(Received for publication, May 16, 1995; and in revised form, June 29, 1995)
From the
The reaction of Factor XIII with fibrin is the last
enzyme-catalyzed step on the coagulation cascade, leading to the
formation of a normal blood clot. The finding that fibrin is preferred
by the cross-linking enzyme about 10-fold over the circulating
fibrinogen suggests the operation of a unique substrate-level control
for the orderly functioning of the physiological process in the forward
direction. An important task is to elucidate the molecular mechanism
for the transmission of the signal generated by the thrombin-catalyzed
cleavage in the central E domain of fibrin to the distant Factor
XIII
-reactive glutamine residues. By focusing on the
substrate sites present in
chain remnants of D type
domains of fibrinogen and by employing the approach of fragment
complementation with the regulatory E domain, which represents
the thrombin-modified portion of fibrin, we have now succeeded in
reconstructing in solution the phenomenon of kinetic enhancement for
the reaction with Factor XIII
.
Two D type
preparations (truncated fibrinogen, 250 kDa and D`,
105 kDa)
were obtained by digestion of human fibrinogen with endo Lys-C. Neither
product could be cross-linked by Factor XIII
, but as shown
by the incorporation of dansylcadaverine, both were acceptor substrates
for the enzyme. The plasmin-derived D (
105-kDa) product,
however, could be cross-linked into DD dimers. In all cases,
the admixture of E fragments exerted a remarkable boosting
effect on the reactions with Factor XIII
. Even with native
fibrinogen as substrate, cross-linking of
chains was
enhanced in the presence of E. Nondenaturing electrophoresis
was used to demonstrate the complex forming potential of E
fragments with fibrinogen, truncated fibrinogen, D`, or D. The GPRP tetrapeptide mimic of the GPRV N-terminal sequence
of the
chains in the E fragments, abolished
both complex formation and the kinetic boosting effect of E on
the reactions of substrates with Factor XIII
. Thus, the
N-terminal
chain sequences seem to act as organizing templates
for spatially orienting the D domains, probably during the
protofibrillar assembly of the fibrin units, for favorable reaction
with Factor XIII
.
Stabilization of the fibrin network during the last phase of
blood coagulation by the Factor XIII system represents a tightly
regulated sequence of events. Though clotting times may be normal,
failures in regard to the functioning of any aspect of this system
could give rise to severe and frequently fatal
bleeding(1, 2) . The introduction of N(
-glutamyl)lysine cross-links by
activated Factor XIII (or Factor XIII
) strengthens the clot (3, 4, 5) and renders it more resistant to
lytic enzymes(6, 7) . Until now, attention was paid
mostly to the biochemical regulation of the conversion of circulating
Factor XIII (or fibrin stabilizing factor A
B
)
to Factor XIII
(or A
*). Activation of this
zymogen occurs in two distinct stages, catalyzed by thrombin and
promoted by Ca
, respectively:
[A
`B
]
A
* +
B
(8, 9) .
Fibrin, which is the
physiological substrate of the cross-linking enzyme, acts as a
``feed forward'' modulator in the conversion (10, 11, 12, 13, 14, 15) ,
ensuring that the transamidating enzyme (A*) is produced
within the physiologically required time frame for efficient clot
stabilization. Factor XIII
reacts first with the
cross-linking sites in the
chains of fibrin and then with those
in the
chains(16, 17, 18) .
The
known preferential reactivity of Factor XIII for fibrin
over fibrinogen (17, 19) is considered to be yet
another regulatory feature for the normal functioning of the
clot-stabilizing system. The thrombin-induced alteration in the central
N-terminal domains of fibrinogen (or, more precisely, the mere cleavage
of fibrinopeptide A from the A
chains(17) ) must generate
the signal for activating the distant, Factor XIII
-reactive
sites in the protein. The purpose of the present work is to shed light
on this unique substrate level modulation in blood coagulation. By
focusing on the
chain cross-linking sites of the substrate, we
succeeded in reconstructing in solution the regulatory mechanism by
complementing fragments of the Factor XIII
-reactive distal D regions of fibrinogen with the central thrombin-modified E domains of fibrin.
