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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Chemistry, Cleveland State
University, and the Department of Molecular Cardiology, The Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland, OH; and
Department of Medical and Surgical Sciences, Second Chair of Internal
Medicine, University of Padua, Padua, Italy.
A 44-year-old woman with a history of severe thrombotic
manifestations presented with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA sequencing of and around the regions
encoding the APC cleavage sites in the factor Va molecule excluded the
presence of the factor VLeiden mutation and of other known genetic
mutations. No antiphospholipid antibodies were present in the
patient's plasma and both prothrombin time and activated partial
thromboplastin time were normal. The total immunoglobulin fraction was
isolated from the patient's plasma and found to induce severe APC
resistance when added to normal plasma and to factor V-deficient
plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total
immunoglobulin fraction purified from the patient's plasma
demonstrated that the antibody recognizes factor V, is polyclonal, and
has conformational epitopes on the entire factor V molecule (heavy and
light chains, and B region). Thus, the immunoglobulin fraction
interferes with the anticoagulant pathway involving factor V. The
inhibitor was isolated by sequential affinity chromatography on protein
G-Sepharose and factor V-Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The
isolated immunoglobulin fraction was also found to inhibit the cofactor
effect of factor V for the inactivation of factor VIII by the
APC/protein S complex. Our data provide for the first time the
demonstration of an antifactor V antibody not related to the presence
of antiphospholipid antibodies, which is responsible for thrombotic
rather than hemorrhagic symptoms.
(Blood. 2002;99:3985-3992) Coagulation factor V circulates in plasma as a
large single-chain protein with a Mr
330 000.1,2 Individuals with a 1691G>A substitution in the factor V
(F5) gene (resulting in an Arg506Gln mutation in the
factor V molecule, factor VLeiden) have a poor
anticoagulant response to APC (APC resistance), which is associated
with a significant increase in risk for deep venous thrombosis (DVT)
(7-fold for the heterozygous and 80-fold for the
homozygous).4-8 APC resistance has been suggested to be
the most common risk factor for developing DVT, most likely because
factor VaLeiden is inactivated by APC at a slower rate than
normal factor Va thus leading to prolonged thrombin
generation.9,10
Antifactor V antibodies are relatively common and are developed usually
after exposure of patients to topical hemostatic agents containing
bovine Case report
Materials and reagents
APC-sensitivity assay The APC-sensitivity ratio (APC-SR) is used to define an individual predisposition to thrombosis because of either genetic or acquired abnormalities. The APC-SR is defined by measuring the clotting time of plasma in the presence or absence of APC as described.4,5,20 The clotting time is registered and the ratio of clotting time in the presence of APC divided by the clotting time in the absence of APC is defined as the APC-SR. Normal APC-SR values are usually higher than 2.0. Any value below 2.0 is considered as APC resistance.Detection of APAs and other routine laboratory determinations for lupus anticoagulant For the determination of APAs, a polyvalent kit IgG, IgA, and IgM (Asserachrom APA, Stago, Asnieres, France), was used as described.21 The enzyme-linked immunosorbent assay (ELISA) kit contains cardiolipin, anionic phospholipid, and 2-glycoprotein I. Tests were considered positive if the
values exceeded 15 phospholipid units (UPL)/mL and borderline when they
were between 5 and 15 UPL/mL. ELISAs for the detection of
anticardiolipin antibodies (Asserachrom Anticardiolipin IgG/IgM,
Stago), anti-2-glycoprotein I antibodies (Asserachrom
anti-2-glycoprotein I IgG/IgM, Stago) and
antiprothrombin antibodies (Asserachrom antiprothrombin IgG/IgM, Stago)
have also been performed using commercially available kits. For the
detection of lupus anticoagulant, the guidelines recommended by the
Subcommittee for Standardization of the International Society on
Thrombosis and Hemostasis were followed,22 using a method reported elsewhere.21 Both PTT-based and dilute Russell
viper venom time-based assays were performed to detect the presence of
lupus anticoagulant as previously described.21
