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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 909-917
Autoimmune Antibody in a Hemorrhagic Patient Interacts With
Thrombin-Activated Factor XIII in a Unique Manner
By
Laszlo Lorand,
Pauline T. Velasco,
S.N. Prasanna Murthy,
Phil Lefebvre, and
David Green
From the Department of Cell and Molecular Biology and the Feinberg
Cardiovascular Research Institute, and the Department of Medicine,
Northwestern University Medical School, Chicago, IL.
 |
ABSTRACT |
Without a prior history of hemorrhagic disease, a 62-year-old man
suffered recurrent episodes of bleeding. Solubility of the patient's
clot in 5 mol/L urea indicated a problem with fibrin stabilization. The
transamidase activity potential of factor XIII, measured by the
incorporation of radioactive putrescine into N,N-dimethylcasein as test
substrate, was 62% of control, close to the normal range of values.
Examination of the patient's clot from recalcified plasma by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis showed that
essentially none of the  chains and only about two thirds of the  chains of fibrin became cross-linked under conditions where both were
fully cross-linked in the controls. An antibody to factor XIII was
isolated which, although recognizing the recombinant rA2
subunits, as well as the virgin A2B2 plasma
ensemble, showed a 100-fold greater affinity for the thrombin-activated
rA2' and A2'B2 forms of
the zymogen, suggesting that the latter would be its main target during
coagulation. Furthermore, the patient's IgG has an ability, never seen
before, for inducing an enzymatically active configuration in the
thrombin-activated zymogen in the absence of Ca2+.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE TERM FIBRIN stabilization
describes the transition of a urea-soluble fibrin clot into a
urea-resistant structure.1-4 The former is produced in
mixtures of thrombin with purified fibrinogen or plasma anticoagulated
by a Ca2+-chelator:
[View Larger Version of this Image (5K GIF file)]
Transition to a urea-insoluble clot requires the presence
of fibrin stabilizing factor (factor XIII), the precursor of the transamidating enzyme (XIIIa) responsible for the cross-linking of
fibrin molecules so that they can no longer be dispersed in 5 mol/L
urea. Activation of the factor XIII zymogen
(A2B2) requires both thrombin and
Ca2+:5-7
[View Larger Version of this Image (5K GIF file)]
The dissociated A2* species is the factor XIIIa enzyme
involved in fibrin stabilization or cross-linking
(xl):
[View Larger Version of this Image (5K GIF file)]
Factor XIIIa reacts in an ordered sequence with fibrin, at a rapid
rate with sites in the chains, and more slowly with those in the
 chains8; the  chains of fibrin do not contribute directly to cross-linking.9
Schemes 1 through 3 represent only the mere outlines of a
complex biological system and do not show the unique orchestration of
regulatory controls6,7,10,11 necessary to ensure clot stabilization within the normally allowed time frame after injury.
Cross-linking of both the and the chains of fibrin are
needed for generating a structure with the firmness required for a
hemostatic plug. Reaction of factor XIIIa with chains causes a
linear end-to-end ligation of fibrin molecules,12-14
whereas chain cross-linking is thought to stiffen the fibrils
cross-wise. Although few in number, generating 4 to 6 mol of
N (-glutamyl)lysine bridges per mole of fibrin causes
a five-fold increase in the viscoelastic modulus (G') of the
clot.15 A urea-soluble thrombus is also more susceptible to
plasmin digestion than its urea-insoluble counterpart.16
The enzymatic cross-linking of 2 plasmin inhibitor to
the chains of some fibrin molecules is thought to provide
protection against lysis.17,18
The search for disorders of fibrin stabilization represents an
interesting chapter in the study of blood coagulation because testing
modalities for identifying and classifying various defects in the
operation of the system were developed well before its relevance to
bleeding conditions became evident. These potentially fatal hemorrhagic
diseases arise if no factor XIIIa activity is generated on the pathway
of coagulation or if the enzyme is unable to cross-link the clot
network within the physiologically permissible time frame.
Characteristically, clotting and bleeding times of the patients are in
the normal range, attesting that immediate hemostasis, which is a
measure of the rate of aggregation of fibrin molecules: n
fibrin  (fibrin)n, proceeds at a regular rate.
Delayed bleeding (oozing) occurs because the hemostatic plug fails on
account of its mechanical weakness and easy lysability. The disorders
are usually diagnosed by the solubility of the patient's clot in 5 mol/L urea,1,2 or in 1% monochloroacetic
acid,19 solvents in which a normal plasma clot cannot be
dispersed. However, a positive finding with this test covers a variety
of molecular disorders of different etiologies.