Human Factor XIII was purified from outdated blood bank
plasma (10, 20, 21) and was stored for up to
3 months at 4 °C at a concentration of 1 mg of protein/ml in 50
mM Tris-HCl, 1 mM EDTA, 1.3 kallikrein-inactivating
units/ml of Trasylol (Miles, Inc., West Haven, CT) at pH 7.5. Human -thrombin (4,570 NIH units/mg), a gift of Dr. J. W.
Fenton, III of the New York State Department of Health, Albany, NY, was
stored at -20 °C as 50 and 1,000 NIH units/ml solutions in 50
mM Tris-HCl, 150 mM NaCl, pH 7.5 (TBS). (
)Hirudin, 650 antithrombin units/mg, was purchased
from Sigma and was stored at -20 °C as 200 antithrombin
units/ml in TBS. Human fibrinogen (a gift from A. B. Kabi,
Stockholm, Sweden; lot 84639) was dissolved in TBS, dialyzed against 2
2 liters of TBS overnight at 4 °C, centrifuged, and stored
at -20 °C as a 4-6 mg/ml solution. Protein
concentration was determined by absorbance at 280 nm using
15.1(22) .
A truncated form of fibrinogen was prepared by digestion with
endoproteinase Lys-C (Promega, Madison, WI) by a procedure similar to
that described for bovine fibrinogen(23) . Human fibrinogen (50
mg) was dissolved in 5 ml of TBS and dialyzed against 2 liters of the
same overnight at 4 °C. Then 25 µl of 1 M CaCl was added to a concentration of 5 mM, and the mixture
was warmed to 37 °C 30 min prior to adding 5 µl of 20 units/ml
endoproteinase Lys-C (final concentration of 0.02 units/ml). Following
incubation at 37 °C for 2.5 h, 50 µl of 100 mMN
-p-tosyl-L-lysine
chloromethyl ketone (Sigma) in TBS was added, to a concentration of 1
mM. The digest was dialyzed against 2 liters of TBS
(overnight, 4 °C), and the truncated fibrinogen was
purified by gel filtration on an Ultrogel AcA 44 column (LKB, Reactifs
IBF, Pharmindustrie, France; 2.6 x 87 cm, equilibrated with 0.1 M NH
HCO
, pH 8.0, and eluted with a flow rate
of 30 ml/h at room temperature). Fractions of 5 ml were collected and
analyzed by SDS-PAGE(24) ; those eluting with the void volume
were pooled, lyophilized, dissolved in TBS, and dialyzed at 4 °C
against the latter overnight. The protein concentration of the product
was determined by the bicinchoninic acid assay (Pierce; (25) )
using human fibrinogen as reference, and the material (8.0 mg/ml) was
stored at -20 °C. The preparation was free of the intact
fibrinogen starting material and comprised one major protein band of
about 250 kDa in SDS-PAGE under nonreducing conditions. In reducing
SDS-PAGE (40 mM dithiothreitol (Sigma) in the sample buffer,
100 °C, 3 min) three components were found with molecular masses of
48, 42, and 28 kDa, respectively. The 48-kDa band was actually a
doublet. When examined by Western blotting (26) the
preparation showed no reactivity to the following monoclonal antibodies
against various regions of the human fibrinogen molecule: F-102,
anti-A
563-578; F-103, anti-A
259-276; 5A2,
anti-A
529-539; 1D4, anti-A
389-402; and
1-8C6, anti-B
1-21 (the first two were kindly provided
by Dr. J. H. Sobel of the College of Physicians and Surgeons, Columbia
University, New York(27) ; the third was raised by G. P.
Samokhin at the National Cardiology Research Center (Moscow); the
fourth and the fifth were gifts from Dr. B. Kudryk of New York Blood
Center, New York(28) ). However, positive immunostaining was
obtained for the 48-kDa band, seen in the reducing SDS-PAGE profile,
with two monoclonal antibodies directed against the C-terminal region
of the
chain in fibrinogen: 4-2, anti-
392-406;
and 4A5, anti-
402-411 (gifts from Dr. B. Kudryk (28) and Dr. G. Matsueda of Bristol-Myers Squibb Pharmaceutical
Research Institute, Princeton, NJ (29) , respectively).
