1. Art and Diseño

From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
Proteolytic Events That Regulate Factor V Activity in Whole Plasma From
Normal and Activated Protein C (APC)-Resistant Individuals During
Clotting: An Insight Into the APC-Resistance Assay
By Michael Kalafatis, Paul E. Haley, Deshun Lu, Rogier M. Bertina, George L. Long, and Kenneth G. Mann
Human factor V is activated t o factor Va by a-thrombin after
cleavages at Arg", Arg"",
and Arg".
Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy
chain. Cleavage at Arg- provides for efficient exposure of
the inactivating cleavages at Arg308and Arg-. Membranebound factor V is also inactivated by APC after cleavage at
Arg308.Resistance t o APC is associated with a single nucleotide change in the factor V gene (G"'+A)
corresponding t o
a single amino acid substitution in the factor V molecule:
Arg-Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of
the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691GG) and individuals homozygous for the factor
V mutation (1691 AA) were diluted in a buffer containing 5
mmol/L CaCI, phospholipid vesicles ( I O pmol/L), and APC.
APC, at concentrations 55.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a
clot was observed in plasma from APC-resistant individuals.
Gel electrophoresis analyses of factor V fragments showed
that membrane-bound factor V is primarily cleaved at Argin both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast
degradation after cleavage at Argm/Argm, in the APC-resistant individuals' plasma, early generation and accumulation
of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg-. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed
in either ptasma from normal or APC-resistant individuals.
Our data show that resistance t o APC in patients with the
ArgW+Gln mutation is due t o the inefficient degradation
(inactivation) of factor Va heavy chain by APC.
0 7996 by The American Society of Hematology.
F
visualize how the events occur in whole plasma during the
performance of the APC resistance assay.
ACTOR V IS A single chain procofactor (molecular
weight [M,] = 330,000) that possesses little or no procoagulant activity.',' At the site of vascular injury, when an
appropriate membrane surface is exposed to the blood flow,
factor V is activated to its active form, factor Va, by a thrombin and factor Xa. The association of factor Va with
factor Xa on the membrane forms prothrombinase, the enzymatic complex that activates prothrombin, with an efficiency
5 orders of magnitude greater than factor Xa acting a10ne.~
Factor V is activated by a-thrombin after cleavage at Arg709,
ArgI0l8, and Arg1s45.495
a-Thrombin-activated factor Va is
composed of a heavy chain (M, = 105,000) containing the
NHz-terminal part of the procofactor (residues 1 through
709, AI-A2 domains) and a light chain (M, = 74,000) containing the COOH-terminal part of the factor V molecule
(amino acids 1546 through 2196, A3-Cl-C2 domains) that
are noncovalently associated in the presence of divalent
metal ions.
Recent data show that the plasma of individuals with an
Arg506+Glnmutation in the factor V molecule have a poor
anticoagulant response to activated protein C (APC), which
in turn is associated with a significant increase in risk of
developing deep-venous thrombosis (7-fold for the heterozygous and 80-fold for the homozygous).6-10
Membrane-bound
factor Va is inactivated by APC after three sequential cleavages of the heavy chain": Arg506,Arg306,and Arg679.Cleavage at ArgSMis necessary for efficient exposure of the inactivating cleavage sites at Arg306and Arg679.Inactivation of the
procofactor, factor V, by APC occurs only in the presence
of a membrane-surface and is associated with cleavages at
Arg3", Arg'", Arg67q,and LysqW.The cleavage at Arg306
occurs first and is the inactivating cleavage site."
We have recently shown that natural purified factor
VaRSMQ
is inactivated by APC at a slower rate than is normal
factor Va." In contrast, the procofactor, factor vRSmQ,
is
inactivated by APC in the presence of a membrane surface
with a rate similar to that observed for normal plasma factor
V." However, our data suggested that cleavage of the mutant
procofactor, factor VRSW,by APC at Arg306and Arg67qoccurs simultaneously. The present study was undertaken to
Blood, Vol 87,No 1 1 (June l), 1996:pp 4695-4707
MATERIALS AND METHODS
Materials, reagents, and proteins. N-[2-Hydroxyethyl]piperazine-N'-2-ethanesulfonicacid (HEPES), 1-palmitoyl-2-oleoyl-phosphatidyl serine (PS) from bovine brain, and 1-palmitoyl-2-oleoyl
phosphatidyl choline (PC) from egg yolk were purchased from
Sigma (St Louis, MO). Activated partial thromboplastin time (a€TT)
reagents used in the present study were purchased from Baxter Diagnostics Inc (Actin and Actin FS; Deertield, IL), Baxter Healthcare
Corp (Actin FSL; Dade Division, Miami, FL), Diagnostica Stago
(PTT Automate; American Bioproducts, Parsippany, NJ), and Organon Technika Corp (Automated a m , Durham, NC). PhosphoT and 25% PS were prepared as
lipid vesicles composed of 75% F
de~cribed.'~
The chemiluminescent substrate, Luminol, was from
DuPont, NEN Research Products (Boston, MA). Hirudin was from
Genentech (South San Francisco, CA) and American Diagnostica
(Greenwitch, CT). Monoclonal antibody (MoAb) aHFVaH,3#6,
which recognizes an epitope located between amino acid residues
307-50612 of the human factor V molecule, and MoAb aHFV#9
directed against the light chain of the cofactor'4 were prepared as
de~cribed.'~,'~
The buffer used in all other experiments was com-
From the Department of Biochemistry, University of Vermont,
College of Medicine, Burlington, VT; Haematologic Technologies,
Inc, Essex Junction, VT; and the Hemostasis and Thrombosis Research Center, University Hospital, Leiden, The Netherlands.
Submitted July 7, 1995; accepted January 25, 19%.
Supported by Merit Award No. R37 HL34575, National Institutes
of Health Grants No. PO1 -HLA6703 and C06-HL39475, and American Heart Association Grant-in-Aid 9201 1860.
Address reprint requests to Kenneth G. Mann, PhD, Department
of Biochemistry, Given Building, Health Science Complex, University of Vermont, College of Medicine, Burlington, VT 05405-0068.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
I -0029$3.00/0
0006-4971/9~/a71
4695
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4696
posed of 20 mmol/L HEPES, 0.15 m o m NaCI, 5 mmoVL CaCl,,
pH 7.4 [HBS(CaZ')].
Preparation of protein C and APC. Human protein C was prepared from fresh frozen citrated plasma as described by Bajaj et
with the following modifications. The protein C-rich pool from the
diethyl aminoethyl (DEAE)-Sepharose column was brought to 75%
saturation with ammonium sulfate and stirred overnight at 4°C. The
protein C was collected by centrifugation at 5,000g for 30 minutes.
The precipitated was suspended in TBS-1 mmoVL benzamidine and
dialyzed versus 2 changes of the same buffer. The sample was
clarified by centrifugation and applied to a 2.5 x 20 cm column of
Q-Sepharose fast flow equilibrated in the same buffer. The column
was washed to baseline and then eluted with a linear gradient of 0
to 40 mmoVL CaCI, in the same buffer. Protein C, which elutes at
15 mmol/L CaCl,, is precipitated by 75% ammonium sulfate and
collected by centrifugation at 5,OOOg for 30 minutes. The precipitate
is dissolved in a minimal volume of 20 mmol/L Tris-Mes, pH 6.0,
1 mmol/L benzamidine. Calcium chloride is added to 2.5 mmol/L
and the sample is clarified by centrifugation at 5,OOOg for 15 minutes.
