(Circulation. 2000;102:1151.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Catalytic Life of Activated Factor XIII in Thrombi
Implications for Fibrinolytic Resistance and Thrombus Aging
From the Harvard School of Public Health (B.R.R., A.K.H., G.L.R.), Harvard Medical School and Massachusetts General Hospital (G.L.R.), Boston.
Correspondence to Guy L. Reed, MD, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave, Boston, MA 02115. E-mail reed{at}cvlab.harvard.edu
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Abstract |
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Background—Because the increased fibrinolytic resistance of older thrombi may be caused by the continuous cross-linking action of fibrin-bound activated factor XIII (FXIIIa), we examined the persistence of FXIIIa catalytic activity in clots of various ages.
Methods and Results—The time-related changes in FXIIIa activity
in clots was measured with (1) 
2-antiplasmin
(2AP), a physiological glutamine
substrate; (2) 2AP13–24, a peptide; and (3)
pentylamine, a nonspecific lysine substrate. The cross-linking of
2AP, 2AP13–24, and
pentylamine into fibrin by clot-bound FXIIIa declined rapidly with
half-lives of 19, 21, and 26 minutes, respectively. Mutational studies
showed that glutamine 14 (but not glutamine 3 or 16) and valine 17 of
2AP13–24 were required for efficient
cross-linking to fibrin. The loss of activity was not due primarily to
FXIIIa proteolysis and was partially restored by reducing agents,
suggesting that oxidation contributes to the loss of the enzyme’s
activity in clots. In vivo, the ability of thrombus-bound FXIIIa to
cross-link an infused 2AP13–24 peptide into
existing pulmonary emboli also declined significantly over
time.
Conclusions—FXIIIa cross-links 2AP and an
2AP peptide, in a sequence-specific manner, into formed
clots with a catalytic half-life of 
20 minutes. This indicates that
FXIIIa activity is a hallmark of new thrombi and that the
antifibrinolytic cross-linking effects of FXIIIa are achieved more
rapidly in thrombi than previously believed.
Key Words: embolism • fibrinolysis • thrombus
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Introduction |
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The successful dissolution or fibrinolysis of blood clots has life-saving effects in patients with thrombotic disease. Yet despite improvements in the design and administration of plasminogen activators, there is evidence that all types of vascular thrombi resist fibrinolysis and that this resistance increases with thrombus age.1 2 Coronary thrombi (>4 hours old) are more resistant to the lytic effects of plasminogen activators, and the success of thrombolysis in venous thromboembolism diminishes significantly with the clinical age of thrombi.3 4 The mechanism(s) responsible for enhanced fibrinolytic resistance of thrombi with age remain unknown.
Experimental studies of pulmonary thromboembolism show that
activated coagulation factor XIII (FXIIIa) makes new thrombi
resistant to lysis by physiological and
pharmacological plasminogen
activators.5 A transglutaminase, FXIIIa
cross-links proteins in the fibrin thrombus by forming an amide bond
between specific glutamine and lysine residues, which produces a
mechanically stronger clot.6 7 8 9 Fibrinolytic resistance is
achieved when FXIIIa covalently links
2-antiplasmin (2AP),
the plasmin inhibitor, to fibrin.10
Cross-linking of fibrin -chains and/or 
-chains by FXIIIa also
increases the resistance to plasmin.11 In vitro,
FXIIIa-mediated cross-linking is initiated at the time of clotting and
appears to be progressive over the course of minutes to hours. Although
there are no direct studies of FXIIIa cross-linking rates in human
thrombi, studies in rabbits suggest that FXIIIa cross-linking of
-chains may continue for 
6 hours after clotting.12
This persistence of FXIIIa cross-linking seemed plausible because there
were no known physiological inhibitors
of FXIIIa in humans and the regulation of the catalytic half-life of
the enzyme was poorly understood. Because more extensively cross-linked
thrombi are less sensitive to degradation by plasmin, the increased
resistance of older thrombi to fibrinolysis could be
mediated, in part, by FXIIIa.
