Originally published In Press as doi:10.1074/jbc.M004142200 on June 2, 2000
J. Biol. Chem., Vol. 275, Issue 33, 25239-25246, August 18, 2000
Recombinant Fibrinogen Studies Reveal That Thrombin Specificity
Dictates Order of Fibrinopeptide Release*
Jennifer L.
Mullin
,
Oleg V.
Gorkun§,
Cameron G.
Binnie§¶, and
Susan T.
Lord§
From the Departments of Chemistry and
§ Pathology and Laboratory Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7525
Received for publication, May 15, 2000
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ABSTRACT |
During cleavage of fibrinogen by thrombin,
fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release.
To examine the basis for this ordered release, we synthesized A'
fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA'
(G14V). Analyses of fibrinopeptide release from A' fibrinogen showed that FpA release and FpA' release were similar; the release of either
peptide followed simple first-order kinetics. Specificity constants for
FpA and FpA' were similar, demonstrating that these peptides are
equally competitive substrates for thrombin. In the presence of
Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB
release from normal fibrinogen was reduced 3-fold, consistent with
previous data; in contrast, the rate of FpA' release from A'
fibrinogen was unaffected. Thus, with A' fibrinogen, fibrinopeptide release from the chain is similar to fibrinopeptide release from the 
chain. We conclude that the ordered release of
fibrinopeptides is dictated by the specificity of thrombin for its
substrates. We analyzed polymerization, following changes in turbidity,
and found that polymerization of A' fibrinogen was similar to that
of normal fibrinogen. We analyzed clot structure by scanning
electron microscopy and found that clots from A' fibrinogen were
similar to clots from normal fibrinogen. We conclude that premature
release of the fibrinopeptide from the N terminus of the chain does
not affect polymerization of fibrinogen.
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INTRODUCTION |
Fibrinogen is a 340-kDa plasma protein that is involved in the
final phase of the coagulation cascade. Fibrinogen consists of two
pairs of three polypeptide chains (A, B, and 
) that fold to
produce a trinodular protein with two distal (D) nodules connected to a
central nodule (E) by coiled-coil regions. The central nodule of the
molecule consists of the N termini of all six polypeptide chains, and
the D nodules consist predominantly of the C termini of the and chains, each folded into a globular domain. To initiate polymerization,
the serine protease thrombin cleaves four specific Arg-Gly bonds at
the N termini of both the A and B chains, releasing
fibrinopeptides A (FpA)1 and
B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the "A" site, which noncovalently interacts with the
"a" site in the chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1). The release of FpB, a 14-residue peptide, exposes the "B" site (2-4), which presumably interacts with a "b" site in the chain of the D nodule of another
molecule (5). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2) and to be
analogous to the A:a interaction; however, the mechanism of this
interaction is not yet well understood. The final product of this
polymerization is a complex, branching network of fibers.
The interactions of thrombin with fibrinogen have been extensively
studied (Refs. 6 and 7; for a review, see Ref. 8). Thrombin contains
three domains that interact with fibrinogen: the active site, an apolar
specificity pocket, and a fibrinogen-binding exosite. The exosite, also
called the fibrinogen recognition site, confers the specificity with
which thrombin binds to fibrinogen. Upon binding, thrombin cleaves FpA
and initiates polymerization. FpA release from fibrinogen follows
first-order kinetics, described by the kinetic constant
k1. In contrast, FpB is released from fibrinogen
at a slow initial rate (3, 9, 10), which is then increased upon
polymerization (2, 9-11) and the depletion of FpA as a substrate for
thrombin. This efficient release of FpB, subsequent to FpA release,
follows first-order kinetics and is described by the kinetic constant
k2, assuming that the release of fibrinopeptides
A and B from thrombin occurs through two successive first-order processes.
Crystallographic data have depicted the contacts between FpA and
thrombin (12); but, to date, similar data are not available for
thrombin and FpB. The sequence of FpB is different from that of FpA
(13) such that FpB should require different contacts with thrombin
(12). Because we do not have structural information, our understanding
of FpB release is indirect and based on kinetic studies that examined
the timing of FpB release. These studies have shown that the majority
of FpB is released from fibrin after FpA has been removed (3, 14) and
that the rate of FpB release is enhanced upon polymerization of des-A
polymers (2-4, 15). To date, however, the mechanism responsible
for the delay in FpB release remains undetermined.