The sample is then chromatographed on dextran sulfate agarose as
previously de~cribed.'~
The resulting protein C is activated to APC
as previously described." It is noteworthy that, whereas all APC
preparations had normal esterase activity when using a chromogenic
substrate, some of the preparations had low to no anticoagulant
activity. Thus, during APC generation, the activity of the enzyme
was continuously monitored for its anticoagulant activity rather than
for its esterase activity. The reason for the variability in the anticoagulant properties of the APC preparations is unknown and is presently
under investigation. Recombinant APC with an alanine instead of a
y-carhxyglutamic acid at position 20 (APCyZoA)
was obtained as
recently described."
APC-resistance assay. The chromogenic substrate activity of
APC was determined by incubating APC (0 to 100 nmoVL) in a
reaction mixture composed of 20 mmol/L Tris/HCl, 0.15 m o m
NaC1, 200 mmoVL Spectrozyme PCa (American Diagnostica), pH
7.4, at room temperature. The initial rates of substrate hydrolysis
were monitored at 405 nm using a Molecular Devices Corp (Sunnyvale, CA) Vmax microtiter plate reader. Control experiments in
which hirudin (25 nmoVL) was incorporated into the reaction mixture ruled out any interference by traces of cy-thrombin. The APCresistance assay was performed as previously detailed.6 Briefly,
plasma samples (100 pL) were incubated with the aPTT reagent
(100 pL) for 5 minutes at 37°C. Coagulation was initiated by adding
100 pL of solution composed of 20 mmol/L Tris/HCI, 50 mmol/L
NaCI, 30 mmol/L CaCI,, 0.1% bovine serum albumin (BSA), pH
7.4, containing varying amounts of plasma APC (0 to 100 nmol/L).
The clot time was determined visually.
Immunoblotting of plasma samples. Citrated plasma (100 pL)
from normal plasma (pool of 30 donors), a normal individual with
a 1691 GG genotype, or 2 APC-resistant individuals with a 1691
AA genotype was diluted 10-fold in HBS(Caz'). Phospholipid vesicles (10 pmol/L) composed of 75% PC and 25% PS were added.
After 2 minutes of incubation at 37"C, purified human plasma APC
was added. The clot formation was detected visually. At selected
time intervals, aliquots of the mixture were withdrawn (120 pL),
mixed with 2% sodium dodecyl sulfate (SDS) and 2% 8-mercaptoethanol, and analyzed on a 4% to 12% (linear gradient) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to the
method of Laemmli." The proteins were transferred to nitrocellulose'' and probed with either an MoAb that recognizes an epitope
located between residues 307 and 506 of the human molecule'z~20
or an MoAb that is directed against the light chain of factor Va.
Immunoreactive fragments were visualized using the chemiluminescent substrate Luminol." The immunoreactivity of fragments derivand factor Va (or factor VaRs06Q)
ing from factor V (or factor VRSOMI)
-
KALAFATIS ET AL
for cyHFVaH&6 after incubation with APC are depicted in Fig 1. In
some experiments, after clot formation, the samples were centrifuged
and aliquots of the supernatant were analyzed by SDS gel electrophoresis.
Factor V inactivation studies. Human factor V was incubated
with either plasma-derived APC or rAPCyZoAin the presence of
PCPS vesicles. The concentrations of all reagents are given in the
figure legends. At selected time intervals, aliquots of the mixture
were assayed for cofactor activity in a clotting time-based assay
using factor V-deficient plasma." At the same time intervals, the
factor V samples were also analyzed by SDS-PAGE. After electrophoresis and transfer to nitrocellulose, factor V fragments were
probed with MoAbs aHFVaH&#6and/or aHFV#9 using the chemiluminescent substrate Luminol. The potential of contamination by athrombin in the APC preparations was controlled for by the addition
of hirudin.
RES U LTS
Variability of the APC-resistance assay. It is well established that factor Va is inactivated after three largely sequential cleavages of the heavy chain," with cleavage at A r c 6
potentiating the inactivating cleavages at Arg306 and at
Arg679.Factor V inactivation occurs after a single cleavage"
at Arg306.During the APC-resistance assay, citrated plasma
is incubated with the aFTT reagent (buffered lipid + contact
activator) to allow activation of the intrinsic pathway of
blood coagulation that ultimately will result in factor V activation. The lipid composition in the aPTT reagents shown
in Fig 2 varies from soybean phospholipid to rabbit brain
phospholipid, whereas the factor XI1 activator varies from
ellagic acid to micronised silica. Thus, if timely activation
of the procofactor does not occur, significant differences in
the clotting times for normal plasma and plasma from APCresistant individuals would not be observed, because in both
cases factor V will be inactivated with similar rates.
To test the reproducibility of the results of the APC-resistance assay, we performed the assay6 using five different
commercially available aPTT reagents. Figure 2A through
E shows an increase in clotting time when increasing concentrations of APC are added to normal plasma. However, the
increase in clotting time (up to approximately 250 seconds
in all cases) does not occur at the same APC concentrations.
For example, in Fig 2B, using normal plasma, a clotting time
of 250 seconds is observed at 25 nmol/L APC. A similar
clotting time is observed in Fig 2A and D at 100 nmoVL
APC. The data obtained using plasma from the 2 APCresistant patients showed similar heterogeneity. Thus, Fig 2
shows that the variability of the assay is dependent in part
on the aPTT reagent itself. The aPTT reagents shown in Fig
2A and D gave results that are comparable to previously
published data.6 The aFTT reagent shown in Fig 2C gave
results in which the patient values were greater than those
reported in the literature. Finally, the reagents shown in Fig
2B and E gave totally different results than those observed
in general for the APC-resistant patients. The data clearly
show that the APC-resistance assay is sensitive to the aFTT
reagent used.
The data presented in Table 1 show the APC ratios when
using the APC resistance assay6 and various aPTT reagents
(displayed in Fig 2) at two APC concentrations (10 and 20
nmolk). No significant differences between the APC ratios
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4697
INSIGHT INTO THE APC-RESISTANCE ASSAY
B
A
A-....
x4
... .
CO'............"
........,.
211
1-101
MoAb aHFVa.c#G?O For simplicity, only the final
product deriving from factor V (M, = 30,000) or factor
V ~ 5 m
(M. = 54.000) is depicted in (C) and (DI.
om-=
IS
obtained for the same plasma sample (from normal or APCresistant individual) were observed when comparing aPTT
reagents at low APC concentrations (except for reagent B).
Thus, the overall variability of the results obtained when
using different aPTT reagents can be overcome by the use
of low concentrations of APC. It is also important to note
that the correlation of the protein concentration to the activity
of the APC preparation is also essential for the sake of
reproducibility of the results between different laboratories.
'$46
I,*
D
C
Fig 1. Specificity of MoAb aHFVa&.
APC
cleaves sequentially the membrane-bound cofactors
(A) normal plasma factor Va and (B) plasma factor
Va-.
APC also cleaves the membrane-bound procofactors (C) normal plasma factor V and (D) plasma
factor VR-.
(a) Regions that are recognized by
. c c ..
2,-
I,*
-
I
m
I
m
1Ilb
en
2l.L
In addition to the type of the aPTT reagent, the quality of
the APC preparations is critical to the standardization of
the APC-resistance clotting assay. All APC preparations,
although having similar activities towards chromogenic substrates, do not cleave and inactivate factor Va similarly. The
data presented in Fig 3 show this point. Two preparations
of APC that were purified from normal pooled plasma (as
protein C) and activated to APC using the same protocol
were assessed for their ability to cleave a chromogenic sub-
n
v)
U
c
0
0
Q)
v)
W
.-i
I-
+
0
0
APC (nM)
Fig 2. APC-resistanca assay as a function of the
type of aPTT reagent. The APC-resistance assay was
performed as described by Dahlback et ala using various, commercially available aPlT reagents (individually assayed in [A] through [El). (0)
Normal plasma.