Protein C and protein S assays Amidolytic and clotting activities of protein C were measured in plasma using Behrichrom protein C and protein C reagent kits, respectively (Dade-Behring, Milan, Italy), as previously described.23 In both methods, Protac was used to activate protein C. The amidolytic method measures the effect of APC on a specific chromogenic substrate (Chromozym-TH), whereas the clotting method measures the prolongation of the aPTT after activation of protein C present in the patient's plasma. For the determination of protein S activity, a commercially available kit, IL PS test (Instrumentation Laboratories, Milan, Italy) was used.24 Protein S cofactor activity for APC is determined after activation of protein C present in the test mixture (including protein S-deficient plasma and diluted patient's plasma) by Protac. Functional tests for protein C and protein S were performed in patient's plasma or after reconstitution of protein C- and protein S-deficient plasmas with protein C and protein S, respectively, isolated from patient's plasma by immunoaffinity chromatography.Protein C and total and free protein S antigen levels were determined as previously described.25,26 Factor V antigen was detected in patient's plasma by an ELISA as previously described.20 Factor V functional assay were performed on ACL 3000 plus (Instrumentation Laboratories) using the PT/fibrinogen reagent (Instrumentation Laboratories) and factor V-deficient plasma (Instrumentation Laboratories). Patient's plasma collected on many occasions was tested repeatedly during 10 years of follow-up using all the above-mentioned tests. Preparation of the factor V-Sepharose 4 Fast Flow Approximately 7 mL swollen activated Sepharose 4 Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) was washed as suggested by the manufacturer. Purified human factor V (~30 mg) was extensively dialyzed against the coupling buffer, sodium citrate, pH 6.5, and added to the beads at 4°C. The percent coupling was monitored every 20 minutes by taking the optical density of the supernatant. Following a 2.5-hour incubation at 4°C the reaction was stopped. The coupling efficiency determined by the absorption at 280 nm of factor V remaining in the supernatant was 89%. The beads were further treated as described by the manufacturer and stored at 4°C in 20 mM Tris, 0.15M NaCl pH 7.4 (Tris-buffered saline [TBS]), and 0.01% NaN3.Isolation of the factor V inhibitor Two different separations were performed. For the APC-SR assays 5 mL plasma was applied to a 1-mL column of protein G-Sepharose (Sigma) in TBS. Following washing, the immunoglobulin fraction was eluted with 0.1 M glycine, 0.5 M NaCl, pH 3. Fractions were collected and neutralized in Tris pH 8.0. Tubes containing the patient's total immunoglobulin fraction were pooled, dialyzed, and used in the mixing experiments for the determination of the APC-SR as described.20For the direct inhibition of the factor V inactivation by APC, a
different procedure was used. Higher volumes of plasma containing the
inhibitor were applied to a 5-mL column of protein G-Sepharose equilibrated in TBS (Amersham Pharmacia Biotech, Uppsala). Following washing, the column was eluted as described above with an acidic solution (pH ~3). Approximately 100 mg total immunoglobulin isolated from patient's plasma was applied on a 7-mL human factor V-Sepharose column (27 mg factor V/7mL beads) equilibrated in the same buffer. Following washing with 20 mM Tris, 2.0 M NaCl, pH 7.4, elution was
performed with either 3M NaSCN or an acidic solution (glycine, NaCl, pH
~2.8). When elution was performed at low pH the fractions from the
column were collected directly in tubes containing Tris buffer (pH 8.0)
to neutralize the acidic solution. The absorbance at 280 nm of the
fractions eluted from the antifactor V column was determined using an
extinction coefficient of Measurement of inhibitory activity and clotting assays Factor V cofactor activity was measured by a clotting assay using factor V-deficient plasma and standardized to the percent of control. The assay end point was determined by visualization of the fibrin clot. Factor V was incubated with the inhibitory immunoglobulin fraction eluted from the factor V column and was assayed directly in such a clotting assay. Control experiments included a commercial preparation of human nonimmune IgG, antibodies purified from normal plasma or buffer or both.Inactivation of factor VIII The effect of factor V on the inactivation of factor VIII was studied as previously described.27 Briefly, purified recombinant factor VIII (60 nM) was incubated with APC (10 nM) and protein S (100 nM) in the presence of PCPS vesicles (10 µM) at 37°C. At selected time intervals factor VIII activity was assayed as described by using a 2-stage clotting assay.27 When the effect of factor V was evaluated, the procofactor (± IgG) was preincubated with factor VIII in the presence of PCPS vesicles and protein S and APC was added to start the inactivation reaction.Immunoprecipitation experiments Human factor V (600 nM) was incubated with -thrombin (15 nM)
for 10 minutes at 37°C. The reaction was stopped with either DFP (2 mM) or hirudin (30 nM). Factor Va (600 nM) was incubated with the
patient total immunoglobulin fraction in TBS, 5 mM Ca++ to
final concentrations of 400 nM factor Va and 1 µM immunoglobulin fraction. Following a 30-minute incubation at room temperature the
sample was split in half. EDTA (50 mM) was added to one solution, whereas the other solution was incubated with buffer in the absence of
EDTA. Following a 10-minute incubation, protein G-Sepharose was added.