To extend our understanding of the mechanism of thrombin on
fibrinopeptide B and its resulting effect on polymerization, we have
designed a variant recombinant fibrinogen (A') that contains a
fibrinopeptide A-like substrate on the N termini of the chains. This fibrinogen was designed to probe the mechanism of fibrinopeptide release and more specifically to determine whether the delayed release
of FpB is a consequence of its affinity for thrombin or of its location
on the N terminus of the chain. Our studies have revealed that it
is the specificity of thrombin for FpB that is responsible for the
order of fibrinopeptide release during fibrin polymerization.
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EXPERIMENTAL PROCEDURES |
Materials--
HEPES, 
-aminocaproic acid, sodium phosphate,
and all other reagents were obtained from Sigma unless otherwise noted.
Human -thrombin was purchased from Enzyme Research Labs (South Bend, IN); two different lot numbers were used for this work: HT1330, 1.85 mg/ml equivalent to 5655 units/ml; and HT1540PA, 1.96 mg/ml equivalent
to 6392 units/ml. Human -thrombin (4300 units/ml, 1.71 mg/ml) was a
generous gift from Dr. Frank Church (University of North Carolina,
Chapel Hill). Recombinant hirudin was purchased from
Calbiochem-Novabiochem. All restriction enzymes were obtained from New
England Biolabs Inc. (Beverly, MA). Glutaraldehyde, osmium tetroxide,
and sodium cacodylate were all electron microscopy-grade materials obtained from Electron Microscopy Sciences (Fort Washington, PA). Eight-well strip polymerase chain reaction tubes were
obtained from NalgeNunc (Naperville, IL). UV-transparent 96-well
microtiter plates (catalog no. 3635) were purchased from Corning
Costar. All plasmid vectors, Chinese hamster ovary cells, bacteria, and culture medium have been previously described (16). Monoclonal antibody
IF-1 (17) was obtained from Iatron Lab, Inc. (Tokyo).
Expression Vector Construction--
Cloning procedures and
vectors were as described previously (16). The expression plasmid for
the variant A' chain (pMLP-A') was assembled from two encoding
fragments and the expression vector p284. A Gly-to-Val substitution at
position 14 was introduced by oligonucleotide-directed mutagenesis of
the human A chain cDNA cloned in a single-stranded phage using
the T7-GEN in vitro mutagenesis kit (U. S. Biochemical
Corp.) and the oligonucleotide 5'-AAC GCG TGG GCC ACG CAC
GAC TC. The resulting clone encoded Val at position 14 of the A
chain and contained a new MluI site (underlined). The
altered segment was isolated from this clone as an 870-bp
PvuII/BglII fragment (40 bp of 5'-untranslated
sequence, the signal sequence, and codons for A' residues 1-174)
and ligated to a 1360-bp BglII/SspI fragment
(codons for A' residues 174-625 plus 10 bp of 3'-untranslated
sequence) isolated from a plasmid containing the A cDNA; the
product fragment (2230 bp) was ligated into expression vector p284
cleaved with SmaI, and the products were transformed into
competent DH5 cells. Plasmid DNAs from individual colonies were
screened by restriction enzyme analysis, including the newly introduced
MluI site, and candidate clones were sequenced as described
(16). The complete A' coding segment was sequenced to demonstrate
that the desired codon change was present, and no unanticipated changes
were introduced.
The expression plasmid for the variant A' chain (pMLP-A') was
prepared by ligation of a polymerase chain reaction fragment encoding
the chain signal sequence and FpA' and a fragment encoding B
residues 15-461 into expression vector p284. The polymerase chain
reaction fragment was prepared by amplification of a segment from
pMLP-A' with oligonucleotides
5'-TCCACAACCCTTGGGCCACGGACG (which introduced a
DsaI site, underlined) and 5'-TTCAGATCTGGCCATACACTT; the
product was cleaved with SalI and DsaI, and the
156-bp fragment was isolated by electroelution following agarose gel
electrophoresis. The fragment encoding B residues 15-461, along
with the stop codon and 10 bp of 3'-untranslated sequence, was isolated
from pMLP-B as a 1350-bp fragment following cleavage with
DsaI and SnaBI. The p284 vector was cleaved with
SmaI and SalI. Following ligation into the
vector, individual colonies were screened by restriction enzyme
analysis, and candidate clones were sequenced. The sequence
demonstrated that the junction between FpA' and B residue 14 was as
intended and that no unanticipated changes were introduced.