(A,0 ) Results found using plasma from 2 patients
homozygous for the ArgSW41nsubstitution'* (patients l and ll).
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
KALAFATIS ET AL
4698
Table 1. APC Ratios for the aPlT Reagents Used in Fig 2
Normal Plasma
Patient No. 1
Patient No. 2
a P r r Reagent
10 nmol/L APC
20 nmollL APC
10 nmol/L APC
20 nmol/L APC
10 nmol/L APC
20 nmol/L APC
A
B
C
2.5
3.4
2.4
D
2.3
E
2
3.1
4.8
3
3
4
1.2
1.3
1.2
1.2
1.5
1.3
1.6
1.4
1.5
1.6
1.2
1.4
1.2
1.2
1.4
1.4
1.7
1.5
1.5
1.4
APC ratios were determined using the method of Dahlback et
strate and to prolong the clotting time of normal plasma.
Both preparations possess full chromogenic substrate activity, but only one was capable of prolonging the clotting time
of normal plasma (Fig 3A and B). Influence of contaminating
thrombin on the chromogenic substrate activity was ruled
out by the inclusion of 25 n m o K hirudin in the incubation
mixture. The example given in Fig 3 is an extreme case of
a commonly observed phenomenon from several commercial
sources and research laboratories. Although slight differences between these two preparations are detectable by SDSPAGE analysis (Fig 3C), the reason for the differences in
the anticoagulant activity between the two APC preparations
is unknown and presently under investigation. It is clear that
elements outside the active site of APC are involved. We
have previously devised a protocol (see the Materials and
Methods) that consistently produces APC with full anticoagulant activity. When these APC preparations are used in the
APC-resistance assay reported by Dahlback et a1,6 APC ratios for normal andor patient plasmas are twofold to threefold higher than those reported. The details of this protocol
lead us to speculate that the variant anticoagulant activity of
APC preparations may be a property of the lipid binding
domain of the APC molecule. The results of these experiments point out the requirement for functional standardization of the APC resistance type assay to confirm the quality
of the APC preparations with respect to factor Va cleavage
and prolongation of the clotting time.
Inactivation of factor v5MQ
by APC. Natural purified
factor VaRSMQ
is inactivated by purified APC at a slower rate
than is normal factor Va." In contrast, the procofactor, factor
VR506Q,
is inactivated by APC in the presence of a membrane
surface with a rate similar to that observed for normal plasma
factor V, with cleavages at Arg306and Arg679occurring at
similar rates." The absence of the APC cleavage site at
ArgSo6of factor Va thus could lead to the accumulation of
active cofactor in APC-resistant patients and may be part of
the reason for their thrombotic tendencies. Although cleavage at kg306will inactivate the procofactor,L1,12
our data also
suggested that factor VR506Q
can also be initially cleaved at
Arg679.Cleavage at that position would generate the major
portion of the heavy chain (M, = 99,000, amino acid residues
1 through 679). In normal factor V, this product is produced
slowly in small amounts and is immediately degraded by
APC cleavages at ArgSo6and Arg306.
To ascertain whether A X can cleave membrane-bound
factor VRSMQ
initially at Arg679,the inactivation of factor V
Leiden7.l2(1 10 n m o n ) was studied. As one would antici-
pate, in the presence of 2.2 nmoK APC (Fig 4A, 0 ) the
inactivation rate of factor VRSMQ
proceeds at a rate faster
than that observed in the presence of 550 p m o a APC (m).
Electrophoretic analyses of samples obtained during inactivation of factor VRSmQ
by 2.2 n m o n APC showed that
inactivation of the procofactor correlates with the transient
appearance of an M, = 99,000 fragment, and the appearance
of an M, = 54,000 fragment that is accumulated during the
inactivation process (amino acid residues 307 through 679,
Fig 4B, lanes 12 through 18). In contrast, at the lower APC
concentrations (550 p m o n ) , electrophoretic analyses of the
samples obtained after inactivation of factor VR506Q
(Fig 4A,
W) showed increased accumulation of the M, = 99,000 fragment and slower formation of the M, = 54,000 fragment
(Fig 4B, lanes 3 through 8), suggesting that, at lower concentrations of APC, a cleavage fragment that represents a portion of the heavy chain of factor Va (M, = 99,000, amino
acid residues 1 through 679) can accumulate. The data show
that, after incubation of factor V Leiden with PCPS vesicles
and low concentrations of APC, there is appearance of a
transient M, = 99,000 fragment (Fig 4B, lanes 4 through 8).
The potential cofactor activity of factor VRsw and the transient M,= 99,000 fragment disappears as a consequence of
cleavage at Arg3%(Fig 4B, lanes 8 and 9).
In
The procoagulant effect of rAPCY2" on factor p5MQ.
the presence of all concentrations of APC, the M, = 99,000
fragment is transient and disappears as a consequence of
cleavage at Arg3%(Fig 4B). However, cleavage at Arg306of
factor Va by rAPCyZoA
is inefficient." Thus, during exposure
of factor VR5MQ
to rAPC y20A, cleavage of the M, = 99,000
fragment at Arg306would be delayed, resulting in an increase
in the M, = 99,000 fragment and in cofactor activity. To
ascertain if membrane-bound factor VRSMQ
is activated by
APC after cleavage at Arg679,factor VRSw cofactor activity
was evaluated using rAPCyZoA
in a clotting time-based assay
(Fig 5A).
Normal plasma factor V is not activated by rAPCYzoA
(Fig
5A, 0).The gel electrophoresis data shown in Fig 5B (lanes
1 through 9) shows that rAPCyZoA
cleaves normal plasma
factor V at ArgsM, generating an M, = 75,000 fragment
containing the region 1 through 506 of factor V. Cleavage
at Arg"' also occurs, resulting in the generation of the M,
= 99,000 fragment. Little activity loss (increase in clotting
time) is associated with appearance of these fragments. Activity loss (increase in clotting time from 32 to 36 seconds)
correlates with the appearance of the M, = 30,000 fragment
(Fig 5B, lanes 7 through 9, arrowhead) showing cleavage at
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
INSIGHT INTO THE APC-RESISTANCE ASSAY
APC (nM)
APC tnMl
1
2
(A)
" c
(0)
Fig 3. APC-resistanceassay as a function of the quality of APC. (A)
The APC-resistanceassay was performed as described by Dahlback et
ale using normal plasma and two different APC preparationsand the
aPlT reagent shown in Fig 2A. (B)The amidolytic activity of the
two APC preparations was tested using the chromogenic substrate
spectrozyme PCa. (C) An SDS-PAGE (5% to 15% linear gradient under
reducing conditions)of the two APC-preparations: lane 1, APC represented as (A)in (A) and IBI; lane 2, APC represented as Dl in (A)
and (B).The position of the molecular weight markers is indicated
at left of (Cl; from the bottom to the top: 14,000, 18,000, 29,000,
43,000, and 68.000).
4699
A$".
Thus, in the presence of rAPC'"'", the order of cleavages of membrane-bound normal plasma factor V is reversed, ie, cleavages at A r s " and
are followed by
slow cleavage at Arg"" and a slow loss in activity.