The solutions were gently mixed and centrifuged for 10 minutes at
14 000g. The supernatant was discarded and the beads were
washed 3 times with 2 M NaCl. Each time the supernatant was discarded.
The final wash was performed in the presence of 2% sodium dodecyl
sulfate (SDS). Following centrifugation, the supernatant was analyzed
on a 5% to 15% SDS-polyacrylamide gel electrophoresis (PAGE). After
transfer to nitrocellulose, factor V fragments were detected using
monoclonal antibody HVaHC#17 (2 µg/mL) that recognizes an epitope on factor Va heavy chain,9,10 monoclonal
antibody HFVaLC#9 (5 µg/mL) that recognizes an
epitope on the light chain of factor Va,10 and a
polyclonal antibody to factor V. Immunoreactive fragments were
visualized with chemiluminescence. In some experiments, factor Va was
treated with APC alone (20 nM) or with APC and PCPS (50 µM) prior to
the addition of the EDTA and protein G-Sepharose.
Gel electrophoresis The SDS-PAGE analyses were performed using 5% to15% and 8% to18% gradient gels according to the method of Laemmli.28 Electroblotting was performing according to the method of Towbin et al.29 NH2-terminal sequencing from polyvinylidene difluoride (PVDF) membranes was performed as described3 in the analytical facility of Dr Alex Kurosky at the University of Texas Medical Branch at Galveston.
Routine coagulation tests The APC-SR of the patient was 1.14. This value is low and consistent with the presence of homozygous factor VLeiden mutation. Genetic analyses5 as well as plasma assays30,31 revealed that the patient has normal circulating factor V and has no mutation at and around the Arg506, Arg306, or Arg679 APC-cleavage sites. Further, the patient's factor V molecule has normal procoagulant activity and both PT and aPTT were within the normal ranges (Table 1). Total and free protein S antigen levels, protein C antigen level, and chromogenic activity were normal (Table 1). However, protein S and protein C anticoagulant activities appeared to be spuriously low in the aPTT- or PT-derived assays. After reconstitution of protein C- and protein S-deficient plasma with protein C and protein S isolated from patient's plasma, normal levels of anticoagulant activity were found. These data demonstrate that all known coagulation proteins involved in the APC pathway (ie, protein C, protein S, or factor V) isolated from the patient's plasma have normal activity when purified from the patient's plasma and tested in vitro with purified reagents. The severe APC resistance observed was most likely due to an inhibitor present in the plasma of the patient that could interfere with factor V inactivation by APC. No lupus anticoagulant or APAs have been detected in patient's plasma.Effect of normal plasma on the patient's APC-SR To ascertain if the patient's plasma contains an inhibitor that is able to disrupt the APC pathway in normal plasma, a mixing experiment was performed. The patient's plasma was mixed with increasing concentrations of pooled normal plasma (PNP) and tested for the APC-SR. Figure 1A demonstrates that the patient's plasma contains an inhibitor that impairs APC anticoagulant activity in normal plasma. The inhibitory activity of the patient's plasma appears to be very strong because the APC-SR of the mixtures remains the same (1.2-1.4) even at proportions 80:20 normal/patient plasma. These data demonstrate that the patient's plasma contains an inhibitor that is able to inhibit the anticoagulant pathway in normal plasma.
