Brief Review
Sol Sherry Lecture in Thrombosis
Research on Clot Stabilization Provides Clues for Improving
Thrombolytic Therapies
Laszlo Lorand
T
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in urea or not has been investigated and debated in the
literature for more than 100 years [see reference 4], with
some researchers answering in the affirmative, others in the
negative. In view of what we now know,2,3 both sides may
have been right on this issue).
An appropriate therapeutic aim for helping individuals
with thrombotic tendencies would be to block the factor
XIIIa-mediated reactions in a well controlled and highly
selective manner without interfering with the primary “clotting time.” This would result in preventing a significant
portion of the clot from progressing to the fully stabilized
state (illustrated by the bottom panel of Figure 1).
his article is a summary of the Sol Sherry Lecture of the
Council on Arteriosclerosis, Thrombosis, and Vascular
Biology, which was presented at the 71st Scientific Sessions
of the American Heart Association in November 1998.1 It
highlights the work from our laboratory, designed to dissect
the intricate reactions and molecular control mechanisms that
operate in the final stages of the coagulation cascade. This
research brought forth the idea that, by selectively blocking
the maturation and accretion of thrombi, we should be able to
achieve a much safer and more efficient thrombolysis at
lower dosages of clot dissolving agents than currently in use.
Fibrin is the fundamental building block of the clot matrix.
Network formation occurs in an orderly sequence, well
separated in time into distinct phases during the course of
coagulation of normal plasma. After the reaction of thrombin
with fibrinogen, a protofibrillar lattice is formed, with fibrin
units lined up in a half-staggered array, reminiscent of laying
bricks without mortar (Figure 1, top panel). Lateral bundling
into filaments and fibers with concomitant entanglements and
branching generates a 3D gel, the appearance of which is a
measure of “clotting time.” Then, under the influence of the
activated fibrin stabilizing factor (factor XIIIa), covalent
bonds are introduced into the structure that causes an irreversible, end-to-end fusion of the fibrin particles (Figure 1,
middle panel). Finally, full maturation of the network is
brought about by forming covalent bonds between the protofibrils and filaments (Figure 1, bottom panel). Clots displaying the features shown in the top panel of Figure 1 can be
readily dissociated into monomeric fibrin in 5 mol/L urea2,3
and on removal of urea, the gel reforms. The equilibrium
between the monomeric or low oligomeric forms of soluble
fibrin and clotted fibrin can be described as:
The Urea-Soluble Clot
The dramatic conversion of fibrinogen to fibrin is caused by
the proteolytic action of thrombin with the release of
N-terminal fibrinopeptide moieties5– 8 (Figure 2a). These
“caps” have to be removed from the protein for unmasking
the self-assembly potential that was built into the parent
fibrinogen molecule by evolution. The demonstration that
thrombin was a protease provided the first example of a
limited proteolytic type of control in the now familiar mode
of regulation for most of the reactions of the coagulation and
immunocomplement cascades. A key feature of the reaction
of thrombin with fibrinogen is the generation of N-termini of
glycine in fibrin9 –11 because the immediate sequences of
amino acids at these newly exposed sites12 can serve as
ligands for promoting the noncovalent assembly of fibrin
molecules. Short peptide analogues of the natural sequences
have been found to inhibit the clotting of purified
fibrinogen.13
The fibrinopeptides (A and B) are located at the ends of the
Aa and Bb chains of fibrinogen in the central E domain of the
large molecule (Mw;340 000), made up of a disulfide
bonded duplex of 3 different chains [AaBbg]2.14 Release of
fibrinopeptides by thrombin sets the train of events in motion
for clotting by allowing the newly unmasked N-termini to
interact with complementary holes (polymerization pockets)
in the distal D domains of the protein.15–17 Actually, as seen
from the activities of some snake venom enzymes, the release
In contrast to this, the factor XIIIa-stabilized networks,
shown in the middle and bottom panels of Figure 1, cannot be
dispersed in urea. (As a historical aside, it is interesting to
observe that the question of whether fibrin could be dispersed
Received Received August 31, 1999; accepted November 2, 1999.
From the Department of Cell and Molecular Biology and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School,
Chicago, Ill.
Correspondence to Laszlo Lorand, Department of Cell and Molecular Biology, Northwestern University Medical School, Searle 4-555, 303 E Chicago
Avenue, Chicago, IL 60611-3008. E-mail [email protected]
(Arterioscler Thromb Vasc Biol. 2000;20:2-9.)
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
2
Lorand
Factor XIII Inhibition Promotes Thrombolysis
3
electronmicroscope with characteristic ca. 230Å repeats.20 –22
Although prior thrombin cleavage is necessary for unmasking
the N-terminal ligands, the polymerization pockets into which
these knobs fit are fully accessible also in the fibrinogen
molecule. Long filamentous assemblies can be obtained by
mixing fibrinogen with fibrinopeptide-denuded E fragments
derived from the plasmin digest of fibrin.23
The Urea-Insoluble Clot
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Figure 1. Stages in the formation and maturation of fibrin clots.