In this study, we examined the catalytic activity of FXIIIa in vitro and in vivo after clotting. These findings have implications for the potential therapeutic use of FXIIIa inhibitors and for diagnostic or treatment strategies that seek to target newly formed thrombi.
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Methods |
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Materials were obtained from the following suppliers: 5-(biotinamido)pentylamine, Pierce;
2AP,
Calbiochem; biotinylated 2AP peptides
(high-performance liquid
chromatography–purified >95%), Advanced Chemtech;
bovine thrombin, DTT, and tissue transglutaminase from guinea pig
liver, Sigma; calcium chloride, Mallinckrodt; purified FXIII, human
fibrinogen, plasmin, and rabbit anti-human fibrin antibody, American
Diagnostica; fresh-frozen human plasma pooled from random
donors, MGH Blood Bank; mouse antifibrinogen antibody 4A5 and mouse
anti-FXIII antibody 309, Brscom; iodoacetamide, Calbiochem;
[125I]NaI, NEN-Dupont; and the
histological preparations avidin-biotin complex
(ABC) and 3,3'-diaminobenzidine (DAB), Vector
Laboratories. Ferrets (0.9 to 1.0 kg) were obtained from Marshall Farms, North Rose, NY; mice (Black C57, 4 to 8 weeks old), Jackson Laboratories, Bar Harbor, Me; ketamine, Fort Dodge Laboratories; acepromazine maleate, Fermenta Animal Health Co; surgical instruments, VWR and Roboz; Bard Parker surgical blades and polyethylene tubing, Becton Dickinson; silk suture, American Cyanamid Co; Surflo IV catheter, 20 gauge, Terumo Medical Corp; and 3-way stopcocks and micro tissue grinder, VWR.
Assays of FXIIIa Catalytic Life and Substrate Specificity
The persistence of FXIIIa cross-linking activity in clots
was examined with various substrates: human
125I-labeled 2AP,
5-(biotinamido)pentylamine, and a peptide,
2AP13–24 (in which
lysine 24 was biotinylated). Clots were prepared (50 µL) in duplicate
by combining fibrinogen (2 mg/mL final), CaCl2
(2 mmol/L final), buffer (Tris-buffered saline, pH 7.4), and
thrombin (1 U/mL) and incubating at 37°C. The FXIIIa substrates
5-(biotinamido)pentylamine (0.5 mmol/L final),
125I-2AP (70 µg/mL
final), and 2AP13–24 (0
to 1 mmol/L final) were added 0 to 240 minutes after clotting with
thrombin. In 1 set of control samples, FXIIIa was inhibited with
iodoacetamide (10 mg/mL) before the addition of substrate; in another
set, no substrate was added. After 2 hours of incubation at 37°C,
iodoacetamide was added to stop the reaction. Clots were then
centrifuged at 14 000 rpm for 2 minutes, washed and compressed
in 1 mL saline, and centrifuged again. The washed clots were
solubilized in 100 µL of 9 mol/L urea, pH 9.0, at 37°C for 60
minutes. Then 100 µL of SDS-reducing sample buffer was added, and
samples were held at 85°C for 30 minutes and examined by SDS-PAGE.
Samples containing cross-linked
2AP13–24 and
pentylamine substrates were electroblotted to polyvinylidine
difluoride (PVDF) membranes and detected by
125I-streptavidin followed by phosphorimaging or
by streptavidin-peroxidase and the developing agents
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium
(NBT-BCIP) as we have described.5 Phosphorimages were
examined under conditions in which there was a linear relationship
between substrate incorporation and pixel volume. In experiments
examining the effect of specific amino acid residues on the
cross-linking of the
2AP13–24 peptide to
fibrin, cross-linking was detected by immunoblotting
with an anti-peptide antibody directed against this epitope followed by
125I-labeled protein A and phosphorimaging as
described.13 Plasma clots were formed by mixing 25 µL
pooled human fresh-frozen plasma (sodium citrate) with 25 µL 30
mmol/L CaCl2 instead of human fibrinogen.