Properties of the isolated inhibitor To ascertain the nature of the inhibitor, patient's plasma was loaded onto a protein G-Sepharose column. Before the protein G-Sepharose column, the APC-SR in patient's plasma was 1.11, whereas the patient's plasma that flowed through the column had a normal APC-SR value of 3.45. Control experiments demonstrated that similar treatment of normal plasma had no influence on the APC-SR values (before protein G-Sepharose, the APC-SR of normal plasma was 3.45, whereas after the passage through the column the value was 3.1). Overall these data demonstrate that the patient's plasma contains an immunoglobulin molecule that is responsible for the inhibition of the anticoagulant pathway and the resulting prothrombotic effect.To test the hypothesis that the inhibitory antibody was directed
against one of the proteins involved in the anticoagulant pathway (ie,
factor V or APC or both) the specificity of the inhibitor was tested in
a Western blot and was also assayed for its effect in normal plasma.
Figure 2 demonstrates that the antibody
only recognizes factor V under nonreducing conditions. The isolated immunoglobulin fraction from the patient's plasma did not recognize factor V under reducing conditions nor factor Va under either reducing
or nonreducing conditions on a Western blot. Also, the antibody
fraction from the patient's plasma did not recognize APC. These data
demonstrate that the antifactor V antibody present in the patient's
plasma most likely recognizes conformational epitopes on the
procofactor.
The specificity of the inhibitor was further investigated by supplementing factor V-deficient plasma with PNP and purified immunoglobulin from either normal plasma (Figure 1B, filled diamonds) or from patient's plasma (Figure 1B, filled triangles). For comparison the factor V-deficient plasma was also supplemented with either normal plasma (Figure 1B, filled squares) or with patient's plasma (Figure 1B, filled circles). No significant differences were observed between the APC-SR of the 4 mixtures at very low concentrations of factor V. However, the APC-SR of normal plasma supplemented with the patient's immunoglobulin fraction (Figure 1B, filled triangles) or the APC-SR in the patient's plasma (Figure 1B, filled circles) was consistently lower than that of normal plasma. Assuming a concentration of 20 nM factor V in normal plasma, the inhibition becomes apparent at about 20% factor V, which corresponds to approximately 4 nM. In fact, addition of patient's immunoglobulin to PNP is able to induce the same effect on the APC-SR as the effect observed with the patient's plasma (Figure 1B, filled triangles). Within physiologic levels of factor V, the inhibitory effect seems to be proportional to factor V concentrations. These data demonstrate that an increase in the concentration of factor V accentuates the inhibitory effect of the reconstituted plasma. The data strongly suggest that the patient's plasma contains an antifactor V antibody (immunoglobulin molecule) that strongly interferes with the ability of the molecule to be inactivated by APC. To determine the location of the conformational epitope(s) recognized
by the inhibitory antibody, an immunoprecipitation method was
developed. Because the antibody recognizes conformational epitopes on
factor V, experiments also need to be performed in the presence of EDTA
(following the addition of
Isolation of the inhibitor by affinity chromatography To study the effect of the inhibitor on factor Va function, a monospecific polyclonal antibody is necessary because the inhibitory antifactor V population is most likely a minor portion of the total immunoglobulin fraction isolated by protein G-Sepharose. Thus, the inhibitor was isolated by sequential affinity chromatography using protein G-Sepharose and factor V-Sepharose. The inhibitory fraction contained within the material eluted from the protein G-Sepharose was subsequently subjected to affinity chromatography on factor V-Sepharose. The flow through of the latter column contained the majority of the patient's immunoglobulins. Following washing of the column with 2 M NaCl, elution was performed with either 3 M NaSCN or with a low pH buffer (pH 2.8).Samples obtained following elution of the factor V column were dialyzed and analyzed by SDS-PAGE. The sample had the electrophoretic mobility of an IgG molecule (Mr 150 000) nonreduced (not shown). However, close examination of the gel revealed a doublet of Mr 150 000. Following transfer to a PVDF membrane and staining with an antihuman IgG (whole molecule), both bands of the doublet were visible. Further, on reduction 3 bands of Mr about 100 000 (HC1), 50 000 (HC2), and 25 000 (LC) were detected. No band of Mr 150 000 remained following complete