Clotting time is defined by the rate of assembly of the protein
into structures as illustrated in the top panel. Such clots,
obtained in mixtures of fibrinogen or Ca21-chelated (citrate, ethylenediaminetetraacetic acid) plasma with thrombin, can be
reversibly dissociated into fibrin monomers, proving that these
clots—in contrast to the covalently linked polymers in the ureainsoluble structures shown in the middle and bottom panels—
are made up of fibrin molecules held together only by weak
secondary bonds.
of fibrinopeptides A from the Aa chains alone is sufficient to
initiate clotting.18,19 In either case, the E domain of one fibrin
molecule makes contact with the D domains of 2 adjacent
fibrins, neatly aligning them end-to-end (Figure 2b). This
mode of assembly gives rise to the half-staggered arrangement of fibrin units in the protofibrils, recognized in the
There is a large family of Ca21-dependent enzymes, widely
distributed in nature and commonly referred to as transglutaminases, which promote the cross-linking of proteins by
intermolecular Ne(g-glutamyl)lysine side chain bridges. As
first documented for the clotting of blood in crustaceans24,25
and also for the process of forming the copulation plug in
rodents,26 such transamidases may have served as the evolutionary prototypes of clotting enzymes. In the clotting of
lobster blood, the enzyme is released from exploding amoebocytes, performing a platelet-like function. In the clotting of
the seminal vesicle secretion proteins of rodents, the transglutaminase is secreted from another lobe of the prostate and
becomes activated only in the vagina of the female as it
encounters its substrates after ejaculation. In both phenomena, proteins are remodelled in a single enzymatic step,
resulting in their transfer directly from a soluble phase into an
insoluble coagulum, extensively cross-linked through covalent bonds. In sharp contrast, with vertebrate blood, crosslinking follows clotting and the factor XIIIa-catalyzed reaction is superimposed on a preformed assembly of an aligned
and organized fibrin network. Thus, in the clotting of human
blood, cross-linking contributes exclusively to the maturation
and rigidification of the gel but does not significantly alter the
morphology of the structure.
The covalent cross-linking of fibrin units occurs through an
amide exchange reaction (transamidation) between select
glutamine and lysine residues of neighboring protein molecules27–31 (Figure 3a). As a result, a few strategically located
Ne(g-glutamyl)lysine side chain bridges are formed. For the
Figure 2. a) Generating the set of new
N-termini of glycine in the limited proteolysis of fibrinogen by thrombin is the
key for initiating the actual clotting reaction. The site of attack by thrombin in
the critical step of releasing the fibrinopeptide A moiety from the Aa chain of
the protein is shown. b) Release of fibrinopeptides (arrowheads) from fibrinogen
triggers the process of self-assembly.
The new N-terminal knobs, unmasked
by thrombin in the central E domains
bind to complementary holes in the D
domains of the protein. Thus the E
domain of 1 fibrin molecule makes noncovalent contacts with the D domains of
2 adjacent fibrins, aligning them
end-to-end.
4
Arterioscler Thromb Vasc Biol.
January 2000
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Figure 3. a) Outline of the amide
exchange stabilizing reaction catalyzed
by factor XIIIa (A2*). Formation of Ne(gglutamyl)lysine bridges between the endto-end aligned fibrin units are shown.
b) Competitive inhibitors can block the
factor XIIIa (A2*)-mediated crosslinking
(ie, reaction 1) without interfering with
the clotting time of the system. Serving
as specific tracers, the inhibitors themselves become incorporated by the
enzyme into the acceptor (reaction 2)
and donor (reaction 3) cross-linking
sites.
sake of simplicity, only 1 of the 2 bridges connecting the g
chains of 2 fibrins with an end-to-end orientation32 is shown
in the figure. One should bear in mind, however, that factor
XIIIa reacts with the g, as well as the a, chains of fibrin in an
ordered sequence.33 Crosslinks spanning more than 2 g chains
and several a chains, giving rise to a variety of g2, g3, g4
structures, homologous an and hybrid apgq chain combinations, play important roles in clot stabilization.34 –38 Furthermore, the factor XIIIa reaction is also responsible for the
covalent attachment of a fraction of the a2 plasmin inhibitor
(a2PI, also referred to as a2 antiplasmin or a2AP) in plasma to
the a chains of some of the fibrin molecules, providing added
protection against lytic agents.39 – 40
Human red cells contain a transglutaminase with a much
broader substrate specificity than factor XIIIa. This enzyme
can modify and crosslink fibrinogen as well as fibrin by ag
and an type of intra- and intermolecular Ne(g-glutamyl)lysine
bonds.37 If the enzyme was released from trapped erythrocytes during the aging of the thrombi, its action would add to
the complex pattern of cross-linking of the network.