Slow activation of factor VR51nQ
by rAPCY"'" occurs during
2 hours of incubation (Fig SA, a),which is associated with
the accumulation of an M, = 99,000 fragment and a reduction in the clotting time (Fig SA1.1 from 34 to 29 seconds;
and Fig SB, lanes 13 through 18). The starting factor VR5IffiQ
solution (factor V Leiden7.") had a cofactor activity of 1.12
U/mL in the absence of rAPCY2"".After 2 hours of incubation with PCPS vesicles and rAPCY2"", the factor VR5(K'Q
solution had a cofactor activity of 1.84 U/mL. Thus, in the
presence of rAPCY"'", the factor VR5'HQ
solution is 1.64 times
more procoagulant than in the absence of rAPCy'"". Electrophoretic analyses (Fig SB) showed generation of an M, =
99,000 fragment. This fragment is most likely a consequence
of cleavage at Arg'"'. In normal factor V, the loss in activity
associated with the slow cleavage at Arg"" and Arg"" by
rAPCY"'" appears to be compensated by activation associated
with the generation of the M, = 99.000 portion of the heavy
chain (amino acid residues I through 679; Fig SR, lanes 4
through 9,*). In contrast, in factor VR5(KQ
there is accumulation of the M, = 99,000 (Fig SB) as a consequence of impaired inactivation of this fragment by rAPCY?"". Overall,
the data suggest that, under certain circumstances, APC can
activate factor VR51KQ
to a factor VaRSlmQ
that would be stable
because of delayed cleavage at ArgUK.Thus, whereaq APCY2""
has impaired capability to cleave normal factor V at Arg'",
the same molecule cannot cleave and inactivate factor V
Leiden because of the absence of the cleavage site at Arg"".
Thus, cleavage at Arg5'" may be required for cleavage of
the M, = 99,000 factor V heavy chain species. It is noteworthy that, whereas cleavage at Argh79in factor Va is lipid
independent" and is accelerated after cleavage at Arg'", no
cleavage of factor V by APC in the absence of a membrane
surface was observed when using Coomassie Blue-stained
gels." The present data obtained using the more sensitive
immunoblotting technique suggest that cleavage of factor V
at Arg"" by APC may occur even in the absence of a membrane surface. However, this cleavage is slow under the
experimental conditions used; hence, the resulting fragments
cannot accumulate sufficiently to be seen on a Coomassie
Blue-stained gel and can only be identified using sensitive
staining methods (ie, immunoblotting).
Proteolyis of factor V and factor
during clotting.
When phospholipid vesicles (10 pmol/L) and Ca" were
added to plasma from either normal or APC-resistant individuals, clotting is observed at approximately I O minutes
(Table 2). Figure 6 depicts the products formed from factor
V during the clotting experiment described above visualized
with a mixture of aHFVaHc#6and aHFV-9. Clot formation
in plasma from a normal individual (1691 GG genotype; Fig
6A, vertical arrow) as well in plasma from an APC-resistant
individual (1691 AA genotype, patient I"; Fig 6B, vertical
arrow), correlates with appearance of the heavy chain portion
of factor Va (Fig 6A, lane 4. and 6B, lane 5 ; HC) and of
the M,= 220,000 fragment containing the light chain portion
of the cofactor. Prolonged incubation of the normal plasma
sample at 37°C results in the appearance of the light chain
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
KALAFATIS ET AL
4700
A
B
i
2
3
4
5
8
7
8
~
m
1 2n 1 3 1 4 1 5 i e 1 7 1 8
’’
200-
41-994
9F
-
41 679)
68BO7-679)
44
24
of factor Va (Fig 6; LC) and of an M, = 75,000 fragment
(amino acid residues 1 through 506; Fig 6A, lanes 7 and 8,
arrowhead). The appearance of the latter is followed by the
appearance of a fragment of M, = 30,000 (amino acid residues 307 through 506; Fig 6, lane 8, star) showing cleavage
of the heavy chain of normal factor Va at Arg””, followed
by cleavage at ArgqW(Fig 6A, lanes 7 and 8). These data
demonstrate, as previously shown,” APC generation in
plasma after clot formation. Furthermore, no M, = 62,000/
Fig 4. Titration of the inactivation of human factor p- by
human plasma APC. (A) Plasma
factor VRm (factor V,,,’* 110
nmol/L) was incubated with
PCPS vesicles (200 PmollL) for 5
minutes at 37°C. Hirudin 120
nmol/L) and APC were then
added 12.2 nmollL IO1 or 550
pmol/L 1.1).
At selected time intervals, aliquots were assayed
for cofactor activity in a clotting
time-based assay using factor Vdeficient plasma. Results are expressed as the percentage of initial clotting activity as a function
of time after the addition of APC.
The inset shows the analysis of
the inactivation reaction during
the first 30 minutes. At the same
time intervals, aliquots were
withdrawn and analyzed by
SDS-PAGE. (B)The samples assayed for clotting activity in (A)
were also analyzed after reduction using 2% pmercaptoethano1 and 2% SDS on a 4% t o 12%
linear gradient SDS-PAGE (300
ng of protein were applied per
lane). After transfer t o nitrocellulose, the immunoreactive fragments were detected using
MoAb aFVa&.
Lanes 1 and
10 represent membrane-bound
factor VR5m control and no APC;
lanes 2 through 9 show membrane-bound factor VRwith
550 pmol/L APC at 1,3,5,10,15,
30,60,and 120 minutes after the
addition of APC; and lanes 11
through 18 depict membranebound factor VRin the presence of 2.2 nmol/L APC at the
same time intervals as shown in
lanes 2 through 9. The position
of the molecular weight markers
is indicated at left. The positions
of the M. = 120,000 fragment
containing amino acid residues 1
through 994 and of the M, =
99,000 fragment containing residues 1through 679 are indicated
on the right.
54,000 fragment (amino acid residues 307 through 709/679)
is observed in plasma from the APC-resistant individual (Fig
6B, lanes 6 through 8), even after extended incubation of
patients plasma at 37°C. The data show activation of factor
V in plasma from normal and APC-resistant individuals after
incubation with Ca” and a membrane surface.
The effect of APC on factor psmQ
in whole plasma. In
studies using either normal plasma (pool from 30 healthy
donors) or plasma collected from an individual with a normal
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
INSIGHT INTO THE APC-RESISTANCE ASSAY
4701
A
Fig 5. Activation of factor
by rAPCmA. Normal plasma factor V (100 nmollL, 0 )and factor VR5(factor VI," 100 nmol/L, 0 )were incubated with rAPCyzoA
(2 nmol/L) in the presence of PCPS vesicles (200
pmollL) and hirudin (20 nmollL) at 37°C. At selected
time intervals, aliquots of the mixtures were assayed
for activity using a one-stage clotting activity using
factor V-deficient plasma. The results are expressed
as clotting time (in seconds) as a function of time
after the addition of rAPCyZoA.
The clotting time of
the factor VR5ffi0
solution before the addition of APC
was 33 seconds (O), whereas the clotting time of
the normal factor V solution was 31.5 seconds (0).
Similar results were found in three separate experiments using factor V from patient I (ie, a shortening
in the clotting time of approximately 4 seconds was
observed after 2 hours of incubation of factor V, and
rAPCYzoA).
The procoagulant effect of rAPCyZoA
is less
pronounced when using factor
from patient
11." (BI The samples assayed for clotting activity in
(A) were also analyzed by SDS-PAGE after reduction
with 2% p-mercaptoethanol on a 4% t o 12% linear
gradient (approximately 250 n g of protein per lane).