The
distal D fragment of fibrinogen was obtained by activation of
endogenous plasminogen with urokinase (109,127 units/mg, Calbiochem, La
Jolla, CA) according to some modifications of a previously published
procedure(30) . To 5 ml of human fibrinogen solution (50 mg of
protein in TBS, exhaustively dialyzed at 4 °C), 25 µl of 1 M CaCl was added. After warming to 37 °C (30
min), 5 µl of 5,000 units/ml human urokinase was admixed, followed
by incubation at 37 °C for 80 min. To terminate plasmic digestion,
Trasylol (100 µl of 10,000 kallikrein-inactivating units/ml) was
added, and the mixture was dialyzed against 500 ml of TBS at 4 °C
for 2 days. Purification of the D fragment was accomplished by
gel filtration on an Ultrogel AcA 44 column as described above for truncated fibrinogen with inclusion of 5
kallikrein-inactivating units/ml of Trasylol in the eluting buffer.
Fractions of 5 ml were collected. When analyzed by nonreducing
SDS-PAGE(24) , the first emerging protein peak was found to
comprise intact fibrinogen, X and Y
fragments(31, 32, 33, 34) , whereas
the early portion of the second major peak showed a mixture of D and E
fragments(32, 33, 34, 35) . The last
three fractions of this peak, however, contained D fragments
alone. These fractions were pooled, lyophilized, and dialyzed as
described for truncated fibrinogen. The preparation (11.8
mg/ml, bicinchoninic acid assay; Pierce(25) ), comprising
approximately equal amounts of D
and D
(36) as judged by nonreducing SDS-PAGE, was stored at
-20 °C.
The central domain E fragment was
purified from cross-linked fibrin according to some modifications of a
previously published procedure(37, 38, 39) .
To 10 ml of human fibrinogen (100 mg of protein in TBS, exhaustively
dialyzed at 4 °C), 50 µl of 1 M CaCl was
added. After warming to 37 °C (30 min), 5 µl of 1 mg/ml of
human Factor XIII and 10 µl of 1,000 NIH units/ml of human
-thrombin was admixed, followed by incubation at 37 °C
overnight. Human plasminogen (20.7 casein units/mg, Chromogenix AB
(Molndal, Sweden)) was activated by mixing 4 µl of 1 mg/ml solution
in TBS with 0.5 µl of 5,000 units/ml of human urokinase followed by
incubation for 1 h at 37 °C. The cross-linked clot was dispersed,
2.5 µl of activated plasminogen solution was added, and the mixture
was incubated overnight at 37 °C on a shaker. Trasylol (25 µl
of 10,000 kallikrein-inactivating units/ml solution) was added, and the
digest was dialyzed against 2 liters of TBS overnight at 4 °C.
Purification of the DD
E complex was accomplished by gel
filtration on an Ultrogel AcA 44 column as described above for truncated fibrinogen. Fractions of 5 ml were collected. When
analyzed by nonreducing SDS-PAGE(24) , the first emerging
protein peak was found to comprise the DD
E complex (37) .
Pooled and lyophilized fractions (57 mg by weight) were dissolved in
5.7 ml of 150 mM sodium citrate, 3 M urea at pH 5.5
and incubated at 37 °C for 5 h. Purification of the dissociated E fragments was accomplished by gel filtration on an Ultrogel
AcA 44 column as described above for truncated fibrinogen;
however, the column was equilibrated with 50 mM sodium
citrate, 3 M urea, pH 5.5. Fractions emerging from the column
in the second peak were pooled, dialyzed against 2
2 liters of
0.1 M NH
HCO
, pH 8.0, overnight at 4
°C, lyophilized, and dialyzed against TBS as described for truncated fibrinogen. The preparation (15.8 mg/ml,
bicinchoninic acid assay; Pierce(25) ) was comprised of 39%
E
, 48% E
, and 13% E
(40) ,
as determined by nonreducing SDS-PAGE(24) . It was stored at
-20 °C.
The distal D-like fragment (D`) of fibrinogen was also obtained by digestion with endoproteinase Lys-C as described for the truncated fibrinogen, with the exception that the digestion time was increased to 4 h at 37 °C, and the fractions emerging from the column in the second peak were pooled and lyophilized. The preparation comprised two protein bands of 110 and 105 kDa in SDS-PAGE under nonreducing conditions. In reducing SDS-PAGE, four components were identified with molecular masses of 45, 42, 16, and 14 kDa, respectively. The purified material (8.6 mg/ml, bicinchoninic acid assay; Pierce(25) ) was stored at -20 °C.
