target of A P C .
After this report was submitted, Kalafatis et a13’ reported
biochemical studies that are related to this study and that
complement the coagulation experiments presented here. Using purified component prothrombinase assays with the fluorescent thrombin inhibitor DAPA, it was shown3’ that purified thrombin-activated Gln5”-FVa was inactivated by APC
by cleavage at ,g3% at a rate slower than that of normal
FVa that was cleaved at ArgM,
and Arg306.
ACKNOWLEDGMENT
We are very grateful for skilled technical assistance of Marissa
Maley and Yolanda Montejano and for DNA analyses for the FT
Arg506Gln mutation performed by Xiao Xu. We appreciate the
ELISA data provided by Dr JosC Fernhdez and the gift of anti-FT
antibodies provided byDrs Jan Rosing, Guido Tans, andHarry
Bakker.
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