Competitive,29,41– 43 noncompetitive,44 and active sitedirected45 inhibitors were shown to be able to selectively
block clot stabilization by factor XIIIa without causing a
delay in the clotting time of the system. The competitive
compounds were useful for exploring the biochemical details
of the cross-linking reaction because, by becoming incorporated into the donor and acceptor cross-linking residues, they
served as specific labels for these sites (Figure 3b). Even
more importantly, they made it possible to establish the
fundamental paradigm for the present article, that selective
interference with the functioning of the factor XIII system
facilitates thrombolysis.46 – 48
Activation and Regulation of the Factor
XIII System
As in the case with all other enzymes of the coagulation
cascade, only the inactive precursor, ie, factor XIII, of the
cross-linking enzyme circulates in plasma. The zymogen has
an A2B2 subunit structure,49 reminiscent of the heterologous
tetrameric composition of hemoglobin. The A subunits possess the catalytic potential and, quite likely, the carrier B
subunits serve the function of protecting them in the circulation. The B subunits also contribute to binding the zymogen
to fibrinogen50 and act as a brake on factor XIII activation.51
Thrombin and Ca21 are required for conversion into the active
factor XIIIa enzyme,52–55 but the thrombin-catalyzed cleav-
Lorand
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Figure 4. Conversion of the factor XIII zymogen to the factor
XIIIa (A2*) enzyme occurs in 2 distinct steps, promoted by
thrombin and Ca21, respectively. The thrombin-modified FXIII9
intermediate is still inactive. Dissociation from the carrier B subunits facilitates the conformational change necessary for the
unmasking of the active center cysteine thiol in the catalytic A*
subunit.
age near the N-termini of the A subunits only weakens the
heterologous association of the subunits56 and still leaves the
zymogen in an inert tetrameric (A29B2) form (Figure 4).
Expression of enzyme activity depends on the Ca21facilitated dissociation of A29 from the carrier B2 subunits and
on a concerted conformational change that unmasks the
hidden sulfhydryl catalytic centers for the A2* (FXIIIa)
cross-linking enzyme.51,57 Although the other trypsin-like
enzymes of the coagulation cascade operate with serine-OH
active centers, factor XIIIa functions with a cysteine-SH
catalytic residue, assisted by the imidazole ring of a histidine.58,59 There are remarkable kinetic60,61 and structural62
similarities with the papain-calpain family of enzymes, suggesting a common evolutionary ancestor.
Correct timing for the activation and operation of the factor
XIII system is of critical significance for efficient hemostasis.
This is reflected in the remarkably complex and tightly
controlled series of interacting mechanisms that regulate the
rate of conversion of the factor XIII zymogen, as well as the
rate of cross-linking of fibrin by the activated enzyme. A key
Factor XIII Inhibition Promotes Thrombolysis
5
feature in synchronizing the clotting and cross-linking events
is that thrombin plays the dual role of converting fibrinogen
to fibrin and also initiates the process of activating the factor
XIII zymogen. In addition, it has become evident that the
fibrin substrate itself acts to coordinate the orderly sequence
of reactions during the late stages of coagulation; as a
feed-forward regulator, 1) it accelerates the cleavage of factor
XIII by thrombin,63 2) it enables the subunits of the thrombinmodified zymogen to dissociate at the 1.5 mmol/L concentration of Ca21 in plasma,64 and 3) the noncovalent assembly
of fibrin units speeds up the end-to-end fusion of the g chains
in the D domains of the protein.65,66 These unique controls
(Figure 5) must have evolved to ensure that in the physiological sequence of events, only fibrin, and not the parent
fibrinogen molecule, should be the target for cross-linking by
factor XIIIa.
Determinants of the Morphological and
Rheological Properties of the Clot Network
In the physiological milieu of pH, ionic strength, and Ca21
concentration in plasma, clot morphology is determined
mainly by the concentration of fibrinogen and by the thrombin activity generated at the site of injury. In addition, some
proteins adhering to fibrin, eg, IgG and albumin, may add to
fiber thickness.67 With all other conditions being equal,
scanning electronmicrographs show considerably tighter networks— characterized by greater fiber and branch point
densities—near the upper end of the physiological range of
fibrinogen concentration. Also, clots formed rapidly at higher
concentrations of thrombin (with shorter clotting times) give
rise to much tighter structures than their slowly forming
counterparts generated at lower concentrations of thrombin
(with longer clotting times).38 Tightly knit clots would, of
course, increase the likelihood of a higher degree of entrapment of blood cells in the network.
Remarkably, stabilization by the factor XIII system does
not seem to affect clot morphology in regard to fiber and
branch point densities, but the fibers become somewhat
thinner and longer,38,68 suggesting that the covalent bonds
introduced by factor XIIIa strengthen the internal architec-
Figure 5. The reactions and controls
that regulate clot stabilization were
examined in purified systems, which then
made possible the reconstruction of the
entire physiological pathway.
6
Arterioscler Thromb Vasc Biol.
January 2000
tures of the fibers themselves. Notwithstanding the morphological similarities, the properties of the network are greatly
different because the clot can no longer be dissolved in
5 mol/L urea.2,3 Another sign of the profound change induced
by the action of factor XIII is the large increase in the
viscoelastic (storage) modulus, a measure of clot stiffness.69,70 To some extent, this quantity also correlates with the
concentrations of fibrinogen and thrombin, but the role of
factor XIII is paramount in this regard, causing an approximately 5-fold increase throughout the physiological range of
fibrinogen concentration.38
What Lessons Can be Learned From Studying
Disorders of Fibrin Stabilization?