Absence of a functional FXIII zymogen in the plasma is responsible for
most of these diseases. The autosomal recessive condition occurs with
an estimated frequency of 1 in 5 million, with about 300 known cases
worldwide in diverse racial and ethnic groups.20-22 On
account of transcriptional or folding problems, most are due to the
lack of synthesis of A subunits, but there are cases also with the
absence of the carrier B subunits without which the A subunits cannot
survive in the circulation.23-25
The present case belongs to a different class of the disorders of
fibrin stabilization in which an acquired inhibitor interferes with one
or more of the biochemical reactions involved in the physiological
pathway pertaining either to the activation of the factor XIII zymogen
(scheme 2) or to fibrin stabilization (scheme 3). Acquired inhibitors,
mostly autoimmune antibodies,26 may arise against any of
the molecular species shown in schemes 2 and 3. According to their
modes of action, three major types can be distinguished: the type I
inhibitor prevents activation of the A2B2,
factor XIII ensemble to A2*; the type II inhibitor
interferes with the transamidating function of the activated
A2*; and the type III inhibitor is directed against the
fibrin substrate itself, blocking the reactivities of the cross-linking
sites for access to the A2*, factor XIIIa enzyme. This
report describes a patient who came to our attention after suffering a
massive hematoma at age 62. Laboratory tests showed the presence of a
circulating antibody with partial characteristics of the type I and II
inhibitors, but with a target specificity not seen in previous
literature reports.26 An abstract of this work was
published earlier.27
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CASE HISTORY |
This man, born in 1927, had a coronary angiogram to evaluate angina in
1989. Excessive hemorrhage occurred from the femoral artery puncture,
necessitating suturing of the vessel. In April 1991 he developed low
back pain. After spinal manipulation, a huge ecchymosis covered his
back and flanks, and hematologic evaluation was advised. He was noted
to be anemic and was treated with transfusions of packed red blood
cells and epsilon aminocaproic acid, and the hematoma subsided. Three
months later another hematoma appeared on his left forearm; this time
there was no history of trauma. Hematuria also occurred, but cystoscopy
showed no obvious cause.
Past medical history showed several prior surgical procedures including
appendectomy, thyroidectomy, and coronary artery bypass grafting at age
53; none of these was associated with excessive bleeding. His
medications included thyroid, propranolol, lasix, potassium, and
atarax. Aspirin had been discontinued after development of the back
bleeding. There was no family history of a hemorrhagic disorder.
On physical examination, a subconjunctival hemorrhage was noted in the
right eye, and large areas of skin discoloration demarcating the
previous hematomas were observed on the back and forearm. There were a
few fading ecchymoses and petechiae at sites of constriction from the
belt and hose. There was no lymphadenopathy or hepatosplenomegaly.
Extensive coagulation studies, including a bleeding time, partial
thromboplastin time, prothrombin time, thrombin time, fibrinogen, and
clotting factors VII, VIII, IX, XI, and XII, were all normal.
The patient was treated with prednisone in a dose of 20 mg daily for 4 weeks, and 20 mg every other day for an additional 4 weeks, and then
the drug was discontinued. However, in December, 1991 he experienced
persistent oozing from a lesion on his cheek, and subsequently
hematomas on the leg and forearm. Minor bleeding episodes continued
over the next 2 years. In March 1993, he presented with unstable
angina. A trial of oral cyclophosphamide, 150 mg daily, was given
before coronary angioplasty. Nevertheless, he developed a large
hematoma at the site of femoral artery catheter insertion, which slowly
resolved over a period of several weeks. In January, 1995 unstable
angina recurred; coronary angiography was again associated with a groin
hematoma. In March, 1995, coronary artery bypass grafting was
performed; hemostasis was secured with the use of a factor XIII
concentrate, as we have previously described.28 Since his
operation he has continued to have minor bleeding, most recently an
extensive ecchymosis of his right upper arm after a venepuncture in May 1997.
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MATERIALS AND METHODS |
Blood was collected into 3.8% citrate with a ratio of 9:1.
Platelet-poor plasma was prepared by centrifuging at 1,360g for 20 minutes. Serum was separated by centrifugation (1,360g for 20 minutes) from blood clotted in glass tubes at room temperature for 2 hours.
IgG was purified from serum on a ZetaChrom 60 D1 amine disk (AMF
Laboratory Products, Meriden, CT) as previously described29 or on a Protein A-Sepharose 4B column (Pharmacia, Piscataway, NJ). In
the latter procedure, normal or patient serum (4 mL) was diluted with 9 vol phosphate-buffered saline (PBS) (10 mmol/L sodium phosphate, 154 mmol/L NaCl, pH 7.4) and passed through the Protein A column (1 × 6.5 cm) at approximately 1 mL/min at room temperature. The
column was washed with a minimum of 200 mL of PBS, the IgG eluted with
0.1 mol/L glycine-HCl, pH 2.5, fractions with pH <5.5 were pooled
(approximately 4 mL) and neutralized to pH 7 to 7.4 with 1 N NaOH.
After dialysis against 2 × 4 L PBS, the IgG was concentrated
using a Centricon 30 microconcentrator (Amicon, Danvers, MA). Protein
concentration was determined by absorbance at 280 nm
(E1cm1% = 13.5).
Fibrinogen (plasminogen-free; American Diagnostica, Greenwich, CT) was
dissolved in 50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 150 mmol/L
NaCl, dialyzed at 4°C overnight against the same buffer, and stored
at  20°C. The protein concentration was determined by
absorbance at 280 nm (E1cm1% = 15.1). Human
-thrombin (a gift from J.W. Fenton III, New York State Department of
Health, Albany) was stored at 70°C in 0.75 mol/L NaCl.
Bovine thrombin (Parke-Davis, Ann Arbor, MI) was dissolved in 25 mmol/L
Tris-HCl, pH 7.5, 25% (vol/vol) glycerol to 500 U/mL and stored at
70°C. Thromstop (American Diagnostica) was dissolved in 50 mmol/L Tris-HCl, pH 7.5 to 0.2 mmol/L and stored at 20°C. Trasylol (10,000 U/mL; FBA Pharmaceuticals, West Haven, CT) was diluted
as needed into 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl.
N,N-dimethylcasein was purchased from Sigma, St Louis, MO or prepared from Hammersten casein (United States Biochemical Corp,
Cleveland, OH) using the procedure of Lin et al30; it was
dissolved in 50 mmol/L Tris-HCl, pH 7.5 and stored at 20°C.