Fragments were visualized following transfer t o nitrocellulose and staining with aHFVAHe#6. Lane l,
normal plasma factor V control, no APC; lanes 2
through 9, normal plasma factor V in the presence
of PCPS vesicles and rAPCYzoA
after 2, 5, 10, 20, 30,
60,90, and 120 minutes of incubation; lane 10, factor
Vnma control no APC; lanes 11 through 18, factor
VRm in the presence of PCPS vesicles and rAPCYzoA
at the same time intervals as shown in lanes 2
through 9. Lane 19, control, a-thrombin activated
factor Va. The positions of the molecular weight
markers are indicated on the left. The arrowheads at
right depict the position of the M,= 75,000 fragment
(amino acid residues 1 through 506) and of the M, =
30,000 fragment that derives from the normal procofactor after cleavage by rAPCYzoA
at Arg5ffifollowed
by slow cleavage at Arg3ffi.The star at the right of
lanes 9 and 18 represents the position of a fragment
that would derive from normal factor V or factor
VR5- after cleavage at Arg"'. The position of the
heavy chain of the cofactor (HC; amino acid residues
1 through 709) is also indicated at the right of (6). It
is noteworthy that the fragments shown in lanes 3
through 9 and 14 through 18 of (6) (indicated by the
star and the arrowheads) are not visible under the
conditions used after either Coomassie Blue or silver
staining. In different experiments after staining of
the gel with silver nitrate, a slight decrease in the
concentration of normal factor V is only observed.
:
vsm0
1
34 t
a/
0
0
0
29
21)
20
0
40
60
80
120
100
Time alter the addition of APC?&I)
B
i
2
a
4
I
e
T
e o m n ~
i
~
u
i
~
n 19 ~
i
r
t
200.
97-
HC
l-SO6l
68-
43-
29
107-
10
1691 GG genotype,' in the presence of a membrane surface,
Ca", and 2.8 and 5.5 nmol/L APC, no clot was observed
(Table 2). Under similar experimental conditions when using
whole plasma from APC-resistant individuals homozygous
(1691 AA) for the ArgC'"-Gln mutation (patients I and U),''
a clot was noticed (Table 2). Figure 7 shows the effect of
APC (2.8 nmol/L) on factor V in whole plasma (pool plasma
from 30 healthy donors [Fig 7A] and an APC-resistant individual [Fig 7B1) in the presence of phospholipid vesicles
and Ca". During the first 4 minutes of incubation, a doublet
of M, = 330,000/280,000 appears in both normal factor V
(Fig 7A, lanes I through 3) and factor VR50hP
(Fig 7B, lanes
1 through 3). showing some cleavage of factor V at Arg"".
Subsequently, in normal plasma, there is the appearance of
an M, = 30,000 fragment (amino acid residues 307 through
506; Fig 7A, lanes 3 through 5). Thus, as shown using purified factor V," in normal plasma, factor V is cleaved by
APC in the presence of PCPS vesicles at Arg"", followed
by cleavage at Arg". The M, --100,000 fragment (Fig 7;
HC) most likely represents the heavy chain portion of factor
V. This product is rapidly cleaved at Arg"" and Arg'Oh by
APC to generate the M, = 30,000 fragment (Fig 7A, lanes
6 through 8)." These data suggest activation of factor V to
factor Va by a-thrombin and/or factor Xa after cleavage at
Arg7'" or potentially by APC cleavage at Arg"' and generation of a heavy chain-like product before factor V degradation by APC.
The addition of APC and a membrane surface to the
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4702
KALAFATIS ET AL
Table 2. Clotting Time in Plasma From Normal and APC-Resistant
Individuals in the Presence of a Membrane Surface and Ca2+
Clotting Time (min)
2.8 nmol/L
Normal plasma
(pool from 30
donors)
1691 GG individualt
1691 AA individual
(patient I #9ON
1691 AA individual
(patient II #173H
*No clot was
No APC
APC
5.5 nmol/L
APC
>5.5 nmol/L
APC
8.9 t 1.7
>30*
>30
> 30
8.1 t 1.4
1 1 % 1.4
>30
17.5 2 2.4
>30
24.7 % 7
230
>30
10.1 t 2
17.7 2 2
30
>30
observed, visually, after 30 minutes of incubation.
tPlasma from a normal individual with an Arg at position 506 in the factor V
molecule.
+The patient number identifies the APC-resistant individuals in the Leiden
thrombophilia study as
plasma from an APC-resistant individual (patient I, APC:SR
ratio 1.13)'",'* results in the accumulation of a fragment containing the majority of the heavy chain portion of factor V
(HC; Fig 7B, lane 4). The appearance of this fragment is
observed earlier in plasma from APC-resistant individuals
than in normal plasma (compare Fig 7A and B, lanes 4 and
5), and no M, = 30,000" or M, = 54,O0Oi2fragments are
observed after 15 minutes of incubation with APC. As a
consequence, this fragment is accumulated during the initial
15 minutes of incubation (Fig 7B, lanes 5 and 6). Coincidentally, clot formation is observed after 18 minutes of incubation (Fig 7B, vertical arrow). Similar results were found
when using plasma from patient I1 (APC:SR 1.14; Table
2). l o . l l
Figure 8 shows that in normal plasma when APC is added
before clot formation the generation of enough heavy chain
to support clot formation is impaired. Figure 8A shows the
accumulation of heavy chain of factor Va (lanes 1 through
5 ) that results in clot formation at approximately 10 minutes.
No clot and no accumulation of heavy chain was observed
in the presence of 2.8 and 17 nmol/L APC (Fig 8B and C).
Furthermore, by increasing the concentration of APC, there
is an increase of the M, = 30,000 fragment containing amino
acid residues 307 through 506. These data show that clotting
in normal plasma is very sensitive to the APC concentration.
Figure 9 shows that under similar experimental conditions
the heavy chain of factor VaRsohQis more resistant to APC
degradation. Clot formation is observed when APC (at 2.7
and 5.5 nmoVL) is introduced into the plasma from APCresistant individuals (Fig 9B and C). Collectively, the data
show fibrin clot formation in plasma from APC-resistant
individuals in the presence of a membrane surface and limited amounts of APC ( ~ 5 . nmoVL).
5
After clot formation
(Fig 9B and C, fragments a and b) or using higher concentrations of APC (Fig 9D and E, fragments a and b), there is
generation of an M, = 62/56,000 fragment that is generated
after cleavage at Arg306/Arg679
of the heavy chain of factor
vaR5MQ
It is noteworthy that clot formation when using patients'
plasma was very sensitive to both the APC preparation and
concentration used in each experiment. Similar results were
observed with two different APC preparations (ie, in the
presence of PCPS vesicles, Ca2+, and 2.8 and 5.5 nmol/L
plasma APC); a clot was observed in plasma from patient 1
and patient I1 (Table 2). No clot formation was observed in
the presence of higher concentrations of APC ( I 1 and 17
nmol/L) in plasma from APC-resistant individuals. Thus, at
high APC concentrations, formation of the active heavy
chain is counterbalanced by cleavage of the procofactor at
Arg"''. As a result, there is no clot formation.
DISCUSSION
After incubation of plasma with PCPS vesicles and Ca",
there is clot formation as a consequence of initiation of the
intrinsic and extrinsic pathways of the blood clotting process,
resulting in the activation of factor Va. Subsequent to clot
formation, there is initiation of the anticoagulant pathway
that regulates APC formation and thus factor V N a inactivation. Thus, the relative rates of activationhnactivation of
factor V determines the outcome, once a membrane surface
and Ca'+ are mixed with plasma. Our data show that the
addition of PCPS vesicles, Ca'+, and controlled amounts of
APC to diluted plasma from APC-resistant individuals results in the accumulation of the factor Va heavy chain (HC)
due to delayed cleavage at Arg3%in the case of membranebound factor VaR506Q.