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Hemorrhaging may occur in some individuals in spite of
the finding that the clotting and primary bleeding times are
within the normal range of values. The hemostatic plug in
these patients seems to fail because of extreme fragility
and gives rise to a delayed oozing of blood at the wound
site. Diagnosis usually rests on the observation that the
recalcified plasma clot of the patient, unlike a normal one,
can be solubilized in 5 mol/L urea.2,3 The hereditary lack
of functional factor XIII zymogen is responsible for the
majority of such cases,71–73 and most of them are due to the
absence of the catalytic A subunits,74,75 although cases are
also known with absence of B subunits.76 Acquired inhibitors constitute yet another class with quite diverse etiologies in this family of disorders of fibrin stabilization.77,78
The natural inhibitors may interfere with 1 or more of the
biochemical steps involved in the activation of the factor
XIII zymogen (A2B2 3 A29B23 A2*1B2) or with the actual
production of the covalent Ne(g-glutamyl)lysine crosslinks
catalyzed by the A2*, factor XIIIa enzyme. Most of these
inhibitors are found to be autoimmune antibodies directed
against some of the molecules participating in the physiological sequence of events depicted in Figure 5.
In addition to the abnormal solubility in urea, the patients’
clots are much softer than normal showing very low viscoelastic moduli and a greatly enhanced susceptibility to
digestion by lytic agents. These features can be illustrated
with the experiments in Figure 6, carried out with factor XIII
deficient plasmas. Panel A shows that supplementation with
the purified A2B2 zymogen restored normal (approximately
5-fold) values to clot stiffness,79 and the data in panel B
demonstrate that supplementation with the zymogen (using
only the recombinant A2 subunits in this experiment) can also
protect the clot against premature lysis (see also reference
80). These findings prove that proper functioning of the factor
XIII system is required for imparting normal stiffness and
adequate fibrinolytic resistance to the clot network.
Therapeutic Possibilities
Detailed understanding of the molecular aspects of clot
stabilization, together with the patient studies, provide the
background for rational approaches aimed at exploring means
for reducing the stiffness and lytic resistance of clots and
thrombi. Results of some prototype experiments, employing
cross-linking inhibitors such as discussed earlier (see Figure
3b) are presented in Figure 7. In a way, the findings are the
mirror images of the data in panel A of Figure 6 because, as
illustrated in Figure 7 (panel A), the inhibitors can actually
Figure 6. Supplementation of factor XIII-deficient plasma
with the missing factor can normalize values for clot stiffness
(panel A) and increases resistance to lysis (right hand panel).
Purified A2B2 plasma factor XIII (0.2 to 10031029 mol/L) was
used in the experiments in panel A, whereas the recombinant
A2 subunits of the zymogen were used (0 to 7 mg rA2) in
panel B. The 0.2 mL mixtures for the latter comprised 0.1 mL
of factor XIII-deficient citrated plasma, 0.6 U of thrombin,
12 mmol/L CaCl2, 25 mmol/L Tris-HCl buffer pH 7.5, and 20
IU TPA at 22°C (Lorand and Velasco, unpublished
data, 1999).
reduce normal clot stiffness to approximately 20% of
normal, corresponding to the value found in factor XIIIdeficient plasma.79 It is even more significant that the color
photographs in panel B of Figure 7 provide decisive
evidence that the specific inhibitors can also greatly
enhance the susceptibilities of thrombi for digestion by
lytic agents.47,48 These thrombi were formed from whole
human blood in rotating plastic tubes (Chandler loop) in
the absence and presence of a cross-linking inhibitor. They
were indistinguishable before the addition of urokinase,
but after 2 hours of exposure to the plasminogen activator,
only a minuscule residue remained of the thrombus that
was formed in the presence of the inhibitor, which was in
Lorand
Factor XIII Inhibition Promotes Thrombolysis
7
more possibilities for enhancing the fibrinolytic susceptibilities of thrombi.90 The Arg364Ala mutant, although no longer
a plasmin inhibitor, competes effectively against the factor
XIIIa-catalyzed incorporation of the wild type of a2PI into
fibrin. This approach is a special version of the competitive
blocking of the donor cross-linking sites in fibrin, as depicted
by reaction 3 in Figure 3b.]
The concept of utilizing inhibitors of clot stabilization as
presented in this article provides a basic framework for
developing new methodologies that, if translated to clinical
practice, could facilitate thrombolysis with much lower doses
of plasminogen activators than currently used. Significantly,
as clotting time would not be lengthened, the extra risk of
hemorrhage could be avoided.
Acknowledgment
Support of the National Institutes of Health (HL-02212 and HL16346) is gratefully acknowledged.
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References
Figure 7. Inhibitors of the factor XIIIa-catalyzed crosslinking
reactions greatly reduce clot stiffness (panel A) and also the
lytic resistance of thrombi (panel B). The ordinate of panel A
indicates clot stiffness at 60 minutes (Gi,609) in the presence
of crosslinking inhibitors, as percentages of the control value
(Go,609) without inhibitors. The color photograph in panel B
shows that, after 2 hours with 300 CTA units of urokinase
(UK) at 37°C, the thrombus formed in the presence of
1 mmol/L tosylcadaverine, an inhibitor of crosslinking (XL), is
completely lysed (d) whereas the control specimen (b) is still
quite undigested. Photographs (a) and (c) are control thrombi
without urokinase.
stark contrast to the essentially undigested thrombus
formed in the absence of the inhibitor.