Human factor XIII was purified from outdated human CPDA-1
plasma31 and stored at 4°C in 50 mmol/L Tris-HCl, pH
7.5, 1 mmol/L EDTA, 10 U/mL Trasylol. Protein concentration was
determined by absorbance at 280 nm (E1cm1% = 13.8). The B2 subunits of factor XIII were isolated from
human plasma according to Lorand et al.31 Protein
concentration32 is expressed in terms of a bovine serum
albumin standard (Pierce Chemical Co, Rockford, IL). The recombinant
factor XIII A subunit (rA2; a gift from Dr P. Bishop,
ZymoGenetics, Seattle, WA)33 was stored at 4°C in 75 mmol/L Tris-HCl, 1 mmol/L glycine, 0.5 mmol/L EDTA and 0.2% sucrose,
pH 7.5. Protein concentration was determined by absorbance at 280 nm
(E1cm1% = 14.9).
Functional assay for factor XIIIa generation.
The patient's plasma was tested for formation of the intrinsic
transamidase upon treatment with thrombin and Ca2+, and
enzyme activity was measured by the incorporation of
14C-putrescine into N,N-dimethylcasein using a filter paper
assay.34 Patient or normal plasma (18 µL) was mixed with
6 µL 50% (vol/vol) glycerol and 6 µL 20 mmol/L Gly-Pro-Arg-Pro (Oz
Chemicals, Jerusalem, Israel) and treated with bovine thrombin (5 µL,
250 U/mL) for 30 minutes at room temperature, followed by the addition
of 5 µL of 0.1 mmol/L Thromstop. Incubation was performed in a total volume of 75 µL which, in addition to the above components, comprised 50 mmol/L Tris-HCl, pH 7.5, 0.11 mmol/L 14C-putrescine (61 µCi/µmol; Amersham, Arlington Heights, IL), 5.3 mg/mL
N,N-dimethylcasein, 13 mmol/L dithiothreitol (DTT), and 10 mmol/L
CaCl2. The incorporation reaction was allowed to proceed for 30 minutes at 37°C and 10 µL aliquots were spotted on filter paper and processed for measuring protein-bound radioactivity. Enzyme
activity is given as 14C-putrescine (cpm) for the 10 µL
mixture for the 30-minute reaction. Activity measured in the absence of
thrombin was used as reference.
Fibrin chain cross-linking profiles of plasma clots.
Patient or normal plasma (100 µL) was mixed (37°C; total volume
of 500 µL) in 40 mmol/L Tris-HCl, pH 7.5, 80 mmol/L NaCl with 6 U of
bovine thrombin and 10 mmol/L CaCl2. Clots were wound onto glass rods as they formed, washed (1 × 3 mL and 1 × 1 mL of 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1 mmol/L EDTA), solubilized (200 µL of 50 mmol/L sodium phosphate, pH 7.1, 2% sodium dodecyl sulfate [SDS], 9 mol/L urea, 40 mmol/L DTT at 37°C for 60 minutes), and 15 to 45 µL aliquots were analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE).35 Gels were
stained with Coomassie brilliant blue R and scanned on an Ultrascan SL
densitometer (LKB, Bromma, Sweden).
Electrophoretic analysis of cross-linked fibrin polymers in
anticoagulated plasma after clotting with thrombin.
Normal or patient plasma (100 µL; with 1 mmol/L EDTA) was treated
with 2.5 U bovine thrombin for 30 minutes at 37°C in a total volume
of 500 µL, which also included 40 mmol/L Tris-HCl, pH 7.5, 80 mmol/L
NaCl, and 10 U/mL Trasylol. Clots were wound onto glass rods as they
formed, washed (2 × 3 mL of 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1 mmol/L EDTA), solubilized (200 µL of 10 mol/L urea,
2% SDS at 37°C for 60 minutes) and centrifuged (16,000g, 10 minutes). Electrophoresis was performed by applying 20 µL aliquots to 2% agarose gels.36 Other 10-µL aliquots of the
urea-SDS solubilized materials were treated with 40 mmol/L DTT at
37°C for 20 minutes, and electrophoresed on SDS-polyacrylamide as
described above.
Effect of patient's IgG on the enzymatic cross-linking of clots
from purified human fibrinogen.
Incubations (37°C, 2 hours) were performed in mixtures (500 µL)
comprising 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.5 mg/mL human
fibrinogen, factor XIII (2.5 µg/mL rA2 or 5 µg/mL A2B2), 2 mg/mL IgG (normal or patient, isolated
on a ZetaChrom 60 disk), 20 U/mL Trasylol, 12.5 U/mL bovine thrombin,
and either 2 mmol/L EDTA or 10 mmol/L CaCl2. Clots were
wound onto glass rods as they formed, washed, solubilized in 200 µL
as described above, and 50-µg aliquots were analyzed by
SDS-PAGE.9
The effect of the patient's IgG on the thrombin-activated
rA2 or rA2'.
Conversion of rA2 (25 µg/mL) to rA2'
was performed at room temperature for 20 minutes in 50 mmol/L Tris-HCl,
pH 7.5 with 1.25 U/mL of human -thrombin. Enzyme activity was
measured by a recently published procedure37 on a CytoFluor
Model 2300 fluorimeter (Millipore, Bedford, MA), upgraded to a model
2350. The rate of increase in fluorescence was monitored at 37°C
for the enzyme-catalyzed incorporation of dansylcadaverine
[N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide] into
N,N-dimethylcasein (excitation filter 360/40 nm; emission filter 490/40
nm; sensitivity 5). Measurements were performed in a 96-well
low-fluorescence CytoPlate (PerSeptive Biosystems, Framingham, MA) in
125-µL mixtures, which comprised 50 mmol/L Tris-HCl, pH 7.5, 2 mg/mL
N,N-dimethylcasein, 0.5 mmol/L dansylcadaverine, 0.25 µg
rA2', and 0 to 40 µg of normal or patient IgG, in
the presence or absence of 1 mmol/L CaCl2. In measuring
enzyme activities, corrections were applied with regard to the
nonenzymatic controls.