Dansylcadaverine fumarate (melting point, 148-150
°C) was prepared by Dr. K. N. P. Parameswaran from a commercially
purchased monodansylcadaverine (Sigma). Concentrations of its solutions
in TBS were calculated from absorbance at 327 nm,
=4,670 M
cm
(41) .
Activation of Factor XIII
to XIII by thrombin and Ca
was carried
out at room temperature for 30 min using 50 NIH units of thrombin/mg of
zymogen. Thus, a typical mixture sufficient for multiple experiments
(36 µl) was comprised of 4 µl of Factor XIII (1 mg/ml stock), 4
µl of thrombin (50 NIH units/ml stock), 26 µl of TBS, and 2
µl of CaCl
(100 mM stock) in TBS. At the end
of the incubation period, in most experiments, thrombin activity was
quenched by addition of 4 µl of hirudin (Sigma; 200 units/ml
stock). Otherwise, 4 µl of thrombin (same stock as above) was added
instead of hirudin.
Reaction of fibrin occurs much faster with Factor XIII than reaction of the parent fibrinogen
molecule(17, 19) . Depending on individual
preparations and clotting conditions, a 4-15-fold increase in
rate is observed for fibrin for the enzyme-catalyzed incorporation of
small primary amine probes, such as glycine ethyl ester, hydroxylamine,
hydrazine, or dansylcadaverine. Placed in the framework of other
physiologically important regulatory processes, this rate differential
(matching, for example, the difference in clotting times between a
normal and a hemophilic plasma) is of obvious significance. The
difference in rates for incorporation of about 2 equivalents of amines
reflects mainly on the reactivities of
chain acceptor
sites(16) , located in the distal D domains of the
protein(45) . The
chain acceptors, themselves capable of
reacting with 4-6 equivalents of amines become engaged
later(16, 17, 46) .
The kinetic advantage
for fibrin over fibrinogen can be seen also when, instead of thrombin,
a snake venom enzyme, Arvin, is employed for clotting(17) .
Since Arvin cleaves only fibrinopeptide A from the N termini of
the A chains, this limited proteolytic alteration of fibrinogen
alone seems to be responsible for triggering the enhanced reactivity of
amine acceptor sites for Factor XIII
. However, the relevant
N-terminal region of the protein, located in its central E
domain, is far removed from the Factor XIII
-reactive
glutamine residues (45, 46, 47) . This poses
the intriguing question as to what the actual mechanism of the long
range communication between the two distant loci might be.
In the present work, we focused exclusively on the reactivities of
the chain acceptor sites in the D domains of
fibrin(ogen). Reactivity to Factor XIII
was probed by the
incorporation of dansylcadaverine; reaction products were measured
quantitatively (42, 43, 44) and were also
documented on the SDS-PAGE profiles of the proteins photographically
under UV illumination(17) . Three partially degraded forms of
human fibrinogen were used as substrates. Digestion of the parent
protein with endo Lys-C yielded a
250-kDa species, referred to as truncated fibrinogen, and also a
105-kDa fragment, called D`, while digestion of fibrinogen with plasmin produced the
known D fragments. The latter comprise the
A
-(105-206)B
-(134-461)
-(63/86-411)
segments of the protein (48) . On the basis of a variety of
tests performed (modification by thrombin, SDS-PAGE, Western blots with
antibodies, and lack of cross-linking by Factor XIII
) (data
not shown), coupled with known amino acid sequences and proteolytic
cleavage sites (49, 50, 51) , it may be
suggested with a certain degree of confidence that the constituent
chain composition of the endo Lys-C truncated fibrinogen may
be represented as
(A
-(1-206/219/230)
-(54/55/59-461)
-(1-406))
,
and that the endo Lys-C-derived D` fragment is comprised of
-(79/82-206/219/230)
-(134-461)
-(63/86-406).