The validity of the notion that interference with any aspect
of fibrin stabilization would produce similar effects is supported by numerous other experiments. Active center directed
inhibitors of factor XIIIa were shown to be highly effective in
reducing the viscoelastic moduli of clots38 and also for the
enhancement of clot lysis.45 A protein isolated from the giant
amazon leech was described to possess similar properties,
although its mode of interaction with factor XIIIa is not yet
known.81– 83
Positive results were obtained with a monoclonal antibody
directed against the thrombin cleavage site of factor XIII,
which blocked the activation of the zymogen.84 Another
interesting approach utilizes a monoclonal antibody against
a2PI.85 As mentioned before, the factor XIIIa-catalyzed
covalent attachment of a2PI to fibrin significantly contributes
to lytic resistance.40,41 In fact, the hemorrhagic manifestations
in patients with a2PI deficiency are being attributed to the
excessive digestibility of the clot.86 The antibody to a2PI was
shown to be effective in promoting clot lysis in vitro and
thrombolysis in vivo in animal models.85,87– 89 [Added in
proof: Recent results from a mutant form of a2PI point to
1. Lorand L. Research on clot stabilization provides clues for improving
thrombolytic therapies. Circulation. 1998;17(Supplement I):ID. Abstract.
2. Lorand L. A study on the solubility of fibrin clots in urea. Hung Acta
Physiol. 1948;1:192–196.
3. Laki K, Lorand L. On the solubility of fibrin clots. Science. 1948;
108:280.
4. Meissner I, Wöhlisch E. Untersuchungen über die Einwirkung hydrotroper Substanzen auf das fibrinogen und die Blutgerinnung. Biochem
Zietschrift. 1937;293:133–141.
5. Lorand L. “Fibrino-Peptide”: aspects of the fibrinogen-fibrin transformation. Nature. 1951;167:992–994.
6. Lorand L. Fibrino-peptide. Biochem J. 1952;52:200 –203.
7. Bettelheim FR, Bailey K. The products of the action of thrombin on
fibrinogen. Biochim Biophys Acta. 1952;9:578 –579.
8. Doolittle RF, Blomback B. Amino acid sequence studies on fibrinopeptides from various mammals: evolutionary implications. Nature.
1964;202:147–152.
9. Bailey K, Bettelheim FR, Lorand L, Middlebrook WR. Action of
thrombin in the clotting of fibrinogen. Nature. 1951;167:233.
10. Lorand L, Middlebrook WR. The action of thrombin on fibrinogen.
Biochem J. 1952;52:196 –199.
11. Lorand L, Middlebrook WR. Species specificity of fibrinogen as
revealed by end-group studies. Science. 1953;118:515–516.
12. Iwanaga S, Wallen P, Grondahl NJ, Henschen A, Blomback B. Isolation and characterization of N-terminal fragments obtained by
plasmin digestion of human fibrinogen. Biochim Biophys Acta. 1967;
147:606 – 609.
13. Laudano AP, Doolittle RF. Studies on synthetic peptides that bind to
fibrinogen and prevent fibrin polymerization. Structural requirements,
numbers of binding sites and species differences. Biochemistry. 1980;
19:1013–1019.
14. Henschen A, Lottspeich F, Kehl M, Southan C. Covalent structure of
fibrinogen. Ann N Y Acad Sci. 1983;408:28 – 43.
15. Kudryk BJ, Collen D, Woods KR, Blomback B. Evidence for localization of polymerization sites in fibrinogen. J Biol Chem. 1974;249:
3322–3325.
16. Côté HCF, Pratt KP, Davie EW, Chung DW. The polymerization
pocket “a” within the carboxyl-terminal region of the gamma chain of
human fibrinogen is adjacent to but independent from the calciumbinding site. J Biol Chem. 1997;272:23792–23798.
17. Spraggon G, Everse SJ, Doolittle RF. Crystal structures of fragment D
from human fibrinogen and its crosslinked counterpart from fibrin.
Nature. 1997;389:455– 462.
18. Nolan C, Hall LS, Barlow GH. Ancrod, the coagulating enzyme from
the Malayan pit viper (Agkistrodon rhodostoma) venom. Methods
Enzymol. 1976;45:205–213.
19. Stocker K, Barlow GH. The coagulant enzyme from Bothrops atrox
venom (Batroxobin). Methods Enzymol. 1976;45:214 –223.
20. Hall C, Slayter H. The fibrinogen molecule: its size, shape and mode
of polymerization. J Biophys Biochem Cytology. 1959;5:11–15.
8
Arterioscler Thromb Vasc Biol.
January 2000
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
21. Cohen C. Weisel JW, Phillips GN Jr, Stauffacher CV, Fillers JP, Daub
E. The structure of fibrinogen and fibrin: I. Electron microscopy and
X-ray crystallography of fibrinogen. Ann N Y Acad Sci. 1983;408:
194 –213.
22. Weisel JW, Phillips GN, Cohen C. The structure of fibrinogen and
fibrin: II. Architecture of the fibrin clot. Ann N Y Acad Sci. 1983;408:
367–379.
23. Veklich Y, Ang EK, Lorand L, Weisel JW. The complementary
aggregation sites of fibrin investigated through examination of
polymers of fibrinogen with fragment E. Proc Natl Acad Sci U S A.
1998;95:1438 –1442.