Cross-linking of the recombinant C30 fragment of fibrinogen by
rA2' without Ca2+ in the presence of the
patient's IgG.
A recombinant 30-kD fragment of -chain of fibrinogen (a
gift from Drs H.C.F. Côté and Earl W. Davie, University of
Washington, Seattle)38 was incubated with
rA2' and EDTA at 37°C for 4 hours. The 25-µL
mixture contained 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, C30
(160 µg/mL; 5.3 µmol/L), 0 to 3 mg/mL normal or patient IgG, rA2' (10 µg/mL; 0.625 µmol/L; quenched by
hirudin after its activation by thrombin), and 1 mmol/L EDTA or 5 mmol/L CaCl2. After the reaction, the samples were analyzed
by SDS-PAGE under nonreduced conditions on 10% acrylamide and the
Coomassie brilliant blue R stained gels were scanned.
Immunoblots (dot blot assay) with the patient's IgG.
One-microliter aliquots of 0.2 mg/mL A2B2, 0.1 mg/mL rA2, 0.2 mg/mL A2'B2
(treated with 30 U/mL human -thrombin at room temperature for 20 minutes), 0.1 mg/mL rA2', 0.1 mg/mL B2,
and 0.2 mg/mL fibrinogen were spotted on nitrocellulose (0.2 µm pore
size; Schleicher & Schuell, Keene, NH). Unbound areas were blocked with
1% Blotto (1% nonfat dry milk [Carnation, Los Angeles,
CA] in PBS) for 20 minutes. The strips were incubated
with patient IgG (0.05 mg/mL in 1% Blotto) or with normal IgG as
control for 60 minutes at room temperature and washed (three times with
PBS). Binding of IgG was identified with a Vectastain ABC kit specific
to human IgG (Vector Laboratories, Burlingame, CA). The biotinylated
secondary antibody was diluted 1:4,000 in 1% Blotto, the strips
incubated for 1 hour, and washed (three times with PBS). After
formation of avidin-biotinylated peroxidase complex (dilution 1:4,000
in 1% Blotto for 30 minutes), the strips were incubated for 1 hour and
washed (three times with PBS). The peroxidase label was developed with
a solution containing 10 mL of 4-chloro-1-naphthol (3 mg/mL in ice-cold
methanol; Sigma), 50 mL PBS and 30 µL of 30% hydrogen peroxide.
Recognition of factor XIII components by the patient's antibody in
solution.
Enzyme-linked immunosorbent assay (ELISA) studies were performed in
96-well Microtest III assay plates (Falcon 3910) at room temperature.
The A2B2 antigen was diluted to 4 µg/mL in 50 mmol/L Tris-HCl, pH 7.5 and 100 µL/well allowed to bind to the plate for 2 hours at room temperature, followed by blocking with 2% Blotto
(2% nonfat dry milk in PBS; 3 × 250 µL). Patient IgG (10 µg
in 2% Blotto) was mixed with varying amounts of factor XIII (A2B2 or rA2) or thrombin-treated
factor XIII (A2'B2 or
rA2') for 30 minutes at room temperature, and the
mixtures were then added to the wells. After overnight incubation at
room temperature, the wells were washed (three times with 2% Blotto),
reincubated for 2 hours with an alkaline phosphatase-conjugated
antibody to human IgG (-chain specific, Sigma; 100 µL/well of a
1:5,000 dilution in 2% Blotto) and washed again (three times with 2%
Blotto; two times with 100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 5 mmol/L MgCl2), and 100 µL of 1 mg/mL p-nitrophenyl
phosphate disodium (Sigma) in the final wash buffer added to each well.
Color was read in a Dynatech MR600 (Dynatech Laboratories, Alexandria,
VA) microplate reader at 410 nm.
Changes in the titer of the antibody in the patient's serum during
the course of disease.
A2'B2 (4 µg/mL in 50 mmol/L Tris-HCl,
pH 7.5; 100 µL/well) was bound to ELISA plates at room temperature
for 2 hours and blocked with 2% bovine serum albumin (BSA) in PBS. The
patient's serum (0.25 µL in 100 µL of the BSA buffer), or normal
serum as control, was added to the wells and incubated overnight at
room temperature. This was followed by washing with BSA and
reincubation for 2 hours with an alkaline phosphatase-conjugated
antibody to human IgG (-chain specific; 100 µL/well of a 1:5,000
dilution in BSA). The plates were washed (three times with BSA; two
times with 100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 5 mmol/L
MgCl2) and 100 µL of 1 mg/mL p-nitrophenyl phosphate
disodium in the final wash buffer was added to each well. Color was
read in the microplate reader as above.
| |
RESULTS |
Malfunctioning of the fibrin stabilizing system in the patient's
plasma.
Initial laboratory findings showed that the recalcified plasma clot of
the patient could be readily dissolved in 5 mol/L urea.1-4 Inasmuch as this indicated a problem with fibrin stabilization, the
susceptibility of the patient's clot to fibrinolysis was also tested.