reduction, suggesting 2 populations of immunoglobulin molecules. An antihuman IgG molecule identified all 3 bands. It is noteworthy that although the bands of Mr 50 000 and Mr 25 000 were readily visible on short exposures of the PVDF membrane to a film, the band of Mr 100 000 was only observed following prolonged exposure of the PVDF membrane to the film. In addition, NH2-terminal amino sequence of the bands of Mr 50 000 and 25 000 fragments revealed complete identity with the heavy and light chains of an IgG molecule (not shown). NH2-terminal amino acid sequence of the third band of Mr 100 000 (HC1) was unsuccessful most likely because of a blocked NH2-terminal amino acid. However, the fact that an antihuman IgG molecule recognizes this band is indicative of a heavy chain of an abnormal immunoglobulin molecule. It is also possible that the Mr 100 000 band is a cross-reactive contaminating band in our preparation. Overall the data demonstrate that the patient's plasma contains 2 populations of antibodies (IgG) eluted from the factor V-Sepharose column, that are directed against factor V. One of these 2 antibodies has a Mr 100 000 heavy chain and a Mr 25 000 light chain, whereas the other antibody is a typical IgG molecule. Analysis of the effect of the immunoglobulin fraction on the anticoagulant pathway involving factor V/Va and APC The anticoagulant pathway involving factor V and APC relates to 2 different mechanisms. The first is the direct inactivation of the membrane-bound cofactor by APC. The second is more indirect and involves the cofactor effect of factor V for the inactivation of factor VIII by the APC/protein S complex.To ascertain the inhibitory potential of the purified monospecific
antifactor V antibodies, the inhibition of the APC-mediated inactivation of membrane-bound factor Va was studied in a system using
purified components. Factor Va activity was monitored with a clotting
assay using factor V-deficient plasma. The results of the clotting
assay are shown in Figure 4A. Under the
conditions used, in the absence of the inhibitor, membrane-bound factor
Va loses approximately 60% of its activity in 1 minute and more than 90% of cofactor activity is eliminated within the first 5 minutes of
incubation with APC (Figure 4A, filled squares). The disappearance of
cofactor activity coincides with cleavage of the heavy chain (a) at
Arg506 and Arg306 and generation of fragments of Mr 75 000
(b) and 30 000 (c) (Figure 4B). At the end of the APC time course
(Figure 4A, 30 minutes) no clotting activity remains. Incubation of
factor Va with equimolar concentrations of the inhibitory molecule, prior to the addition of APC, results in a membrane-bound factor Va
molecule that loses approximately 10% of its activity within 1 minute
(Figure 4A, filled circles), whereas approximately 60% of its clotting
activity remains following a 30-minute time period (Figure 4A, filled
circles). The resulting activity profile demonstrates a continuous,
slow decrease in activity, which persists following extensive
incubation of membrane-bound factor Va with APC in the presence of the
inhibitor (not shown). Gel electrophoresis analyses of samples taken
during the time course of the inactivation of factor Va by APC in the
presence of the inhibitor (Figure 4C) revealed slow cleavage of factor
Va at both Arg306 and Arg506 (Figure 4C, lanes 4 and 5). The increase
in density of the Mr 75 000 and 30 000 fragments is
visible in lanes 4 and 5 (Figure 4C). There is also a slight increase
in the concentration of a Mr 60 000 fragment (representing
amino acid residues 307-679/709 of the heavy chain of the cofactor,
Figure 4C, open arrowhead). Cleavage of the cofactor at Arg506 is
membrane independent, whereas cleavage at Arg306 requires the presence
of a membrane surface.2,3 Because both cleavages are
delayed in the presence of the inhibitor, the data demonstrate that the
inhibitor impairs efficient factor Va-APC interaction on the membrane
surface and as a consequence proper inactivation of the cofactor. It is
also possible that following binding to the inhibitor the conformation
of the cofactor is altered, making the APC-cleavage sites of the heavy
less accessible for APC. Because APC and factor Xa compete for the
binding to factor Va,32 it is possible that the inhibitor
may interfere with prothrombinase assembly. It is noteworthy that
normal PT and aPTT values were observed in the patient (Table 1). To
verify the effect of the antibody on the interaction of the cofactor with factor Xa, the purified immunoglobulin fraction was tested for
direct inhibition of prothrombinase complex assembly and function. Under the conditions used (10 nM factor Xa, 4 nM factor Va, 8 nM IgG,
and 20 µM PCPS) no inhibition of prothrombinase assembly and function
was observed.