24. Lorand L, Doolittle RF, Konishi K, Riggs SK. A new class of blood
coagulation inhibitors. Arch Biochem Biophys. 1963;102:171–179.
25. Bruner-Lorand J, Urayama T, Lorand L. Transglutaminase as a blood
clotting enzyme. Biochem Biophys Res Comm. 1966;23:828 – 834.
26. Williams-Ashman HG, Notides AC, Pabalan SS, Lorand L. Transamidase reactions involved in the enzymic coagulation of semen:
isolation of g-glutamyl-e-lysine dipeptide from clotting secretion
protein of guinea pig seminal vesicle. Proc Natl Acad Sci U S A.
1972;69:2322–2325.
27. Lorand L, Ong HH. Studies on fibrin crosslinking. Nature of the
acceptor groups in transpeptidation. Biochem Biophys Res Comm.
1966;23:188 –193.
28. Lorand L, Ong HH, Lipinski B, Rule NG, Downey J, Jacobsen A.
Lysine as amine donor in fibrin crosslinking. Biochem Biophys Res
Comm. 1966;25:629 – 637.
29. Lorand L, Rule NG, Ong HH, Furlanetto R, Jacobsen A, Downey J,
Oner N, Bruner-Lorand J. Amine specificity in transpeptidation. Inhibition of fibrin crosslinking. Biochemistry. 1968;7:1214 –1223.
30. Matacic S, Loewy AG. The identification of isopeptide crosslinks in
insoluble fibrin. Biochem Biophys Res Commun. 1968;30:356 –362.
31. Pisano JJ, Finlayson JS, Peyton MP. Crosslink in fibrin polymerized
by factor XIII:e(g-glutamyl)lysine. Science. 1968;160:892– 893.
32. Chen R, Doolittle RF. Isolation, characterization and location of a
donor-acceptor unit from crosslinking fibrin. Proc Natl Acad Sci
U S A. 1970;66:472– 479.
33. Lorand L, Chenoweth D. Intramolecular localization of the acceptor
crosslinking sites in fibrin. Proc Natl Acad Sci U S A. 1969;63:
1247–1252.
34. McKee PA, Mattock P, Hill RL. Subunit structure of human
fibrinogen, soluble fibrin and crosslinked insoluble fibrin. Proc Natl
Acad Sci U S A. 1970;66:738 –744.
35. Mosesson MW, Siebenlist KR, Amrani DL, DiOrio JP. Identification
of covalently linked trimeric and tetrameric D domains in crosslinked
fibrin. Proc Natl Acad Sci U S A. 1989;86:1113–1117.
36. Mosesson MW, DiOrio JP, Siebenlist KR, Wall JS, Hainfeld JF.
Evidence for a second type of fibril branch point in fibrin polymer
networks, the trimolecular junction. Blood. 1993;82:1517–1521.
37. Murthy SNP, Lorand L. Crosslinked Aa z g chain hybrids serve as
unique markers for fibrinogen polymerized by tissue transglutaminase. Proc Natl Acad Sci U S A. 1990;87:9679 –9682.
38. Ryan, EK, Mockros LF, Weisel JW, Lorand L. Structural origins of
fibrin clot rheology. Biophys J. 1999;77:2813–2826.
39. Sakata Y, Aoki N. Significance of crosslinking of a2-plasmin inhibitor
to fibrin in inhibition of fibrinolysis and in hemostasis. J Clin Invest.
1982;69:536 –542.
40. Ichinose A, Tanaki T, Aoki N. Factor XIII-mediated crosslinking of
NH2 terminal peptide of a2-plasmin inhibitor to fibrin. FEBS Lett.
1983;153:369 –371.
41. Nilsson JLG, Stenberg P, Ljunggren C, Hoffman KJ, Lunden R,
Eriksson O, Lorand L. Fibrin-stabilizing factor inhibitors. Ann N Y
Acad Sci. 1972;202:286 –296.
42. Parameswaran KN, Velasco PT, Wilson J, Lorand L. Labelling of
e-lysine crosslinking sites in proteins with peptide substrates of factor
XIIIa and transglutaminase. Proc Natl Acad Sci U S A. 1990;87:
8472– 8475.
43. Lorand L, Parameswaran KN, Velasco PT, Murthy SNP. Biotinylated
peptides containing a factor XIIIa or tissue transglutaminase-reactive
glutaminyl residue block protein crosslinking phenomena by
becoming incorporated into amine donor sites. Bioconjugate Chem.
1992;3:37– 41.
44. Samama M, Soria J, Soria C, Maamer M, Otera AM. A new inhibitor
of factor XIII and platelet aggregation INO 3178. In: Davidson JF,
Cepelak V, Samama MM, Desnoyers PC (eds). Prog Chem Fibrinolysis Thrombolysis. New York: Churchill Livingstone; 1979;4:
235–240.
45. Freund KF, Doshi KP, Gaul SL, Claremon DA, Remy DC, Baldwin JJ,
Pitzenberger SM, Stern AM. Transglutaminase inhibition by 2-[(2oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa
inactivation. Biochemistry. 1994;33:10109 –10119.
46. Lorand L, Jacobsen A. Accelerated lysis of blood clots. Nature.
1962;195:911–912.
47. Bruner-Lorand J, Pilkington TRE, Lorand L. Inhibitors of fibrin
crosslinking: relevance for thrombolysis. Nature. 1966;210:
1273–1274.