This accumulation of a procoagulant
HC leads to clot formation and possibly to the thrombotic
tendency manifested in these individuals.
The level of accumulation of HC is regulated by the rate
of cleavage at Arg'06 on the membrane-bound factor VRS"6Q/
V(a)R5"6Qand its effect can be observed in plasma in the
presence of all proteins that participate in clot formation at
a concentration of APC that is compatible with accumulation
of enough HC to promote clot formation. Furthermore, the
inactivating cleavage at Ar2(I6 occurs first on membranebound factor V, whereas this cleavage is potentiated by prior
cleavage at Arg'"' on membrane-bound factor Va. It is noteworthy that whereas empirically determined low concentrations of APC allowed clot formation in plasma from APCresistant individuals, similar concentrations of APC abolish
clot formation when introduced in normal plasma before the
initiation of clotting.
In plasma from APC-resistant individuals in the presence
of PCPS vesicles and Ca2' at low APC concentrations (55.5
nmol/L, Fig 10A) there is early formation of factor Va heavy
chain that results in clot formation. Thus, under these conditions, cleavages of factor VRsf'hQat Arg67' and Arg"" occur
at similar rates, resulting in delayed cleavage at Arg"'" and
the transient accumulation of HC. No clot formation was
observed in normal and APC-resistant individuals at high
APC concentrations (>5.5 nmol/L, Fig IOB). Thus, at high
APC concentrations, cleavage at Arg'"', which is responsible
for inactivation of factor VR5'"Q, is faster than cleavage at
Arg67'. As a consequence, the plasma is rapidly depleted of
the factor V(a) pool with procoagulant potential; consequently, there is no generation of HC and no clot formation.
Because factor Va and not factor V is procoagulant, the data
suggest that, at high APC concentrations (>5.5 nmol/L),
cleavage and inactivation of membrane-bound factor V by
APC is accelerated and becomes faster than cleavage of the
procofactor by a-thrombin and/or factor Xa and generation
of active cofactor.
Our present data show that the use of an active APC
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4703
INSIGHT INTO THE APC-RESISTANCE ASSAY
B
A
1
2
3
4+s
6
7
8
1
2
3
4
5 + 6
7
8
M,.Io-~
-"HC"
97-
6843-
2918Fig 6. Proteolysis of factor V during clot formation. (A) Normal plasma; (8)plasma from an APC-resistant patient (patient 1, APCSR, 1.13).
Lanes 1 through 8, plasma in the presence of phospholipid vesicles after incubation at 37°C at 0, 2, 4, 7, 10, 12, 17, and 22 minutes. At 9
minutes and 55 seconds in (AI and at 10 minutes and 30 seconds in (B), a clot was observed (indicated by the vertical arrow at the right of
lane 4 in [AI and at the right of lane 5 in [BJ).After centrifugation, the supernatant was further analyzed by SDS-PAGE, followed by transfer
t o nitrocellulose. After transfer t o nitrocellulose, the immunoblot was probed with a mixture of t w o MoAbs, ie, aHFV-9, which recognizes an
epitope on the light chain of the cofactor," and aHFVaHc#6, which recognizes an epitope on the heavy chain of factor Va between amino acid
residues 307 and 506.'' The position of the molecular weight markers is indicated on the left of (A). The HC at the right of (BI indicates the
position of the heavy chain of human factor Va, whereas LC indicates the position of the light chain of factor Va. The arrowhead at the right
of (A) indicates the M, = 75,000 fragment deriving from factor Va heavy chain after cleavage at Arg- (amino acid residues 1 through 5061,
whereas the star depicts the M. = 30,000 fragment that derives from the M, = 75,000 fragment after cleavage at Arg306(amino acid residues
307 through 506). HC and LC are given in quotes because the identity of these fragments is inferred from the following: (1) lmmunostaining
is performed with a mixture of MoAbs. When used in separate blots, individually, aHNam#6 recognizes an epitope between amino acid
residues 307 and 506 of factor V," whereas aHFV-9 recognizes the light chain of the cofactor. Thus, aHFVaHc#6recognizes factor V, the M.
-100,000 heavy chain (amino acid residues 1through 7091, the M, = 75,000 fragment (amino acid residues 1 through 5061, and the M, = 30,000
fragment (amino acid residues 307 through 506);'
whereas aHFV-9 reacts with factor V, the M, = 220,000 fragment intermediate, and the
light chain of factor Va (aHFV-9 has also a slight cross-reactivity with the M, = 150,000 activation peptide of factor V); and (2)in separate
experiments using normal plasma or plasma from APC-resistant individuals, the fragment of M, -100.000 migrates at a position that is
consistent with the heavy chain of factor Va, whereas the fragment greater than the M, = 75,000 fragment migrates as the light chain of the
human cofactor; (3) the appearance of the M. -100.000 fragment is followed by clot formation (see also Fig 7).
preparation is essential for factor V/factor Va inactivation
analysis in whole plasma. In our hands, all APC preparations,
although having similar chromogenic activities, do not express similar inactivating properties with respect to factor
V N a (shown in Fig 3). During the standard APC-resistance
assay, 100 pL of plasma is incubated with the aPTT reagent
for 5 minutes, followed by incubation with APC at concentrations varying from 5 to 100 nmoI/L.' aPTT is composed
of a membrane surface and an activator of the intrinsic pathway of blood clotting (activator of factor XII). The inactivation of factor Va is a sequential phenomenon with cleavage
at Arg" potentiating cleavage at Arg"Oh,'l.12whereas membrane-bound factor V is inactivated after cleavage at Arg"'*.
Thus, if the surface is not adequate for factor V binding and/
or if activation of factor V does not occur at high APC
concentrations using a fully active APC molecule, both normal plasma factor V and factor VRsMQwould be inactivated
with similar rates as a consequence of cleavage at Arg".
Thus, an APC-resistant individual will escape detection. In
contrast, under similar experimental conditions (ie, in the
presence of a less sensitive aPTT reagent and high APC
concentrations) using an APC preparation with reduced anticoagulant activity, a difference in the inactivation rates may
be observed between normal factor V and factor VRsw.
However, this difference will be augmented if the aPTT
reagent favors production of factor Va. Thus, the need for
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
KALAFATIS ET AL
4704
A
2
a
M r X l 0 3 ~
3
4
5
6
7
8
1
2
3
97-
08-
43-
291%
74-
standardization of both the a m reagents and the APC preparations becomes critical with respect to the reproducibility
of the results from one laboratory to another. It is noteworthy
that, during the standard APC-resistance assay (1/3 diluted
plasma as compared with 1/10 diluted plasma in the present
study), in plasma from patients homozygous for factor VLeidcn
clots are formed even at high APC concentrations."' This
phenomenon might be related to both differences in the concentration of the clotting factors and/or the presence of a
strong activator of factor XI1 (in the a P l T reagent).
Under our experimental conditions, in the absence of
APC, the clotting time in our assay is 8 to 10 minutes. This
longer clotting time as compared with the standard a m
assay (Fig 2) is most likely due to the absence of an activator
of the intrinsic pathway of blood coagulation. The longest
clotting time shown in Table 2 using limited amounts of
APC is greater than 30 minutes. Thus, the clotting time
ratio for normal individuals observed using our experimental
protocol (>3.0) is not significantly different than that observed using the standard a m - b a s e d screening assay (2.0
to 4.0; Koster et al,24Vooberg et al,2sand Table I). However,
our assay uses a membrane surface of known composition
(phospholipid vesicles composed of 75% PC and 25% PS),
whereas the a m reagents lipid composition is not always
defined or described. Thus, in the presence of a bioactive
APC preparation (defined in Fig 3). high reproducibility between different plasma samples would be obtained.