The patient's plasma and a normal control plasma sample were spiked
with I125-fibrinogen, mixed with 20 ng/mL r-tPA, and
clotted with 0.5 U/mL thrombin and Ca2+ for 2 hours at room
temperature. The samples were then incubated at 37°C and assayed in
duplicate at 30, 60, 90, and 120 minutes. The time to reach 50% lysis
(LR50) for the patient was 27 minutes, versus >120
minutes for the control. This experiment was repeated with the
patient's purified IgG (5 mg/mL), mixed with 30% normal plasma. A
mixture of normal IgG and normal plasma was used as a control. Samples
were treated as above. The LR50 for the patient IgG was 22 minutes, compared with 63 minutes for the control.
The catalytic potential of the factor XIII zymogen in the patient's
plasma was evaluated for transamidation after its conversion to XIIIa
with thrombin plus Ca2+, in the
14C-putrescine:N,N-dimethylcasein substrate
system.34,39 Results from these amine incorporation
experiments (Table 1) excluded the
possibility of a hereditary factor XIII deficiency.
Analysis of the cross-linking profile of the patient's clot by
SDS-electrophoresis in polyacrylamide after reduction with DTT
(Fig 1A) or in agarose without treatment by
DTT (Fig 1B), showed an abnormal feature never seen before in this
family of diseases. A significant proportion (measured in the gel scan
as 61%) of the monomeric chains of fibrin in the patient's clot were found to become cross-linked to dimeric - structures when clotting was induced only by the addition of thrombin alone without Ca2+ (ie, in the presence of EDTA; lane 3, Fig 1A).
Normally, such cross-linked - dimers in plasma can be generated
only with a combination of thrombin plus Ca2+, which also
brings about the formation of high molecular weight, cross-linked
n polymers (lane 2, Fig 1A). By contrast, even the combination of thrombin and Ca2+ proved to be rather
ineffective in promoting the cross-linking of chains in the
patient's clot (lane 4, Fig 1A); moreover, with the added presence of
Ca2+, there was a rather insignificant increase in the
amount of cross-linked - dimers (from 61% to 67%) or in the chains remaining as monomers (compare lanes 3 and 4, Fig 1A).
Altogether, a comparison of the normal electrophoretic profile with
that of the patient's clot (lanes 2 and 4, Fig 1A) showed that, even
under the forced conditions for clotting used in these experiments,
cross-linking of the patient's fibrin was greatly inhibited.
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| Fig 1.
Abnormal fibrin cross-linking profiles in clots obtained
from the patient's plasma in the presence and absence of
Ca2+. (A) Normal (lanes 1 and 2) or patient (drawn
7/31/91; lanes 3 and 4) plasma (100 µL) was clotted in the presence
of either 1 mmol/L EDTA (lanes 1 and 3) or 10 mmol/L CaCl2
(lanes 2 and 4) with 6 U bovine thrombin for 30 minutes at 37°C in
a total volume of 500 µL, which also included 40 mmol/L Tris-HCl, pH
7.5, 80 mmol/L NaCl, and 10 U/mL Trasylol. The washed clots were
reduced with DTT and analyzed by SDS-PAGE. (B) Normal (lanes 1) or
patient (drawn 9/12/91; lanes 2) plasma (100 µL) was clotted in the
presence of 1 mmol/L EDTA with 2.5 U bovine thrombin for 30 minutes at
37°C in a total volume of 500 µL, which also included 40 mmol/L
Tris-HCl, pH 7.5, 80 mmol/L NaCl, and 10 U/mL Trasylol. The washed
clots were dissolved in urea-SDS for electrophoresis on 2% agarose
(lanes 1b and 2b) and also, after treatment with DTT, by SDS-PAGE
(lanes 1a and 2a). Normal controls were collected from two different
donors for the experiments shown in (A) and (B).
|
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Inasmuch as the fibrin molecule is a disulfide-linked, symmetrically
duplex structure of three constituent chains []2
connected to each other by disulfide bonds, SDS-electrophoresis after
reduction with DTT does not provide any information about the true
nature and the extent of the end-to-end ligation with factor XIIIa. The pattern in lane 3, Fig 1A only shows that clotting with thrombin in the
absence of Ca2+ could generate sufficient enzyme activity
in the patient's plasma to bring about the cross-linking of
approximately 61% of the chains. This observation, however, sheds
no light on the size of the covalently fused fibrin units, and the
observation would be just as compatible with the formation of 61% of
2x()2 dimerically cross-linked fibrin molecules as
with the cross-linking of a lower percentage of the fibrin into
covalently linked higher polymeric structures. To examine this issue,
an SDS-agarose electrophoretic procedure was used without prior
treatment of the samples by DTT.36 Lane 1b in Fig 1B shows
that the clot, produced by the addition of thrombin to normal citrated
plasma (containing also 1 mmol/L EDTA), could be readily dissociated to
monomeric fibrin units (marked F1), mixed in with only
negligible amounts of lower forms (<F5) of cross-linked
oligomers. In sharp contrast to this, the patient's plasma, clotted
under the same conditions with thrombin, yielded an extensive array of
covalently fused fibrin molecules (F3, F4,
F5 ... Fn), some of which correspond to
structures of molecular mass much higher than 3 × 106
(lane 2b, panel B). Inasmuch as all of these polymers were generated by
chain ligation, we may conclude that they represent linearly assembled, end-to-end ligated fibrin filaments.14
The clotting abnormality is due to a unique autoimmune antibody
present also in the patient's serum.