The new plasmids, named pMLP-A' and pMLP-A', were used for
synthesis of A' and A' fibrinogens, respectively, in
Chinese hamster ovary cells. pMLP-A' was cotransfected with pMSVhis
into cells previously transfected with plasmids pMLP-B, pMLP-,
and pRSV-neo as described (16). Similarly, pMLP-A' was cotransfected with pMSVhis into cells previously transfected with plasmids pMLP-A, pMLP-, and pRSV-neo. Clones were selected based on neomycin and histidinol resistance and screened for fibrinogen secretion by enzyme-linked immunosorbent assay.
Recombinant Fibrinogen Synthesis and Purification--
The
normal, A', and A' clones secreting the highest fibrinogen
concentration were selected, and fibrinogens were synthesized as
described previously (18). Briefly, Chinese hamster ovary cell lines
expressing fibrinogen were grown in roller bottles in serum-free
medium, and the medium was harvested and stored at 
70 °C with
protease inhibitors (16). Of note, the expression of A' fibrinogen
was approximately one-tenth of the expression of normal or A'
fibrinogen. These proteins were purified as described (19). In brief,
the medium was thawed at 37 °C, and fibrinogen was precipitated with
a 40% ammonium sulfate cut. The precipitate was redissolved in buffer
containing 10 mM CaCl2 and applied to a
Sepharose 4B column coupled with monoclonal antibody IF-1, specific for
fibrinogen. Fibrinogen was eluted with buffer containing 5 mM EDTA; dialyzed against 20 mM HEPES (pH
7.4), 150 mM NaCl, and 1 mM
CaCl2 for one change and then extensively against 20 mM HEPES (pH 7.4) and 150 mM NaCl; and stored
at 70 °C. The fibrinogen concentration was determined at
A280 using the extinction coefficient 280 = 1.506 for a 1 mg/ml fibrinogen solution (20).
Purity of the proteins was analyzed by 9% reducing SDS-polyacrylamide gel electrophoresis following the method of Laemmli (21).
Thrombin-catalyzed Fibrinopeptide Release--
The
thrombin-catalyzed release of fibrinopeptides A, B, and A' from normal,
A', and A' fibrinogens was performed essentially as described
(18). Fibrinogen solutions were diluted to 0.3 µM in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM -aminocaproic acid, and 1 mM
CaCl2. -Aminocaproic acid was included to inhibit any possible plasmin contaminant activity. Thrombin was added to the fibrinogen solutions to a final concentration of 0.005 units/ml (0.054 nM). The tubes were mixed by inversion, and the digests were aliquoted into 440-µl portions for the 1-, 2-, and 5-min time
points and into 220-µl aliquots for the 10-, 40-, 80-, 120-, and
180-min time points. All of the manipulations following thrombin addition were completed within 1 min. The aliquots were incubated at
ambient temperature, and the reaction was halted by immersion in
boiling water for 15 min. The samples were kept on ice for the
remainder of the time course. At the end of the reaction, all samples
were centrifuged for 15 min at 4 °C, and the supernatants were
removed and stored at 70 °C prior to analysis by reversed-phase HPLC. Zero-time point controls were prepared by boiling 430 µl of the
fibrinogen solution in the absence of thrombin.
To increase the sensitivity at early time points in the reaction, the
assays were also performed in larger volumes, with less thrombin. This
macroscale assay on normal and A' fibrinogens was performed as
described above, with the following modifications. The thrombin
concentration was 0.002 units/ml (0.018 nM), and the
fibrinogen solution was diluted in 20 mM HEPES (pH 7.4) and 150 mM NaCl. For this assay, the aliquots at the 0-, 1-, 2-, and 5-min time points each contained 2.2 ml; those at the
10-, 15-, and 30-min time points each contained 1.1 ml; and those at
the 45-, 60-, and 180-min time points each contained 220 µl.
These reactions were stopped by the addition of 1 µl of 2000 units/ml hirudin. The samples were boiled and centrifuged, and the supernatants were stored for later HPLC analysis as described above.
Similar assays were performed in the presence of Gly-Pro-Arg-Pro to
examine the role of polymerization in the kinetics of fibrinopeptide
release from normal and A' fibrinogens. Fibrinogen was diluted to a
final concentration of 0.3 µM in 20 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM Gly-Pro-Arg-Pro
acetate salt peptide (GPRP); this concentration of GPRP was
sufficient to abolish an increase in turbidity at 350 nm, due to
polymerization, for 3 h. Thrombin was added to a final
concentration of 0.002 units/ml (0.017 nM), and the samples
were mixed by inversion and aliquoted into 440-µl aliquots for 2- and
5-min time points and into 220-µl aliquots for 10-, 15-, 30-, 45-, 60-, and 180-min time points. The samples were set in boiling water for
15 min, centrifuged, and stored for later HPLC analysis as described above.