-'HC"
Fig 7. The effect of APC on
plasma from normal and APC-resistant individuals. (A) Lane 1,
normal plasma in the presence of
phospholipidvesicles and no APC
after 2 minutes of incubation at
37°C; lanes 2 through 8, normal
plasma incubated with phospholipid vesicles and APC at 2, 4, 6,
10, 15, 20, and 30 minutes after
the addition of purified plasma
APC (2.8 nmollL). (B) Lane 1,
plasma from an APC-resistantpatient (patient l, APCSR, 1.13)'0.'2
in the presence of phospholipid
vesicles and no APC after 2 minutes of incubationat 37°C; lanes 2
through 6, plasma incubatedwith
PCPS and APC (2.8 pmollL) at 2,
4, 6, 10, and 15 minutes. At 18
minutes, a clot was observed
when usingplasma from the APCresistant patient (indicatedby the
vertical arrow in [BI). Lanes 7 and
8, after centrifugation, the supernatant was analyred at 25 and 30
minutes. After transfer to nitrocellulose, the immunoblot was
probed with MoAb aHFVa&.
The position of the molecular
weight markers is indicated on
the left of (A). HC at the right of
(B) indicates the position of the
heavy chain of human factor Va.
Our data also suggests that premature cleavage at Argh79of
the membrane-bound factor VR.WhQ
combined with delayed cleavage at Arg30hcould result in the accumulation of a shorter heavy
chain (amino acid residues 1 through 679). Accumulation of the
latter heavy chain portion that most likely possesses procoagulant
activity will promote systemic coagulation. These conclusions
are partially supported by our data using factor VMw and the
AFCymAmolecules (Fig 5). This hypothesis would also explain
the shortening of clotting time in plasma from patients homozygous for the Ar$06+Gln mutation in the presence of APC during
the recently described new AFC-resistance assay?' The Le et
al" suggested that the mechanism of the shortening of the clotting time in patients homozygous for the factor V Leiden mutation may be related to the fact that APC must cleave more than
one peptide bond (in the right order) in the factor Va molecule
for complete inactivation. Because cleavage at A
r
p is necessary for efficient exposure of the inactivating cleavage site at
Arg- in factor Va heavy chain, we can speculate that production
of a portion of the heavy chain of factor Va (by cleavage of
factor V R m at Arg6'9) before cleavage at Arg- may generate
an APC-resistant heavy chain species in plasma from individuals
with factor V Leiden that would require cleavage at Ar$" for
inactivation. Whether activation of factor V Leiden by APC is
a phenomenon related to cleavage at Arg679alone or is a combination of events that involves as yet unidentified phenomena that
would be potentiated by the Arg%-Gln substitution in factor V
remains to be identified.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
INSIGHT INTO THE APC-RESISTANCE ASSAY
4705
A
B
-
C
1
- 301-
c
506
‘
I
a
&
Fig 8. Titration of plasma from normal individuals by APC. Plasma from normal individuals (100 pL) was diluted 10-fold in a buffer containing
5 mmol/L CaCI, and treated with APC in the presence of phospholipid vesicles as described in the Materials and Methods and in the legend
t o Fig 7. Following transfer t o nitrocellulose fragments were revealed after staining with MoAb aHFVaH&6. (AI Control plasma incubated with
PCPS, no APC. (B) Plasma incubated with 2.8 nmol/L APC. (C) Plasma incubated with 17 nmol/L APC. The position of the M. = 30,000 fragment
spanning the amino acid region 307 through 506 of the factor V molecule as well as the position of factor V and factor Va heavy chain are
also depicted. The arrowhead at the bottom of (AI indicates the time of clot formation. (A) Lanes 1 through 8, the same time points as in Fig
6A. (B and C) Same time points as in Fig 7A. The blots in this figure as well as in Fig 9 (see below) are overexposed for the sake of identification
of trace amounts of heavy chain or heavy chain-derived proteolytic fragments.
B
A
I
W b d
C
D
s*l’
L-
1- HC
Fig 9. Titration of plasma from APC-resistant individuals by APC. Plasma from APC-resistant individuals identified in Table 2 (100 pL) was
diluted 10-fold in a buffer containing 5 mmol/L CaCI2 and treated with APC in the presence of phospholipid vesicles, as described in the
Materials and Methods and in the legend t o Fig 7. Fragments were visualized after staining with MoAb aHFVaHc#6. (A) Control plasma
incubated with PCPS and no APC. IBI Plasma incubated with 2.8 nmol/L APC. (C) Plasma incubated with 5.5 nmol/L APC. (D) Plasma incubated
with 11 nmol/L APC. (€1 Plasma in the presence of 17 nmol/L APC. la1 depicts the M, = 54,000/60,000 doublet deriving from the heavy chain
of factor VaRSm after cleavage at Arg30sand or Arg”’. (b) identifies the M, = 54,000 fragment that derives from factor V after cleavage at
Arg’w/Arg”g. These fragments are difficult t o see because of the albumin and Igs present in plasma. However, these fragments are clearly
visible in lanes 7 and 8 of (Cl and (El.The positions of factor V and of the heavy chain of factor Va are also indicated. The arrowhead at the
bottom of (AI through (CI indicates the time of clot formation. (A) Lanes 1 through 8, same time points as in Fig 6B. (6)through (El, same
time points as in Fig 78.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4706
KALAFATIS ET AL
/
k.
+XPS
B
HzN
(>55nMAPC)
- .
H
AI
.B ....._.WQr(WIQ)
;
m, I,,
I44
\,..\
L
;k&
/
7”
Argsn
H,NJ*I]I
A2
H
,
J
.
4”
7
HN
AI
1“
4sm
t
\4
/+AX\
T
l
H
N
4-
9
9
.
m
T
I
um
-
1
I * I
[”
H,N
4”
ACKNOWLEDGMENT
We thank Shaw Henderson for technical assistance and Dr William Church for the ascites fluid.
ik.
r
mentioned above that cleavage at Arg6” of factor V may
increase its cofactor activity needs further investigation and
eventually the use of a recombinant factor V molecule with
only one available cleavage site for APC (at Arg679).
In conclusion, in APC-resistant individuals, factor VR506U
may be rendered procoagulant as a result of cleavage by
APC at Arg679.These data, in addition to the data showing
delayed inactivation of membrane-bound factor VaRSmQ
by
reason of slow cleavage at Arg306resulting in the prolongation of factor Va cofactor activity after clot formation, may
explain the thrombotic tendency in individuals with the
ArgSo6+Glnsubstitution. Thus, factor V N a inactivation by
APC is a complicated phenomenon governed totally by the
kinetics of cleavage at each site as are all clotting reaction~.’~.’~
Recently, a study by Nicolaes et a13’ attempted to
evaluate the kinetic constants of APC at each cleavage site.