To examine the potential degradation of FXIIIa in clots, samples from these experiments were immunoblotted with a monoclonal antibody directed against the A subunit of FXIII as described.14
Cross-Linking of 2AP13–24 Into Fibrin
by FXIIIa and Tissue Transglutaminase
Fibrin clots (50 µL) were prepared in duplicate by mixing
FXIII-free fibrinogen (2 mg/mL final),
2AP13–24 (0.5
mmol/L final), CaCl2 (2 mmol/L final), human
plasma FXIII (100 nmol/L final), and guinea pig tissue transglutaminase
(100 nmol/L final) and thrombin (1 U/mL final) and incubating at 37°C
for 2 hours. The cross-linking of
2AP13–24 was detected
as described above.
Cross-Linking of 2AP13–24 Into
Pulmonary Emboli In Vivo
We have previously used a pulmonary embolism model in
male ferrets (1.0 kg).15 All in vivo experiments were
approved by the Harvard Medical Area Standing Committee on Animals.
Clots were formed with human fresh-frozen plasma (45 µL) mixed with
trace amounts of 125I-labeled fibrinogen
(100 000 cpm/clot), 2.5 µL of 0.4 mol/L
CaCl2, and 2.5 µL thrombin (1000 U/mL) for 20
minutes at 37°C. After 3 washes, 6 clots were embolized into
2 animals in each of 3 groups: 2 groups had normal clots, and 1 group
received clots washed in iodoacetamide (10 mg/mL) and EDTA (10
mmol/L final) to inhibit clot-associated FXIIIa. After embolization,
2AP13–24 (final
concentration of 0.5 mmol/L) was infused (except in control
animals). Four hours after embolization, the ferrets were euthanized by
CO2 inhalation, and the pulmonary emboli
were isolated.
Samples were weighed and homogenized in a 200-µL solution (24 mmol/L Tris Base, 476 mmol/L Tris HCl, 50 mmol/L MgCl2, 1 mg/mL DNAse I, and 0.25 mg/mL RNAse A), incubated on ice for 10 minutes, and centrifuged at 12 000 rpm at 4°C for 20 minutes. The pellet was dissolved in 200 µL of sample buffer, heated until dissolved, and analyzed by SDS-PAGE as described above.
Mouse Pulmonary Embolism Experiments
Clots were formed with 22.5 µL of whole mouse blood in
polyethylene tubing after mixing with trace amounts of
125I-labeled fibrinogen, 1.25 µL of 0.4 mol/L
CaCl2, and 1.25 µL thrombin (1000 U/mL) at
37°C for 15 and 30 minutes. The right and left internal jugular veins
were exposed in anesthetized mice. Before embolization, 100
U/kg of heparin and 1.5 mg/kg of P-PACK were injected
intravenously to inhibit endogenous thrombin
activity. One vessel was used to embolize the clot, and the
other was used for infusion of
2AP13–24 immediately
after embolization. Two hours after embolization, the thrombi were
isolated and subjected to SDS-PAGE as described above.
Histology
Lung tissue was immersed in 30% sucrose at 4°C overnight,
embedded in OCT, cut into 10-µm sections, and fixed in 100% methanol
(5 minutes). Samples were pretreated with 3%
H2O2 (20 minutes), washed
in PBS 3 times, and treated with 10% normal goat serum (20 minutes).
Samples were then incubated with rabbit anti-human fibrin antibody
(1:400 dilution, 2.5 µg/mL) at 4°C overnight, washed twice (in
high-salt PBS and regular PBS), and incubated with goat anti-rabbit
peroxidase-labeled antibody (1:100, 1 hour). After 3 washes in PBS,
sections were developed in DAB, counterstained in 0.1% methyl green,
and mounted. For the detection of
2AP13–24, sections were
developed with the ABC reagent and developed as described
above.
Statistical Tests
Data were analyzed with either an unpaired Student’s
t test or a 1-way ANOVA with the Bonferroni-Dunn procedure
for testing multiple comparisons.