Figure 1:
Exposure of endo Lys C-truncated
fibrinogen to thrombin increases the rate of the Factor
XIII-catalyzed incorporation of dansylcadaverine. The
Factor XIII zymogen was preactivated with thrombin and Ca
and, upon complete conversion to XIII
, it was mixed
(to yield 0.06 µM Factor XIII
) with truncated fibrinogen (4 µM), dansylcadaverine (2
mM), CaCl
(5 mM), and EDTA (0.1
mM) in TBS at 37 °C. Hirudin (in 4-fold excess) was added
to quench thrombin activity in the controls (opencircles), whereas thrombin (maintained at 2 NIH units/ml)
was allowed to interact with the truncated fibrinogen substrate in the
experimental mixtures (solidcircles). The insets show the corresponding SDS-PAGE (12.5% gel, reducing) profiles for
the 30- and 60-min time points of experimental (solidsymbols) and control (opensymbols)
mixtures. Profiles obtained by staining with Coomassie Brilliant Blue
are on the left, and those taken under UV illumination,
revealing the dansylcadaverine-modified protein bands, are on the right. The
` and
" designations show the positions
of the A
chain remnants of truncated fibrinogen before
and after cleavage by thrombin.
The next issue was to test whether the Factor
XIII reactivity of sites could be enhanced without actually
modifying the truncated fibrinogen substrate, itself, by
thrombin. In these experiments, a fixed concentration (4
µM) of truncated fibrinogen was mixed with
varying concentrations (0-16 µM) of the preparation
of E fragments, representing the thrombin-modified central
domains of fibrin. As seen in Fig. 2, admixture of E
produced a dramatic enhancement in the rate of dansylcadaverine
incorporation into the Factor XIII
-reactive sites of truncated fibrinogen. This finding, coupled with the
observation that the aggregation-inhibitory (52) tetrapeptide
GPRP (5 mM; Oz Chemical Co., Israel), abolished the effect,
may be taken to indicate that a direct contact with the E
domain was required for activating the substrate for Factor
XIII
. Maximal rate enhancement was obtained at an
approximate 1:1 molar ratio of truncated fibrinogen to E fragment added. The results predicted that truncated fibrinogen would form a complex with the E
fragments. Indeed, the existence of such a complex in solution could be
readily demonstrated by nondenaturing electrophoresis (Fig. 3).
Figure 2:
Complementation with E fragments,
representing the thrombin-modified central domains in fibrin, promotes
the reaction of truncated fibrinogen with Factor XIII for
incorporation of dansylcadaverine. Endo Lys C-truncated
fibrinogen was incubated with CaCl
, dansylcadaverine, and
varying concentrations of E. The control mixtures contained
also the tetrapeptide GPRP. Factor XIII
, which was
preactivated by thrombin plus Ca
(and with thrombin
quenched by addition of 4-fold excess of hirudin) was added (to 0.06
µM) at 40 min (37 °C). Final concentrations of the
components in the experimental (solidcircles) and
control (opencircles) mixtures were as follows: truncated fibrinogen, 4 µM; E,
0-16 µM; CaCl
, 5 mM;
dansylcadaverine, 2 mM; EDTA, 0.1 mM. In addition,
GPRP (5 mM) was present in the control mixtures. Reactions
were allowed to proceed for 60 min at 37 °C in TBS. The insets show the SDS-PAGE (12.5% gel, reducing) profiles pertaining to the
60-min time points for the experimental (left, solidcircles) and control (right, opencircles) mixtures, with Coomassie Brilliant Blue-stained
patterns on the left and UV illumination on the right for each.
Figure 3:
The endo Lys C-truncated fibrinogen can
form stable complexes with the E fragments of fibrin in solution. Truncated fibrinogen (t; 4 µM) was
incubated with 0-16 µM of E fragments in
TBS containing 5 mM CaCl
for 60 min at 37 °C.
The control mixtures contained also the tetrapeptide GPRP (5
mM). Glycerol (68% (v/v) in water) was added to a
concentration of 6% (v/v), and the samples were analyzed by
nondenaturing electrophoresis (4-20% gradient gel, pH 8.8,
Bio-Rad, Laemmli (24) system without SDS and stacking gel).
Gels were stained with Coomassie Brilliant Blue. Positions of the
complexes between truncated fibrinogen and E are
marked as t
:E, whereas the positions of the free E species are indicated as E
, E
, and E
.
If E fragments had a similar ability to bind to native
fibrinogen, reactivity of the latter substrate for Factor XIII might also be up-regulated. While some researchers could not find
evidence for complex formation between native fibrinogen and E
fragments(53) , others were able to document the slow
production of complexes of fibrinogen with E and also with
N-DSK fragments (a near equivalent of E) growing into large
aggregates(54, 55, 56, 57, 58, 59) .