It was possible to reproduce both features of the anomaly observed in
Fig 1 with the patient's plasma by clotting normal plasma or
fibrinogen in the presence of the patient's serum or purified IgG
fraction. The findings illustrated by Fig 2
indicate that a circulating antibody, present in apparent excess in the
patient's serum, acts (1) as a strong inhibitor of cross-linking of
chains in a complete clotting mixture composed of fibrinogen,
recombinant rA2 zymogen, Ca2+, and thrombin
(compare lane 6 with lane 4 in Fig 2A), and (2) that the interaction of
the antibody with the factor XIII zymogen can somehow induce the
generation of enzyme activity even in the absence of Ca2+
to give rise to substantial amounts of cross-linked - dimers (lane 5 in Fig 2A and lane 6 in Fig 2B). Rheological measurements (not
shown; kindly performed by Dr E. Ang, Northwestern
University) showed that the viscoelastic storage modulus
(G' at 120 minutes), an index of clot stiffness, was about 90 Pa
with inclusion of normal IgG (such as in lane 4, Fig 2A), whereas that
of the clot with the patient's IgG (as in lane 6, Fig 2A) was only
about half as much (50 Pa).
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| Fig 2.
Effect of the patient's IgG on the cross-linking of
clots obtained in mixtures with purified fibrinogen. (A) Incubations
were performed at 37°C for 2 hours in 0.5 mL mixtures containing 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.5 mg/mL human fibrinogen,
2.5 µg/mL rA2, 20 U/mL Trasylol, either no IgG (lanes 1 and 2) or 2 mg/mL normal IgG (lanes 3 and 4) or patient IgG (purified
on a ZetaChrom 60 disk from serum drawn on 7/31/91; lanes 5 and 6),
either 2 mmol/L EDTA (lanes 1, 3, and 5) or 10 mmol/L CaCl2
(lanes 2, 4, and 6), and 12.5 U/mL bovine thrombin. Washed clots were
analyzed by SDS-PAGE after reduction with DTT. (B) Incubations were
performed at 37°C for 2 hours in 0.5 mL mixtures containing 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.5 mg/mL human fibrinogen,
20 U/mL Trasylol, 2 mmol/L EDTA, either no IgG (lanes 1 and 2) or 2 mg/mL normal IgG (lanes 3 and 4) or patient IgG (lanes 5 and 6), in the
absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 5 µg/mL human factor XIII (A2B2), and 12.5 U/mL
bovine thrombin. Clots were processed and analyzed as in (A).
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Additional experiments were conducted to confirm the unique attribute
of the antibody for activating the thrombin-modified rA2' zymogen in the absence of Ca2+.
Figure 3 shows that the finding holds true
also when the transamidase activity is monitored by the incorporation
of dansylcadaverine into N,N-dimethylcasein.37 Although the
inclusion of Ca2+ (1.2 mmol/L) in the reaction mixture
caused some enhancement in the rate of amine incorporation, enzyme
activity still stayed well below that found with control normal IgG
(Fig 4).
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| Fig 3.
Mixing the patient's IgG with thrombin-modified factor
XIII (rA2') generates transamidase activity even in
the absence of Ca2+. Recombinant factor XIII
A2 subunits (25 µg/mL rA2 in 50 mmol/L
Tris-HCl, pH 7.5) were treated with 1.25 U/mL of human -thrombin at
room temperature for 20 minutes. Reactions were performed at 37°C
in a total volume of 125 µL with 40 µg of normal ( ) or varying
concentrations of patient ( ) IgG (isolated on a Protein A column
from serum drawn 9/12/91), 50 mmol/L Tris-HCl, pH 7.5, 2 mg/mL
N,N-dimethylcasein, 0.5 mmol/L dansylcadaverine, and 0.25 µg
rA2'. The increase in fluorescence intensity
accompanying the amine incorporation reaction at 60 minutes is shown.
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| Fig 4.
Comparing the effect of the patient's IgG on the
incorporation of dansylcadaverine into N,N-dimethylcasein by
rA2' in the presence and absence of
Ca2+. Reactions were performed at 37°C in a total
volume of 125 µL with no IgG (,), 25 µg normal IgG ( ,  )
or patient IgG ( , ; isolated on a Protein A column from serum
drawn 9/12/91), 50 mmol/L Tris-HCl, pH 7.5, 2 mg/mL N,N-dimethylcasein,
0.5 mmol/L dansylcadaverine, and 0.25 µg rA2', in
the absence (open symbols) or presence of 1 mmol/L CaCl2
(closed symbols). Fluorescence was measured as in Fig 3.
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Another proof that the interaction of the patient's IgG with the
thrombin-modified rA2' zymogen could generate an
enzyme (IgG:rA2o) with cross-linking activity
in the absence of Ca2+ was provided by experiments with the
recombinant C-terminal C30 fragment of chains of human
fibrinogen.38 As shown in Fig 5, incremental doses of the patient's IgG in the presence of EDTA gave
rise to increasing amounts of cross-linked C30 dimers, eventually matching that obtained with Ca2+ in the mixture. Inasmuch
as the reaction of the IgG:rA2o enzyme with the
C30, in contrast to that with the preassembled fibrin substrate,
occurs in solution, it was expected that the overall cross-linking
efficiency measured for the dimerization of C30 in Fig 5 would fall
well below that of the formation of - dimers in fibrin (Fig 1A,
lane 3).
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| Fig 5.