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Results |
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To examine the catalytic half-life of FXIIIa in thrombi with physiological substrates, we measured the cross-linking of
2AP to fibrin. The
cross-linking of
125I-2AP was greatest
when it was added synchronously to fibrinogen with thrombin to initiate
clotting (Figure 1
). When added to
older clots, the amount of 2AP cross-linking
dropped off rapidly to the levels of controls in which FXIIIa had been
inhibited with iodoacetamide, or in lanes containing no
125I-2AP. The
cross-linking of
125I-2AP to fibrin
declined exponentially with a half-life of 19 minutes
(r2=0.92, Figure 1, bottom,
semilog plot).
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One potential explanation for the decline in the cross-linking of
2AP to fibrin was that the size of
2AP (70 000 Da) inhibited its diffusion into
the assembling fibrin meshwork. To examine this possibility, we used a
small 2AP peptide
(2AP13–24, 1600 Da),
which should readily permeate the developing thrombus. We and others
have verified that this peptide5 or related
peptides16 17 can compete with cross-linking of
2AP to fibrin when added during clotting. This
peptide was biotinylated at lysine 24 to permit detection. The peptide
cross-linked into fibrin clots as a function of dose (Figure 2). Compared with control clots, the
cross-linking of the
2AP13–24 peptide was
seen at concentrations as low as 1.3 µmol/L. Cross-linking was
half of maximum at 110 µmol/L and saturated at concentrations of
0.33 to 1 mmol/L. A similar dose-response curve was found for the
cross-linking of the
2AP13–24 peptide to
fibrin in human plasma (data not shown).
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Although FXIIIa can cross-link a number of lysine analogues to
glutamine sites in macromolecules such as fibrin, little is known about
its specificity for cross-linking glutamine-containing substrates (acyl
donors), such as 2AP, to fibrin. To examine
this, a panel of 2AP peptides (residues 1 to
24) was created with mutations in residues that could represent
other potential cross-linking sites (Q7, Q14) or residues that are
conserved among different 2APs from different
species (V17, L22; Figure 3, top).
Compared with clots containing no peptide or the wild-type peptide
2AP1–24, peptides
containing mutations Q14A and V17N were not efficiently cross-linked to
fibrin (Figure 3, bottom). In contrast, mutations Q7A, Q16A, and
L22N were cross-linked into fibrin at rates not markedly different from
the wild-type 2AP1–24
peptide. Similar results were seen when these peptides were used as
inhibitors of the cross-linking of
2AP13–24 into fibrin:
all peptides but Q14A and V17N competed for cross-linking (data not
shown). Taken together, these results indicate that
2AP1–24 and
2AP13–24 are specific
glutamine substrates for FXIIIa and suggest that Q14 and V17 are
necessary for efficient fibrin cross-linking.
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Although FXIIIa is the primary transglutaminase found in plasma, other
transglutaminases are found in cells that do not require thrombin for
activation. We examined whether the
2AP13–24 peptide could
be efficiently cross-linked by tissue transglutaminase. In studies with
FXIIIa-deficient fibrinogen, there was little, if any, significant
cross-linking of
2AP13–24 to fibrin with
clotting (Figure 4, top). Addition
of purified tissue transglutaminase (100 nmol/L) caused an increase in
cross-linking that was not statistically significant
(P>0.05). Equivalent amounts of purified exogenous FXIIIa
caused significantly more cross-linking of
2AP13–24 to fibrin,
which was significantly blocked by the thrombin inhibitor
hirudin (P<0.01, Figure 4, bottom). Because FXIIIa
is the only thrombin-dependent transglutaminase, this result indicates
that FXIIIa was responsible for the cross-linking of the
2AP13–24 peptide in
these fibrinogen preparations.
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The 2AP13–24 peptide
was used to measure the catalytic life of FXIIIa in formed or forming
clots. Consistent with experiments performed with
125I-2AP (Figure 1
), the cross-linking of
2AP13–24 to fibrin was
greatest when it was present at the initiation of clotting (Figure 5) and fell rapidly over the course of an
hour to nearly undetectable levels. The half-life calculated for
2AP13–24 cross-linking
was 21 minutes (r2=0.935), which was
comparable to that found for the
125I-2AP (Figure 1). We also found that FXIIIa showed a similar catalytic
half-life for a lysine (acyl acceptor) mimic, pentylamine-biotin, an
even smaller substrate (330 Da) than
2AP13–24. In these
studies, pentylamine-biotin was cross-linked into fibrin clots with a
half-life of 26 minutes (r2=0.744),
which was consistent with the cross-linking of
2AP and
2AP13–24.