48. Lorand L, Nilsson JLG. Molecular approach for designing inhibitors
to enzymes involved in blood clotting. In Ariens EJ (ed). Drug
Design. New York: Academic Press. 1972;3:415– 417.
49. Schwartz, ML, Pizzo SV, Hill RL, McKee PA. Human factor XIII
from plasma and platelets. Molecular weights, subunit structures,
proteolytic activation, and crosslinking of fibrinogen and fibrin. J Biol
Chem. 1973;248:1395–1407.
50. Siebenlist KR, Meh DA, Mosesson MW. Plasma factor XIII binds
specifically to fibrinogen molecules containing gamma chains. Biochemistry. 1996;35:10448 –10453.
51. Lorand L, Gray AJ, Brown K, Credo RB, Curtis CG, Domanik RA,
Stenberg P. Dissociation of the subunit structure of fibrin stabilizing
factor during activation of the zymogen. Biochem Biophys Res
Commun. 1974;56:914 –922.
52. Lorand L, Konishi K. Activation of the fibrin stabilizing factor of
plasma by thrombin. Arch Biochem Biophys. 1964;105:58 – 67.
53. Mikuni Y, Iwanaga S, Konishi K. A peptide released from plasma
fibrin stabilizing factor in the conversion to the active enzyme by
thrombin. Biochem Biophys Res Commun. 1973;54:1393–1403.
54. Nakamura S, Iwanaga S, Suzuki T, Mikuni Y, Konishi K. Amino acid
sequence of the peptide released from bovine factor XIII following
activation by thrombin. Biochem Biophys Res Commun. 1974;58:
250 –256.
55. Takagi T, Doolittle RF. Amino acid sequence studies on factor XIII
and the peptide released during its activation by thrombin. Biochemistry. 1974;13:750 –756.
56. Radek JT, Jeong J-M, Wilson J, Lorand L. Association of the A
subunits of recombinant placental factor XIII with the native carrier B
subunits from human plasma. Biochemistry. 1993;32:3527–3534.
57. Curtis CG, Brown KL, Credo RB, Domanik RA, Gray A, Stenberg P,
Lorand L. Calcium-dependent unmasking of active center cysteine
during activation of fibrin stabilizing factor. Biochemistry. 1974;13:
3774 –3780.
58. Parameswaran KN, Lorand L. New thioester substrates for fibrinoligase (Coagulation factor XIIIa) and for transglutaminase. Transfer of
the fluorescently labeled acyl group to amines and alcohols. Biochemistry. 1981;20:3703–3711.
59. Micanovic R, Procyk R, Lin W, Matsueda GR. Role of histidine 373
in the catalytic activity of coagulation factor XIII. J Biol Chem.
1994;269:9190 –9194.
60. Curtis CG, Stenberg P, Brown KL, Baron A, Chen K, Gray A,
Simpson I, Lorand L. Kinetics of transamidating enzymes. Production
of thiol in the reactions of thiol esters with fibrinoligase. Biochemistry. 1974;13:3257–3262.
61. Stenberg P, Curtis CG, Wing D, Tong YS, Credo RB, Gray A, Lorand
L. Transamidase kinetics. Amide formation in the enzymic reactions
of thiol esters with amines. Biochem J. 1975;147:155–163.
62. Pedersen LC, Yee VC, Bishop PD, Teller DC, Stenkamp RE. Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink
macromolecules. Protein Sci. 1994;3:1131–1135.
63. Naski MG, Lorand L, Shafer J. Characterization of the kinetic
pathway for fibrin promoting of a-thrombin-catalyzed activation of
plasma factor XIII. Biochemistry. 1991;30:934 –941.
64. Credo RB, Curtis CG, Lorand L. a-Chain domain of fibrinogen
controls generation of fibrinoligase (Coagulation factor XIII a ).
Calcium ion regulatory aspects. Biochemistry. 1981;20:3770 –3778.
65. Samokhin GP, Lorand L. Contact with the N-termini in the central E
domain enhances the reactivities of the distal D domains of fibrin to
factor XIIIa. J Biol Chem. 1995;270:21827–21832.
66. Lorand L, Parameswaran KN, Murthy SNP. A double-headed
GlyProArgPro ligand mimics the functions of the E domain of fibrin
for promoting the end-to-end crosslinking of g chains by factor XIIIa.
Proc Natl Acad Sci U S A. 1998;95:537–541.
67. Carr MF. Fibrin formed in plasma is composed of fibers more massive
than those formed from purified fibrinogen. Thromb Haemostasis.
1988;59:535–539.
68. Glover CJ, McIntire LV, Brown CH, Natelson EA. Rheological properties of fibrin clots. Effects of fibrinogen concentration, factor XIII
Lorand
69.
70.
71.
72.
73.
74.
75.
76.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
77.
78.
79.
deficiency, and factor XIII inhibition. J Lab Clin Med. 1975;86:
644 – 656.
Roberts WW, Lorand L, Mockros LF. Viscoelastic properties of fibrin
clots. Biorheology. 1973;10:29 – 42.
Mockros LF, Roberts WW, Lorand L. Viscoelastic properties of
ligation-inhibited fibrin clots. Biophys Chem. 1974;2:164 –169.