The patient's IgG can also promote the cross-linking of
the recombinant C30 fragment of fibrinogen by rA2'
in the absence of Ca2+. The 25-µL reaction mixtures
contained 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, C30 (160 µg/mL; 5.3 µmol/L), and either no IgG (solid bar) or patient IgG
(0.5 to 3 mg/mL; shaded bars; IgG isolated on a Protein A column from
serum drawn 9/12/91), rA2' (thrombin-activated and
hirudin-quenched rA2, 10 µg/mL; 0.625 µmol/L) and 1 mmol/L EDTA. For controls, either 5 mmol/L CaCl2 replaced
EDTA in the absence of IgG (open bar), or 2 mg/mL normal IgG was used
instead of patient IgG (not shown). After the cross-linking reaction (4 hours, 37°C), the samples were analyzed by SDS-PAGE under
nonreduced conditions on 10% acrylamide. The percentage of dimerically
cross-linked C30 products was obtained from densitometric scanning
of the Coomassie brilliant blue R stained gel bands, calculated
as
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Specificity of the patient's abnormal antibody.
In view of the complexities of the biochemical reactions involved in
fibrin stabilization (schemes 1 through 3), it is not surprising to
find that the autoimmune antibodies examined thus far for blocking this
phase of blood coagulation, show a great variety of target
specificities. In this case, immunoblots (dot blots on nitrocellulose)
showed that the patient's IgG recognized the purified plasma factor
XIII zymogen (A2B2), the thrombin-modified allozymogen ensemble (A2'B2), and also
the recombinant rA2 subunit and its thrombin-modified
rA2' form. However, negative results were obtained
with the purified carrier B2 subunits of plasma factor XIII
and with fibrinogen as the antigens (data not shown). These findings
suggest that the antibody is entirely A subunit-directed, recognizing
both the virgin and the thrombin-cleaved zymogens. Further ELISA tests,
in which either A2B2 or
A2'B2 were plated, indicated
significantly higher adsorption of the IgG from the patient's serum to
the thrombin-modified A2'B2 species (data
not shown). However, a more rigorous evaluation of specificity was obtained by allowing increasing amounts of antigen to bind to a fixed
amount of the patient's IgG in the liquid phase and measuring the
amount of residual free antibody by ELISA. The results of these assays,
which probably best reflect on the antigen recognition by the
patient's IgG in plasma, are illustrated in
Fig 6 and can be summarized as follows: (1)
competition by rA2 for the antibody in solution was similar
and perhaps slightly better than that by A2B2;
(2) competition by rA2' was indistinguishable from
that of A2'B2 and, most importantly, (3)
the affinity of the IgG for either of these thrombin-modified zymogens
was shown to be about two orders of magnitude greater than their virgin
counterparts.
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| Fig 6.
ELISA competition assays show that thrombin-modified
factor XIII' is the prime target of the patient's antibody.
Patient IgG (isolated on a Protein A column from serum drawn 9/12/91;
10 µg in 2% Blotto) was mixed with varying amounts of factor XIII
(A2B2 or rA2) or thrombin-treated
factor XIII (A2'B2 or
rA2') for 30 minutes at room temperature and the
mixtures were then added to wells previously coated with 0.4 µg
A2B2. After overnight incubation at room
temperature, the human IgG bound to the immobilized antigen in the
wells was detected with an alkaline phosphatase-conjugated antibody
(-chain specific). For experimental details, see Materials and
Methods.
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The antibody titer in the patient's serum diminished gradually with
time.
During the follow-up period, several serum samples were collected and
were tested for assessing changes in the titer of the circulating
antibody. Thrombin-modified A2'B2 was
used as the antigen, and the extent of IgG binding from the patient's
serum was measured by ELISA. The results presented in
Fig 7 show a gradual decrease in the titer
of the antibody and essentially normal values were recorded for the
samples for the past 18 months. Not surprisingly, disappearance of the
circulating inhibitor allowed for a near-normal cross-linking of the
chains of fibrin upon clotting recent specimens of the patient's
plasma with thrombin plus Ca2+ (lane 4, inset to Fig 7).
Also, unlike clots from the earlier period of the disease, those from
the recent samples could not be dissolved in 5 mol/L urea. However,
despite the fact that the antibody could no longer be detected by ELISA
in the sera recently collected, it still revealed itself, probably on
account of being tightly bound to factor XIII in association with
fibrin(ogen), by causing activation of the zymogen and the
cross-linking of chains when clotting was induced with thrombin
alone in the absence of Ca2+ (lane 3, inset to Fig 7).
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| Fig 7.
Changes in the titers of the antibody in the patient's
serum over the course of the disease. Normal (solid bar) and patient
(open bars) serum samples were diluted (0.25 µL of serum in 100 µL
of 2% BSA in PBS) and incubated in wells coated with 0.4 µg of
thrombin-modified factor XIII (A2'B2).
After overnight incubation at room temperature, the human IgG bound to
the antigen was detected with an alkaline phosphatase-conjugated
antibody (-chain specific). For experimental details, see Materials
and Methods. Values shown are averages for duplicate samples. Inset:
Normal (lanes 1 and 2) and patient plasmas (lanes 3 and 4; drawn
5/03/97) were treated with thrombin in the presence (lanes 2 and 4) and
absence (lanes 1 and 3) of Ca2+ and the washed clots were
analyzed by SDS-PAGE as in Fig 1A.