Fibrinopeptide release was monitored by reversed-phase HPLC as
described (18). Briefly, the samples were loaded onto an Isco HPLC
system with a Vydac C18 column equilibrated with buffer A
(25 mM
NaH2PO4/Na2HPO4 (pH
6.0)). Samples containing 200 µl, 400 µl, 1 ml, or 2 ml were loaded
onto this column, depending on the experiment and the time point.
Fibrinopeptides were eluted with a linear gradient from 100% buffer A
to 40% buffer B (25 mM
NaH2PO4/Na2HPO4 (pH
6.0) with 50% acetonitrile) and monitored by absorbance at 206 nm. The
retention times for FpA' differed from those for FpA and FpB (22). The
differences in molar absorption for FpA, FpA', and FpB were not part of
our analysis, even though the molar absorption of FpA and FpB is
slightly different at A206 (23). Fibrinopeptide
peak area was determined using the accompanying Isco software
(Chemresearch Version 2.4). Fibrinopeptide release curves were prepared
by plotting the percent release versus time as described
(18).
All FpA and FpA' data were fitted with a simple first-order equation.
The FpB data from normal and A' fibrinogens were fitted to a
standard equation describing two consecutive first-order processes as
described (23). The curves described by these equations were plotted
using Delta Graph (DeltaPoint, Inc., Monterey, CA). Specificity
constants (kcat/Km) were
determined by dividing first-order rate constants by the thrombin
concentration as described (18).
Polymerization of Recombinant Fibrinogens--
Polymerization
was monitored at 350 nm in a SpectraMax-340PC 96-well microtiter plate
reader (Molecular Devices, Sunnyvale, CA) at ambient
temperature. Three separate experiments were performed in quadruplicate
for each polymerization condition. For each row used in the plate, four
wells contained normal fibrinogen, and four wells contained A'
fibrinogen. To each reaction well was added 90 µl of normal
recombinant or A' fibrinogen in 20 mM HEPES (pH 7.4),
150 mM NaCl, and 1 mM CaCl2. To
initiate the polymerization reaction, 10 µl of thrombin (4 or 1 units/ml) was added to all reaction wells with a multichannel
pipette such that all reactions began simultaneously.
Immediately after the addition of enzyme, the samples were automixed by
the instrument for 5 s. Turbidity was monitored every 10 s
for 1 h. All turbidity readings were normalized to a 1-cm path
length by the PathCheck sensor within the instrument. Final
concentrations for each set of reactions were 1.2 µM
fibrinogen (0.4 mg/ml) with 3.6 nM thrombin (0.4 units/ml) and 0.3 µM fibrinogen (0.1 mg/ml) with 0.8 nM
thrombin (0.1 unit/ml).
Lag time and Vmax were determined for each
polymerization reaction. Lag time was measured as the time elapsed
until an increase in turbidity was seen, and
Vmax was calculated as the slope of the steepest
part of the polymerization curve.
Scanning Electron Microscopy--
Clots were formed under the
same conditions as described for polymerization. For each condition,
scanning electron microscopy was performed on two clots each with at
least two separate microscopy preparations. The scanning electron
microscopy preparation was performed as described (24) with minor
modifications. In short, clots were polymerized in caps of eight-well
strip polymerase chain reaction tubes from which the bottoms had been
cut. One side of each cap was sealed with Parafilm, and 45 µl of
fibrinogen solution was added. A 5 µl-sample of enzyme was added to
each well, and the samples were mixed with the pipette tip.
Polymerization proceeded in a moist environment at ambient temperature
for 4 h. The Parafilm was gently removed, and the caps were rinsed
in 0.05 M sodium cacodylate buffer (pH 7.3) for 15 min with
three changes. The clots were then fixed in 2% glutaraldehyde
overnight, rinsed again in sodium cacodylate with three changes, and
stained with 2% osmium tetroxide for 30 min. The clots were rinsed
with distilled water and dehydrated with a series of increasing
concentrations of ethanol for 10 min each, up to 100% ethanol.
The samples were critical point-dried in a Balzers CPD020 for ~1 h,
mounted, sputter-coated with ~20 nm of gold-palladium, and viewed on
a Cambridge StereoScan S200 (LEO Electron Microscopy, Thornwood, NY).