However, the results of the latter study are compromised by
the high concentrations of enzyme used in their experiments;
hence, differences in the inactivation rates when varying the
concentration of the reagents are most likely due to the quality and quantity of APC used rather than to the factor Va
concentration, as ~uggested.~’
Thus, the evaluation of the
kinetic constants at each cleavage site in the factor V and
factor Va molecule using a bioactive APC preparation and
natural and recombinant factor V at physiologic conditions
(ie, at low enzyme concentrations) will help the elucidation
of the phenomenon of APC resistance.
nm
ka>>kb >kb’ >k,
Fig 10. Schematic representation for the cleavage of membranebound human factor Vby APC. The heavy chain portion of the
human cofactor (containing 709 amino acids) is composed of two A
domains (Al-A2; A1 spanning region 1 through 303 and A2 spanning
region 317 through 658) associated through a connecting region
(amino acids 304 through 3161.“ The portion of 657 through 709 at
the COOH terminus of the heavy chain portion of factor Va, denoted
by a minus, contains a cluster of addk amino acids. Normal plasma
factor V is rapidly inactivated in the presence of a membrane wrfaw
after cleavageat Arg-. Membraneboundplasmafactor ?is also
inactivated after cleavage at Argas (k.).”,“ However, at low APQ
concentrations, cleavage at k g m (b)occurs almost simuttclneously
on membrane-bound factor
(A), resulting in accumulation of
an M. = 99,OOO fragment which may require deavage at Arg- before
inactivation. As a conaequence, deavage of the M, = 99.000 fragment
at ArgSwIkJ is delayed as compared with cleavage of factor V at
Arg- (It,,) and a clot is formed. At hlgh APC concentrations, cleavage
at Arg- occurs faster than does deavage at Argm. As a consequence, the plasma is rapidly depleted of the factor V pool with
potentlal procoagulant activity and no clot is obaerved (6).
Recently, Bakker et alZ7reported that cleavage of factor
Va at His684results in a factor Va molecule with diminished
cofactor activity. Combination of the data suggests that a
more careful characterization of APC-cleaved factor V (at
Arg679)is warranted. Thus, verification of the hypothesis
REFERENCES
I . Mann KG, Jenny RJ, Krishnaswamy S: Cofactor proteins in
the assembly and expression of blood clotting enzyme complexes.
Annu Rev Biochem 57:915, 1988
2. Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy
S: Surface-dependent reactions of the vitamin K-dependent enzyme
complexes. Blood 76:1, 1990
3. Nesheim ME, Taswell JB, Mann KG: The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol
Chem 254:10952, 1979
4. Kane WH, Davie EW: Cloning of a cDNA coding for human
factor V, a blood coagulation factor homologous to factor VIII and
ceruloplasmin. Proc Natl Acad Sci USA 83:6800, 1986
5. Jenny RJ, P i t ” DD, Toole JJ, Kriz RW, Aldape RA, Hewick
RM, Kaufman RJ, Mann KG: Complete cDNA and derived amino
acid sequence of human factor V. Proc Natl Acad Sci USA 84:4846,
1987
6. Dahlback B, Carlsson M, Svensson PJ: Familial thrombophilia
due to a previously unrecognized mechanism characterized by poor
anticoagulant response to activated protein C. Proc Natl Acad Sci
USA 90:1004, 1993
7. Bertha RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven
RJ,de Ronde H, van der Velden PA, Reitsma PH: Mutation in blood
coagulation factor V associated with resistance to activated protein
C. Nature 369:64, 1994
8. Sun X, Evatt B, Griffin JH: Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous
thrombophilia. Blood 83:3 120, 1994
9. Koeleman BPC, Reitsma PH, Allaart CF, Bertina RM: Activated protein C resistance as an additional risk factor for thrombosis
in protein C-deficient families. Blood 84: 1031, 1994
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
INSIGHT INTO THE APC-RESISTANCE ASSAY
10. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH:
High risk of thrombosis in patients homozygous for factor V Leiden
(activated protein C resistance). Blood 85:1504, 1995
11. Kalafatis M, Rand MD, Mann KG: The mechanism of inactivation of human factor V and human factor Va by activated protein
C. J Biol Chem 269:31869, 1994
12. Kalafatis M, Bertina RM, Rand MD, Mann KG: Characterization of the molecular defect in factor VRSW.J Biol Chem 270:4053,
1995
13. Barenholz Y, Gibbs D, Litmann BJ, Go11 J, Thompson T,
Carlson D: A simple method for the preparation of homogeneous
phospholipid vesicles. Biochemistry 16:2806, 1977
14. Foster BW, Tucker MM, Katzmann JA, Miller RS, Nesheim
ME, Mann KG: Monoclonal antibodies to human coagulation factor
V and factor Va. Blood 61:1060, 1983
15. Bajaj SP, Rapaport SI, Prodanos C: A simplified procedure
for purification of human prothrombin, factor IX and factor X. Prep
Biochem 11:397, 1981
16. Kisiel W, Davie EW: Protein C. Methods Enzymol 80:320,
1981
17. Lu D, Bovill EG, Long GL: Molecular mechanism for familial
protein C deficiency and thrombosis in protein CYemont
(Glu'' + Ala
and V a P + Met). J Biol Chem 269:29032, 1994
18. Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680, 1970
19. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl
Acad Sci USA 76:4350, 1979
20. Kalafatis M, Lu D, Bertina RM, Long GL, Mann KG: Biochemical prototype for familial thrombosis. A study combining a
functional protein C mutation and factor V Leiden. Arterioscler
Thromb Vasc Biol 152181, 1995
21. Nesheim ME, Katzmann JA, Tracy PB, Mann KG: Factor V.
Methods Enzymol 80:249, 1981
4707
22. Lu D, Kalafatis M, Mann KG, Long GL: Loss of membranedependent factor Va cleavage: A mechanistic interpretation of the
Blood 84:687, 1994
pathology of protein C,,.
23. Jackson DE, Mitchell CA, Salem HH: Protein C is responsible
for the rapid inactivation of factor Va following blood clotting in
vitro. Thromb Haemost 72:70, 1994
24. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM: Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study.
Lancet 342:1503, 1993
25. Vooberg J, Roesle J, Koopman R, Buller H, Berends F, ten
Cate JW, Mertens K, van Mourik JA: Association of idiopathic
venous thromboembolism with single point-mutation at ArgSWof
factor V. Lancet 343:1535, 1994
26. The Le D, Griffin JH, Greengard JS, Mujumdar V, Rapaport
SI: Use of a generally applicable tissue factor-dependent factor V
assay to detect activated protein C resistant factor Va in patients
receiving warfarin and in patients with a lupus anticoagulant. Blood
85:1704, 1995
27. Bakker HM, Tans G, Thomassen MCLGD, Yukelson LY,
Ebberink R, Hemker HC, Rosing J: Functional properties of human
factor Va lacking the Asp683-Arg709 domain of the heavy chain. J
Biol Chem 269:20662, 1994
28. Lawson JH, Kalafatis M, Stram S, Mann KG: A model for
the tissue factor pathway to thrombin. I. An empirical study. J Biol
Chem 269:23357, 1994
29. Jones KC, Mann KG: A model for the tissue factor pathway
to thrombin. 11. A mathematical simulation. J Biol Chem 269:23367,
1994
30. Nicolaes GAF, Tans G, Thomassen MCLGD, Hemker HC,
Pabinger I, Varadi K, Schwarz HP, Rosing J: Peptide bond cleavages
and loss of functional activity during inactivation of factor Va and
factor VaRSWQ
by activated protein C. J Biol Chem 270:21158, 1995
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1996 87: 4695-4707
Proteolytic events that regulate factor V activity in whole plasma from
normal and activated protein C (APC)-resistant individuals during
clotting: an insight into the APC-resistance assay
M Kalafatis, PE Haley, D Lu, RM Bertina, GL Long and KG Mann
Updated information and services can be found at:
http://www.bloodjournal.org/content/87/11/4695.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.