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| |
DISCUSSION |
The urea-solubility of the patient's recalcified plasma clot was a
positive sign of a defect with fibrin stabilization. Although the onset
of bleeding at the age of 62 years in our patient made a diagnosis of
hereditary deficiency quite unlikely, we have previously found that a
patient who presented with bleeding for the first time at age 70 actually suffered from inherited deficiency of factor XIII
(unpublished). Enzyme activities for amine incorporation vary about
10-fold within the normal population and, in the absence of a family
history of bleeding, it would be impossible to decide on the basis of
the assay alone whether the measured value for a given individual fell
into the category of low normals or heterozygotes.20,21 Thus, the somewhat reduced, 62% activity of factor XIII in the patient's plasma (Table 1) would not, by itself, be an indicator of a
major deficiency. Values as low as 0.36% of controls were recorded
with the same assay procedure34,39 in families with a mild
phenotype of factor XIII deficiency, not even requiring maintenance
therapy.40 In addition, as expected, the measured concentrations of the A and B antigens for the subunits of plasma factor XIII were also within the normal range of values during the
entire period of our study from 1991 through 1997 (data not shown).
After excluding a hereditary deficiency, our attention was focused on
the presence of an inhibitor found in the plasma and also in the serum,
but not in the platelets, of the patient. It could be purified as an
IgG immunoglobulin and showed some unusual properties. Based on the
data in Table 1, the potential factor XIII activity, measured in the
patient's plasma by the incorporation of a synthetic amine
(putrescine) into a casein substrate,34 was not
significantly affected by the presence of the antibody. Yet, by
examining changes in the fibrin chain profiles during the factor
XIIIa-catalyzed cross-linking in the presence of
Ca2+, it was obvious that we were dealing with a potent
inhibitor. As shown in Figs 1 and 2, the patient's antibody completely
blocked the cross-linking of the chains and partially the chains of fibrin.
The patient's antibody seems to be specifically directed against the
thrombin-activated form of the factor XIII zymogen:
A2'B2, recognizing this species with much
greater affinity than the virgin A2B2 factor
(Fig 6). Moreover, the antibody binds equally well to
A2'B2 and rA2', showing
that it is entirely A subunit oriented. Over the years, we have
examined several autoimmune inhibitors against factor XIII in our
laboratory, but found none displaying the same degree of selectivity
for the thrombin-modified subunits of the zymogen as this patient's IgG.
A most unusual characteristic of this antibody is that, in binding to
A2'B2 or rA2', it is
capable of inducing a significant amount of enzyme activity even in the
presence of EDTA, whereas it is known that conversion to the factor
XIIIa enzyme would obligatorily require Ca2+. Thus, we
conclude that the binding of the patient's IgG to either of the two
thrombin-activated zymogens: eg, IgG + rA2' IgG:rA2' IgG:rA2o or IgG + A2'B2 IgG:A2'B2 IgG:A2o + B2 (we have evidence for
the dissociation of B subunits; not shown), opens up access for
accommodating the substrates, such as casein and dansylcadaverine in
Figs 3 and 4 or the chain crosslinking sites of fibrin in Figs 1,
2, and 5. This allows for substantial catalysis, even though the
turnover is limited because it does not match either the rate or the
extent of the reactions seen in the presence of Ca2+
without the antibody. Otherwise, the catalytic center in the IgG:A2o complex must be the same or very
similar to that found in the Ca2+-activated
A2*, because both enzyme activities could be
abolished by addition of the active site-directed inhibitor of factor
XIIIa (a gift from Dr Andrew M. Stern, Merck Research Laboratories,
West Point, PA):41
1,3,4,5-tetramethyl-2[(2-oxopropyl)thio]imidazolium chloride (data
not shown).
In a recent review,26 22 published cases of acquired
inhibitors of fibrin stabilization were compiled and analyzed. The severity of this class of hemorrhagic diseases is borne out by the fact
that in 6 of the 22 cases bleeding was reported as the cause of death,
ie, a mortality of 27%. The condition was shown to affect males and
females equally (45% and 55%) and, as with other hemorrhagic
disorders due to acquired inhibitors against coagulation factors, the
disease is more prevalent with advancing age; 16 of the 22 patients
surveyed (73%) were in the age group older than 50 years. Also, in the
majority of patients (15, ie, 68%) the acquired inhibitor was
identified as an immunoglobulin. Two of the affected individuals had
hereditary factor XIII deficiencies and developed the neutralizing
antibody after repeated therapeutic infusions of plasma or plasma
fractions. History of treatment by drugs with potential autoimmunizing
side effects (isoniazid, procainamide) or a prior, very strong allergic
reaction may often contribute to precipitating the disease.
Nevertheless, the nature of the actual causative agent in most of these
cases, as in the present one, remains unknown. Omitting the two
hereditary factor XIII-deficient patients from consideration, in 6 of
13 cases with inhibitory IgGs, the antibody eventually disappeared from
the circulation either on account of the cessation of the drug
suspected to have caused the condition or because of immunosuppressive
treatments (cyclophosphamide, prednisone, plasmapheresis, infusion of
factor XIII concentrate) or spontaneously. As illustrated in Fig 7, the inhibitor can no longer be detected in the serum of our patient; nevertheless, the abnormal production of - crosslinked dimers without Ca2+ (inset to Fig 7) still shows the presence of
this antibody in association with the fibrinogen:factor XIII complex.
This unique abnormality, though also progressively diminishing with
time, remains a distinguishing hallmark of this patient's clotting
profile to date.
| |
FOOTNOTES |
Submitted April 6, 1998; accepted September 30, 1998.
Supported by Grants No. HL-16346 and HL-02212 from the National
Institutes of Health (NIH), Bethesda, MD.
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.
Address reprint requests to Laszlo Lorand, PhD, Department of
Cell and Molecular Biology, Northwestern University Medical
School, Searle 4-555, 303 E Chicago Ave, Chicago, IL 60611;
e-mail: l-lorand{at}nwu.edu.
| |
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Top
Abstract
Introduction