The effect of the antibody on the cofactor activity of factor V for the
APC-mediated inactivation of factor VIII was next analyzed (Figure
5). Factor V was found to enhance the
inactivation rate of factor VIII by the APC/protein S complex by 2-fold
as previously described27,33 (Figure 5, filled triangles)
when compared to the inactivation of factor VIII in the absence of factor V (Figure 5, filled circles). The acceleration of the
inactivation of factor VIII was inhibited by the immunoglobulin
fraction eluted from the factor V-Sepharose column (Figure 5, filled
diamonds). In contrast, under similar experimental conditions, a
nonimmune human IgG commercial preparation had no inhibitory effect on
the acceleration of factor VIII inactivation by the APC/protein
S/factor V complex (Figure 5, filled inverted triangles). These data
suggest another prothrombotic mechanism of the immunoglobulin fraction isolated from patient's plasma that could explain the APC resistance. Overall, the functional data indicate that the presence of the inhibitor in the patient's plasma is displayed in vivo by severe thrombotic manifestations.
We have isolated and characterized an antifactor V antibody from plasma of a patient with recurrent thrombotic manifestations. Our data clearly demonstrate the presence of an antifactor V antibody that impairs proper inactivation of the molecule by APC. The antibody also interferes with the cofactor activity of factor V for the inactivation of factor VIII by the APC-protein S complex. Both, impaired inactivation of the cofactor by APC, as well as impaired inactivation of factor VIII, are most likely responsible for the strong APC resistance and the severe thrombotic manifestations observed in the patient plasma. To our knowledge, this the first time that an autoantibody that inhibits the factor V cofactor activity for the inactivation of factor VIII is reported, providing a physiologic relevance for this antithrombotic effect of factor V.27,33 This antibody appears to have occurred spontaneously because no diseases were identified during the follow-up of this patient nor were common causes for the development of antifactor V inhibitor such as exposure to plasma derivatives, antibiotics, or bovine thrombin preparation present. This antibody is not related to APAs because lupus anticoagulant and all other APAs have tested negative during 10 years of follow-up. The majority of the antifactor V antibodies described thus far in the literature have been found to produce a bleeding syndrome in the affected individuals.12 Only 3 cases of patients with antibodies to factor V have been associated with thrombotic events. However, 2 of the 3 cases had an autoimmune syndrome where lupuslike anticoagulant activity was demonstrated in association with anticardiolipin antibodies,13,14 whereas the antifactor V antibody in the third case was associated with Sjögren syndrome.15 It is interesting to note that in all 3 cases prolongation of the basal clotting time was present, suggesting an interference with the procoagulant function of factor V. It has been demonstrated in one case (Sjögren syndrome15) that although the antibody in vitro did not inhibit activated protein C activity toward factor Va, it was able to inhibit the procoagulant function of factor V. It is well established that the binding to a membrane surface is necessary for both normal procoagulant expression of factor Va cofactor activity as well as efficient down-regulation of its activity by APC.3 Thus, the latter 3 antibodies most likely inhibited the interaction of the cofactor with the membrane surface, resulting in both prolongation of clotting time and impaired APC inactivation. Two recent studies using recombinant factor V and factor V C2 domain have demonstrated that the majority of factor V inhibitors are antibodies directed to the C1-C2 domain of factor V and interfere with its binding to the PS-containing membranes.34,35 To our knowledge, this is the first report of an antifactor V antibody that directly inhibits the factor V-APC interaction inducing resistance of the cofactor to APC inactivation, without affecting the proper interaction of factor Va with the membrane surface. It is still unknown how and when the patient described in our report first developed antifactor V antibodies. Antifactor V antibodies are known to