Lorand L, Urayama T, DeKiewiet JWC, Nossel HL. Diagnostic and
genetic studies on fibrin-stabilizing factor with a new assay based on
amine incorporation. J Clin Invest. 1969;48:1054 –1064.
Lorand L, Urayama T, Atencio AC, Hsia DY-Y. Inheritance of deficiency of fibrin-stabilizing factor (factor XIII). Am J Hum Genet.
1970;22:89 –95.
Lorand L, Losowsky MS, Miloszewski KJM. Human factor XIII:
Fibrin stabilizing factor. In: Spaet TH (ed). Progress in Hemostasis
and Thrombosis. New York:Grune & Stratton; 1980;5:245–290.
Ichinose A, Kaetsu H. Molecular approach to structure-function relationship of human coagulation factor XIII. Methods Enzymol. 1993;
222:36 –51.
Mikkola H, Yee VC, Syrjälä M, Seitz R, Egbring R, Petrini P, Ljung
R, Ingerslev J, Teller DC, Peltonen L, Palotie A. Four novel mutations
in deficiency of coagulation factor XIII: consequences to expression
and structure of the A-subunit. Blood. 1996;87:141–151.
Izume T, Hashiguchi T, Castaman G, Tosetto A, Rodeghiero F,
Girolami A, Ichinose A. Type 1 factor XIII deficiency is caused by a
genetic defect of its b subunit: insertion of triplet AAC in exon III
leads to premature termination in the second Sushi domain. Blood.
1996;87:2769 –2774.
Lorand L. Acquired inhibitors of fibrin stabilization: a class of hemorrhagic disorders of diverse origins. In: Green D (ed). Anticoagulants, Physiologic, Pathologic and Pharmacologic. Cleveland:
CRC Press; 1994;169 –191.
Lorand L, Velasco PT, Murthy SNP, Lefebvre P, Green D. Autoimmune antibody in a hemorrhagic patient interacts with thrombinactivated factor XIII in a unique manner. Blood. 1999;93:909 –917.
Shen L, Lorand L. Contribution of fibrin stabilization to clot strength.
Supplementation of factor XIII-deficient plasma with the purified
zymogen. J Clin Invest. 1983;71:1336 –1341.
Factor XIII Inhibition Promotes Thrombolysis
9
80. Edwards MW, deBang E, Strout J, Bishop PD. Recombinant factor
XIII supplemented clots resist lysis by plasmin and leucocyte elastase.
Fibrinolysis. 1993;7:211–216.
81. Seale L, Finney S, Sawyer RT, Wallis RB. Tridegin, a novel peptidic
inhibitor of factor XIIIa from the leech, Haementeria ghilianii,
enhances fibrinolysis in vitro. Thromb Haemost. 1997;77:959 –963.
82. Wallis RB, Seale L, Finney S, Sawyer RT, Bennett GM, Ross-Murphy
SB. Reduction of plasma clot stability by a novel factor XIIIa inhibitor
from the giant Amazon leech, Haementeria ghilianii. Blood Coagul
Fibrinolysis. 1997;8:291–295.
83. Finney S, Seale L, Sawyer RT, Wallis RB. Tridegin a new peptidic
inhibitor of factor XIIIa, from the blood-sucking leech Haementeria
ghilianii. Biochem J. 1997;324:797– 805.
84. Reed GL III, Lukacova D. Generation and mechanism of action of a
potent inhibitor of factor XIII function. Thromb Haemostasis. 1995;
74:680 – 685.
85. Reed GL III, Matsueda GR, Haber E. Synergistic fibrinolysis: Combined
effects of plasminogen activators and an antibody that inhibits a2 antiplasmin. Proc Natl Acad Sci U S A. 1990;87:1114 –1118.
86. Aoki N, Saito T, Kamiya T, Koie K, Sakata Y, Kobakura M. Congenital deficiency of a2-plasmin inhibitor associated with severe hemorrhagic tendency. J Clin Invest. 1979;63:877– 884.
87. Reed GL III, Matsueda GR, Haber E. Inhibition of clot bound
a2-antiplasmin enhances in vivo thrombolysis. Circulation. 1990;82:
164 –168.
88. Butte AN, Houng AK, Jang I-Kyung, Reed GL. a2-Antiplasmin causes
thrombi to resist fibrinolysis induced by tissue plasminogen activator
in experimental pulmonary embolism. Circulation. 1997;95:
1886 –1891.
89. Reed GL, Houng AK. The contribution of activated factor XIII to
fibrinolytic resistance in experimental pulmonary embolism. Circulation. 1999;99:299 –304.
90. Lee KN, Tae WC, Jackson KW, Kwon SH, McKee PA. Characterization
of wild-type and mutant a2-antiplasmins: fibrinolysis enhancement by
reactive site mutant. Blood. 1999;94:164 –171.
KEY WORDS: Fibrin
n Factor XIII n
inhibitors
n
thrombolysis
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Sol Sherry Lecture in Thrombosis: Research on Clot Stabilization Provides Clues for
Improving Thrombolytic Therapies
Laszlo Lorand
Arterioscler Thromb Vasc Biol. 2000;20:2-9
doi: 10.1161/01.ATV.20.1.2
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