In reexamining this issue by nondenaturing electrophoresis, using
either the Pharmacia PhastSystem gels (Fig. 4) or the Bio-Rad
gradient gel system (data not shown), native fibrinogen could
definitely be shown to bind E fragments in solution. Moreover,
in tune with the observations on truncated fibrinogen (Fig. 2), the rate of the Factor XIII
-catalyzed
reaction with native fibrinogen could also be significantly increased
by admixture of E fragments without the necessity of including
thrombin in the medium (Fig. 5). Again, maximal enhancement of
chain cross-linking for the native fibrinogen was obtained by
supplementation with equimolar E, and the GPRP tetrapeptide
abolished the augmenting effect of E fragments.
Figure 4:
Fibrinogen can form stable complexes with
the E fragments in solution. Fibrinogen (2 µM) was mixed
with CaCl (5 mM) and varying concentrations of E fragments (0-8 µM) in TBS. After 60 min
of incubation (37 °C), samples were applied for nondenaturing
electrophoresis (4-15% gradient gel, pH 8.8, 15 °C, 300 V-h;
Pharmacia PhastSystem). Gels were stained with Coomassie Brilliant Blue
and scanned. Complexes between fibrinogen (
) and E appeared near the site of sample application (marked as
E
).
The percentage of bound fibrinogen was calculated from the decrease in
the areas representing free fibrinogen in the
gel.
Figure 5:
The
presence of E fragments accelerates the cross-linking of fibrinogen by
Factor XIII. Fibrinogen was mixed with varying
concentrations of E fragments in the presence of
CaCl
; after a 40-min incubation at 37 °C, Factor
XIII
(preactivated and then mixed with hirudin) was added
to 0.06 µM. The cross-linking reaction was allowed to
proceed for 60 min (37 °C). Final concentrations of components with
TBS in the experimental (solidcircles) and control
mixtures (opencircles) were as follows: fibrinogen,
4 µM; E, 0-16 µM;
CaCl
, 5 mM; EDTA, 0.1 mM. The control
mixtures contained 5 mM GPRP in addition. The insets show the SDS-PAGE (10% gel, reducing), Coomassie Brilliant
Blue-stained profiles pertaining to the 60-min time points for both the
experimental (left) and control (right) mixtures. The
gels were scanned, and the degree of
chain cross-linking was
calculated as 100%
(
-
)/((
-
)+(
)).
Figure 6:
Complementation with E fragments greatly
enhances the reactivity of D` fragments to Factor XIII. The
experimental protocol and procedures were identical to those in Fig. 2, except that endo Lys C-derived D` (8
µM) was used instead of truncated fibrinogen as
substrate. The insets show the SDS-PAGE (12.5% gel, reducing) profiles
pertaining to the 60-min time points for the experimental (upper) and control mixtures (lower), with Coomassie
Brilliant Blue staining on the left and with UV illumination
on the right.
Figure 7:
Admixture of E fragments enhances the
cross-linking of plasmin-derived D fragments by Factor
XIII. The experimental protocol and procedures were
identical to those in Fig. 5, except that D fragments
(8 µM) were used instead of fibrinogen. The insets show the SDS-PAGE (7.5% gel, nonreducing) profiles pertaining to
the 60-min time points for the experimental (solidcircles) and control mixtures (opencircles). Molecular mass markers are shown on the right. Gels were scanned, and the percentage of D
cross-linking was calculated as 100%
DD/(DD +
D)].
Employing the
plasmin-derived D fragment of fibrinogen as substrate, the
effect of complementation by E could be examined for the
formation of -
chain cross-linked D-dimers under the
influence of Factor XIII
. This reaction, too, was greatly
promoted by the admixture of E fragments (Fig. 7). As
with the reaction involving D` (Fig. 6), the GPRP
tetrapeptide abolished the positive modulatory influence of E,
indicating that a direct contact with E was necessary to
activate the Factor XIII
-reactive sites present in both D` and D fragments.