All images were taken at ×16,200 with a 17.0-mm working distance and
20.0-kV accelerating voltage. Fiber diameters were calculated using
ScionImage (Scion Corp., Frederick, MD). Statistical analysis comparing
fiber diameters was carried out by unpaired t test using
StatView. A difference is significant when p < 0.05.
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RESULTS |
Characterization of Recombinant Fibrinogens--
We synthesized
two variant fibrinogens with altered fibrinopeptides A' where glycine
at position 14 was changed to valine. We chose the G14V substitution
based on the previous result that the specificity of thrombin for the
A-(1-51) fusion substrate with this substitution was not
significantly altered compared with normal FpA (22). In A'
fibrinogen, FpA' replaced FpA on the two chains, and in A'
fibrinogen, FpA' replaced FpB on the two chains. SDS-polyacrylamide
gel electrophoresis analysis run under reducing conditions showed that
each variant had the expected bands corresponding to the A, B,
and chains of fibrinogen (Fig. 1) and
that no contaminating proteins were detected. The expected
fibrinopeptides were released following digestion with thrombin,
confirming the presence of the intended sequence substitutions. The
HPLC chromatograms shown in Fig. 2
demonstrated that the G14V substitution in FpA altered the HPLC
retention time relative to that for FpA or FpB as described previously
(22). As expected, the fibrinopeptides from A' fibrinogen had
retention times characteristic of FpA' and FpB (Fig. 2B),
and the fibrinopeptides from A' fibrinogen had retention times
characteristic of FpA and FpA' (Fig. 2C).
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Fig. 1.
SDS-polyacrylamide gel of recombinant
proteins. A 9% reducing gel was used following the method of
Laemmli (21) and stained with Coomassie Blue. Lane 1, normal
fibrinogen; lane 2, A' fibrinogen;
lane 3, A' fibrinogen. Molecular masses (in
kilodaltons) are indicated to the left.
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Fig. 2.
HPLC chromatograms depict retention times of
fibrinopeptides released from normal (A),
A' (B), and
A' fibrinogens
(C).
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Thrombin-catalyzed Fibrinopeptide Release--
We examined the
rate of thrombin-catalyzed fibrinopeptide release by measuring the peak
areas of FpA, FpA', and FpB as detected by HPLC and plotting the data
as the percent fibrinopeptide release with time. Representative curves
are shown in Figs. 3-5; specificity constants from multiple averaged
experiments are presented in Table I.
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Table I
Specificity constants of normal, A', and A' fibrinogens under
microscale conditions (part A), macroscale conditions (part
B), and in the presence of 1 mM GPRP (part
C)
All units are 106 M 1 s1
· kcat/Km values for FpA and FpA'
were calculated as k1/[thrombin].
kcat/Km for FpB was calculated as
k2/[thrombin]. Each set of experiments was
performed with a different thrombin stock and different thrombin
dilutions, which may contribute to the differences in specificity.
These values represent the mean ± S.D.
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A' fibrinogen was synthesized to test the effect of introducing the
G14V substitution on FpA release. When comparing A' fibrinogen with
normal fibrinogen, FpA and FpA' release data were fit to a first-order
rate equation, whereas FpB release data were fit to a standard equation
describing two consecutive first-order processes (23). Specificity
constants (kcat/Km) were calculated as the rate constant (k1 for FpA and
FpA' and k2 for FpB) divided by the thrombin
concentration as described previously (23). Representative data,
presented in Fig. 3A, showed
that the release of FpA' from A' fibrinogen was modestly slower than FpA release from normal fibrinogen. Nevertheless, the specificity constants, presented in Table I (part A), were similar
(kcat/Km for normal FpA = (14.7 ± 4.5) × 106
M1 s1
and for FpA' = (9.7 ± 1.3) × 106
M1 s1).
These results demonstrated that FpA' was comparable to normal FpA as a
substrate for thrombin. We found that the rate of FpB release from
A' fibrinogen was faster than FpB release from normal fibrinogen
(Fig. 3A). The specificity constant for FpB release from
A' fibrinogen ((8.7 ± 1.1) × 106
M1 s1)
was twice that from normal fibrinogen ((4.6 ± 1.4) × 106 M1
s1). The significance of this finding remains
unresolved.
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Fig. 3.
Thrombin-catalyzed fibrinopeptide release
from normal, A', and A'
fibrinogens. Shown are representative curves of
fibrinopeptide release with 0.005 units/ml thrombin and 0.3 µM fibrinogen in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM -aminocaproic acid, and 1 mM CaCl2. All FpA and FpA' release curves were
fit to a first-order rate equation, whereas FpB release curves were fit
to an equation describing two consecutive first-order equations.