develop in individuals secondary to blood transfusions, antibiotic exposure, or autoimmune disorders. However, antibodies to factor V have frequently been found in individuals exposed to topical thrombin36,37 or to bovine proteins (bovine thrombin or fibrin sealant) during major surgery.38-40 The isolated antifactor V antibody found in our patient has some peculiarity. It is not able to induce prolongation of the clotting time (no neutralizing effect), and because the concentration of factor V found in the patient's plasma was normal, it does not appear to interfere with the clearance of factor V. These characteristics make the antibody presented herein different from other antifactor V antibodies, which are often associated with bleeding manifestations and are directed against the C2 domain of the cofactor.34,35 Our experimental data strongly suggest that the antibody recognizes a conformational epitope on the factor V molecule. This concept is not new for neutralizing antibodies. Conformational epitopes were defined in autoantibodies to recoverin in cancer-associated retinopathy,41 in Graves disease where autoantibodies have been associated with a prionlike shift of a thyrotropin receptor A-subunit molecule,42,43 and in human autoimmune thyroiditis.44,45 Conformational epitopes only recognizing factor Va and not factor V were also developed in mice.46-48 Interestingly, the latter epitopes were only expressed on thrombin activation of the cofactor.48 The factor Va-APC interaction involves both chains of factor Va, whereas the B region of the procofactor alone appears to be involved in its anticoagulant effect.49 Our data suggest that the inhibitor developed in this patient is polyclonal with multiple epitopes all over the factor V molecule. The patient's plasma does not appear to have any other procoagulant defects. The presence of antifactor V antibodies with a selective effect on the inhibition of factor V interaction with APC represents a new mechanism responsible for APC resistance and possibly a new, though rare, acquired cause of thrombotic manifestations.
We wish to thank Dr Ken Mann from the University of Vermont for the monoclonal antibodies to human factor V and Dr Lisa Regan from Bayer Corporation for the recombinant factor VIII. We wish to thank Dr Alex Kurosky and Steve Smith from the University of Texas Medical Branch at Galveston, for amino acid sequencing, and Michael Ungham and Robert Dura for technical assistance.
Submitted August 31, 2001; accepted January 24, 2002.
Supported by start-up funds from the Department of Chemistry, Cleveland State University (M.K.), by a grant from the Department of Medical and Surgical Sciences, University of Padua (P.S.), and by grant HL34575 from the National Institutes of Health. Portions of this work were presented in abstract form at the XVIIth Congress of the International Society on Thrombosis and Hemostasis, Washington, DC, August 14-21 1999.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael Kalafatis, Chemistry Department, Cleveland State University, Science Building, 2351 Euclid Ave, Cleveland, OH 44115; e-mail: m.kalafatis{at}csuohio.edu.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
80°C
until use.
) at 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 30 minutes following
the addition of APC; (
) factor Va was incubated with the purified
immunoglobulin inhibitor (64 nM) and the inactivation by APC was
performed under similar experimental conditions as described for normal
factor Va. (B) Some of the samples assayed for cofactor activity in
panel A were also analyzed on a 5% to 15% linear gradient SDS-PAGE as
described in "Patient, materials, and methods." Lane 1, factor Va
control, no APC; lanes 2 through 5, factor Va incubated with APC at 5, 10, 20, and 30 minutes, respectively. (C) Prior to the addition of APC
and PCPS vesicles, factor Va was incubated with the purified
immunoglobulin solution from the patient's plasma. The arrows indicate
the heavy chain of factor Va (a), the Mr 75 000
intermediate (b), that results from cleavage at Arg506 of the heavy
chain, and the Mr 30 000 fragment (c) that derives from
cleavage of the Mr 75 000 intermediate at Arg306.
The open arrowhead indicates cleavage at Arg306 first and generation of
a fragment containing the region 307-6791709 of the heavy chain of
the cofactor.