A review of the literature
suggests that, in previous efforts, considerable difficulties have been
encountered for directly demonstrating complex formation between
monomeric D fragments (from the plasmic digest of fibrinogen)
and E in solution. However, in solid phase assays, D
could be shown to bind E. Moreover, in mixtures of D
with E, products corresponding to 1D1E
and 2D
1E could be generated through the action
of a bifunctional chemical cross-linking agent(30) . As
presented in Fig. 8, with nondenaturing electrophoresis using
the Bio-Rad gradient gel system, we found definite evidence for the
existence of complexes of D`
E and D
E in solution. Similar findings were obtained
with the Pharmacia Phast System (data not shown).
Figure 8:
The endo Lys C-derived D` fragments as
well as the plasmin-derived D fragments of fibrinogen can form stable
complexes with the E fragments of fibrin in solution. Either the D` (panels A and B) or the D fragments (panelsC and D) were
incubated at a concentration of 8 µM with 0-16
µM of E fragments in TBS containing 5 mM CaCl for 60 min at 37 °C. Control mixtures
contained the tetrapeptide GPRP (5 mM) in addition. Prior to
electrophoresis, glycerol (68% (v/v) in water) was added to a
concentration of 6% (v/v), and the samples were analyzed by a
nondenaturing procedure (4-20% gradient gel, pH 8.8, Bio-Rad,
Laemmli (24) system without SDS and stacking gel). Gels were
stained with Coomassie Brilliant Blue. The positions of complexes
between D` and E and between D and E are marked as D`:E and D:E, respectively.
The positions of free D`, D, E
,
E
, and E
are also
marked.
The specific
interactions of substrates with the E fragments must be
critical for functionally up-regulating their Factor
XIII-reactive sites. It is important to point out that the
addition of E fragments had no influence whatever on the
Factor XIII
-catalyzed incorporation of dansylcadaverine
into Ndimethylated casein as a test substrate (data not
shown).
Formation of the copulation plug in rodents represents
another example of a transglutaminase-driven biological clotting
phenomenon in which a specific substrate level regulation was found to
play a major role. The postejaculatory clotting of proteins discharged
from the seminal vesicles is brought about by the
Ca-dependent enzyme secreted simultaneously from the
anterior lobe of the prostate. Acid glycoproteins derived from the
bulbo-urethral (Cowper's) gland greatly accelerate formation of
the coagulum(60, 61, 62) . In the
reconstruction of this clotting system with purified components,
macromolecular polyanions could substitute for the Cowper's gland
secretion. The polyanions seem to exert a positive modulatory effect by
interacting specifically with the seminal vesicle secretion proteins so
as to enhance their proclivity for acting as transglutaminase acceptor
substrates(63) . The situation is quite analogous to the effect
exerted by the E fragments for specifically promoting the
reactions of the fibrinogen-derived substrates for Factor
XIII
.
In tracing the path for the transmission of the
up-regulating signal from the thrombin-modified central E
domain of fibrin to the Factor XIII-reactive distal
glutamine residues, the following must be borne in mind. The GPRP
tetrapeptide, a mimic for the GPRV N-terminal sequence of the
chains of fibrin generated by the cleavage of fibrinopeptide
A(52) , in our experiments abolished both the complex-forming
ability and the kinetic boosting effect of E on the reactions
of D type fragments with Factor XIII
. Thus, the
GPRV ligand in E may be construed to be the initiating signal
for the observed phenomenon. This N-terminal
chain sequence in E is suggested to act as an organizing template to spatially
orient the bound D domains for favorable reaction with Factor
XIII
. The E domain, in a tether-like fashion,
would promote the noncovalent dimerization of D domains. Such
interaction of E with the D domains is known to take
place (64) during the protofibrillar assembly of fibrin units
into half-staggered arrays (with a periodicity of 46:2 = 23
nm;(64) ). The combining site for the GPRV ligand is likely to
be in the vicinity of D domain residues involved in binding
the competing GPRP tetrapeptide, i.e. near Tyr-363 in the
chains(65, 66) . How the effect of the binding
of E to D at this site is further transmitted down
the line within the D domain to boost the reactivity of
Gln-398 in the
chains still remains to be elucidated. It is
clear, however, that the E to D contact carries a
significant functional consequence for the entire clotting process by
boosting the catalytic efficiency for the Factor
XIII
-reactive glutamine residues by about an order of
magnitude.