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We examined whether proteolysis of FXIIIa by thrombin accounted for the
loss of FXIIIa activity in clots (Figure 6). Quantitative
immunoblotting showed that after 1 hour, there was an
10% decrease in the amount of FXIIIa catalytic subunit A', and by 4
hours, there was an 20% decrease. In contrast,
125I-2AP–fibrin
cross-linking had declined by 80% within 1 hour and by >95%
within 4 hours (Figure 1). Because proteolysis alone did not
explain the loss of FXIIIa activity, we examined whether oxidation of
the multiple reduced cysteines of FXIIIa (including its active-site
residue) could account for the loss in catalytic activity of the
molecule. Under normal circumstances, when pentylamine or
2AP13–24 was added to
2- or 4-hour-old clots, there was little cross-linking of the
substrates to fibrin by FXIIIa (Figure 7). The addition of a reducing agent,
DTT, restored the cross-linking of pentylamine or
2AP13–24 to fibrin
clots, which suggested that reversible oxidation of FXIIIa was
responsible for the loss of cross-linking activity.
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To confirm the physiological significance of these
findings, we examined the ability of FXIIIa to cross-link
2AP13–24 to existing
pulmonary emboli in vivo. Three experimental groups were
studied: (1) a control group that received no peptide after
embolization, (2) a group that received a 0.5 mmol/L dose of
2AP13–24 peptide after
embolization, and (3) a group that received a 0.5 mmol/L dose of
2AP13–24 peptide after
embolization of clots that were pretreated with iodoacetamide and EDTA
to inhibit residual clot-associated FXIIIa activity. Figure 8A shows a pulmonary embolus
(stained with anti-fibrin antibody) that has occluded a
pulmonary arteriole. Compared with a control thrombus (8B) from
an animal not receiving
2AP13–24, the thrombus
from an animal receiving the peptide infusion (8C) showed specific
peroxidase staining indicating incorporation of the
2AP13–24 into the
thrombus.
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To confirm that the peptide was covalently incorporated into
fibrin in the thrombus, the lung tissue samples were subjected to
SDS-PAGE, blotted with 125I-streptavidin to
detect biotinylated
2AP13–24 (Figure 9, top), and stained with Coomassie blue
(Figure 9, bottom). Lung tissue from control animals (normal
emboli and no 2AP13–24)
showed 2 nonspecific avidin-binding bands at 70 and 120 kDa in
the tissues with and without the embolus (Figure 9A, lanes 1 and
2). In animals receiving the
2AP13–24 peptide, there
were similar nonspecific staining bands at 70 and 120 kDa in the lung
tissue without emboli (Figure 9C, lane 5). However, the lung
tissue containing pulmonary emboli showed a new broad band at
65 to 70 kDa and additional bands at 60, 100, and 140 kDa
(Figure 9C, lane 6). The intensity of the bands was diminished
in the lung tissue from an animal with a pulmonary embolus in
which FXIIIa had been inhibited by iodoacetamide and EDTA (Figure 9B, lane 4). Taken together, the studies in Figures 8 and 9 show that
2AP13–24 is
cross-linked into preexisting pulmonary embolus in vivo and
that cross-linking is attenuated by inhibition of the FXIIIa in the
thrombus.
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Additional experiments were performed in mice to investigate whether
FXIIIa cross-linking ability also declined in formed pulmonary
emboli in vivo. Compared with control mice (no peptide infused), there
was a significantly greater amount of
2AP13–24 cross-linking
in the 15-minute-old emboli. This cross-linking declined significantly
in the 30-minute-old emboli (P<0.02, Figure 10).
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Discussion |
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The finding that FXIIIa cross-linking caused fibrinolytic resistance in new pulmonary emboli suggested that continued FXIIIa activity may contribute to the increase in fibrinolytic resistance of thrombi with age.5 We found that the catalytic ability of FXIIIa to cross-link its physiological substrate (
2AP, 70 000 Da) into fibrin clots declined
in an exponential fashion, with a half-life of 19 minutes. Similar
half-lives for FXIIIa-mediated cross-linking were also found in much
smaller substrates, such as
2AP13–24 (1600 Da), 21
minutes, and pentylamine-biotin (330 Da), 26 minutes. This argued that
the decline in FXIIIa activity was not due to a size-related limitation
in the permeation of the substrate into the fibrin clot. The decline in
catalytic activity of FXIIIa could not be ascribed to proteolysis of
the catalytic subunit of FXIIIa (Figure 6) but rather appeared
to be related to potential oxidation (Figure 7). This is
consistent with the fact that FXIIIa contains a cysteine
residue in its active site that may be vulnerable to inhibition by
oxidation. Finally, in vivo studies confirmed our in vitro findings by
demonstrating that formed pulmonary emboli covalently
incorporated 2AP13–24
in a manner that declined significantly with time. This incorporation
was due to the enzymatic activity of the thrombus-bound FXIIIa, because
it was significantly attenuated in thrombi in which FXIIIa had been
inhibited immediately before embolization.
FXIIIa makes thrombi resistant to plasmin by modifying the
fibrin matrix through intermolecular cross-links. FXIIIa-mediated
fibrin -chain polymerization5 12 18 and
-chain multimerization11 contribute to this
fibrinolytic resistance. In addition, the cross-linking of the plasmin
inhibitor 2AP to fibrin plays a
clear role in neutralizing
fibrinolysis.5 10 13 In vitro, the
cross-linking of 2AP to fibrin and the
formation of -dimers occur rapidly, within 2 to 5 minutes.
Experimental studies of rabbit thrombi formed in vivo indicate that
-chain dimerization and -chain polymerization are readily seen
within 7 to 9 minutes of thrombus formation, although small increments
in -chain polymerization were detected after 90 to 320
minutes.12 In one of the few studies of the fibrin
structure of human thrombi, all thromboemboli examined showed extensive
-chain polymerization (although the ages of these thrombi were not
specified).19 Although previous studies have examined the
clearance of circulating FXIII zymogen in deficient
patients,20 to the best of our knowledge, these data
represent the first estimates of the catalytic life of FXIIIa
in clots.
Cross-linking by FXIIIa is highly specific, because only a
minority of potential glutamines and lysines in proteins are actually
substrates.21 The primary structure, charge, and
conformation around the respective glutamine residues determine the
suitability of a particular glutamine as a substrate for
FXIIIa.9 22 23 Still, no consensus sites have been defined
to identify potential FXIIIa substrates. Of the 3 potential glutamine
sites in the amino-terminus of
2AP13–24, only
glutamine 14 is required. In addition, Val17,
which is highly conserved in 2AP from other
species, also appears to be necessary.
Our limited in vivo studies confirm that FXIIIa can cross-link a
specific substrate
(2AP13–24) into formed
emboli and that this cross-linking activity declines significantly with
the age of the clot. More extensive studies with imaging techniques
(nuclear medicine, MRI, etc) will be required to definitively
establish, in vivo, the time-related incorporation of specific FXIIIa
substrates into thrombi of different ages and to determine whether
significant species differences exist.
Recently, a polymorphism was described in the FXIII gene that modifies a patient’s risk of thrombosis.24 It is possible that this or other individual genetic differences in FXIIIa structure or the relative "oxidative" environment of the blood might also modify FXIIIa half-life in vivo. However, the fact that FXIIIa remains catalytically active only in recent thrombi could be exploited to detect or target therapies to "new" thrombi. The ability to distinguish new from old thrombi might significantly improve our selection of patients for fibrinolytic therapy and could lead to the design of agents that are directed against recently formed clots.
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Acknowledgments |
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This work was supported in part by NIH grants HL-57314 to Dr Reed and HL-58496 to Dr Robinson. The authors are grateful to Dorothy Zhang for expert histology.
Received October 13, 1999; revision received March 23, 2000; accepted April 4, 2000.
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