A, fibrinopeptide release from normal (solid
lines) and A' (dashed lines) fibrinogens.  and
 , FpA and FpB release, respectively, from normal fibrinogen;  and
 , FpA' and FpB release, respectively, from A' fibrinogen.
B, fibrinopeptide release from normal (solid
lines) and A' (dashed lines) fibrinogens. and
, FpA and FpB release, respectively, from normal fibrinogen; and
, FpA and FpA' release, respectively, from A' fibrinogen.
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A' fibrinogen was synthesized to determine the effect of
substituting FpA' for FpB on the kinetics of fibrinopeptide release. The fibrinopeptide release data showed that the shape of the curve describing FpA' release from A' fibrinogen more closely resembled an
FpA release curve than a FpB release curve (Fig. 3B).
Residual analyses of curves fit (25) to either a first-order rate
equation or two consecutive first-order equations revealed that the
release of FpA' from A' fibrinogen was best described by a
first-order rate equation. Thus, all specificity constants were
calculated with first-order equations for FpA and FpA' and with two
consecutive first-order equations for FpB. Comparing the specificity
constants (Table I, part A) for the release of FpA ((8.1 ± 2.4) × 106 M1
s1) and FpA' ((6.9 ± 2.1) × 106 M1
s1) from A' fibrinogen showed that the
release of these fibrinopeptides was similar. Representative curves
describing FpA and FpA' release were similar (Fig. 3B);
however, the amount of FpA' release was always less than the
amount of FpA released. In comparison with normal fibrinogen, the
specificity constant for FpA cleavage from A' fibrinogen ((8.1 ± 2.4) × 106
M1 s1)
was about half that of FpA release from normal fibrinogen ((14.7 ± 4.5) × 106
M1 s1)
(Table I, part A). FpA' cleavage from A' fibrinogen ((6.9 ± 2.1) × 106 M1
s1) was similar to FpB cleavage from normal
fibrinogen ((4.7 ± 1.4) × 106
M1 s1).
Representative curves describing the release of FpA' from A' fibrinogen and FpB from normal fibrinogen are qualitatively different from one another, as depicted in Fig. 3B, despite their
similar specificity constants.
To better examine the early time points, we increased the sensitivity
of the assay by 1) slowing down the reaction with lower thrombin
concentrations and 2) performing the assay on a macroscale, by
increasing the reaction volume and thus increasing the amount of
fibrinopeptides measured. The results of this assay (Fig.
4) revealed that fibrinopeptides from
both fibrinogens were released from the beginning of the reaction,
although the amount of FpB released was much lower than the amount of
each of the other fibrinopeptides (Fig. 4B). Specifically,
we noted that there was not a delayed enhancement of the rate of FpA'
release from A' fibrinogen, as seen in FpB release from normal
fibrinogen. Comparing the specificity constants (Table I, part B), the
rate of FpA release from A' fibrinogen ((16.5 ± 6.7) × 106 M1
s1) was less than the rate of FpA release
from normal fibrinogen ((20.2 ± 2.9) × 106
M1 s1),
but greater than the rate of FpA' release from A' fibrinogen ((8.6 ± 0.1) × 106
M1 s1).
The rate of FpA' release from A' fibrinogen was similar to the rate
of FpB release from normal fibrinogen ((9.3 ± 0.2) × 106 M1
s1), although the curve describing FpA'
release from A' fibrinogen was qualitatively different from the
curve describing FpB release from normal fibrinogen.
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Fig. 4.
Thrombin-catalyzed fibrinopeptide release
from normal and A' fibrinogens. Shown are
representative curves of macroscale fibrinopeptide release with 0.002 units/ml thrombin and 0.3 µM fibrinogen in 20 mM HEPES (pH 7.4) and 150 mM NaCl. All FpA and
FpA' release curves were fit to a first-order rate equation, whereas
FpB release curves were fit to an equation describing two consecutive
first-order equations. and , FpA and FpB release, respectively,
from normal fibrinogen; and , FpA and FpA' release,
respectively, from A' fibrinogen. A, entire time course
of reaction of normal (solid lines) and A' (dashed
lines) fibrinogens; B, magnification of the early time
points of this reaction. The volume applied to the HPLC column for
these points was 5-fold higher than in the early time points for Fig.
3.
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To examine whether polymerization influences the release of
fibrinopeptides from A' fibrinogen, as it does from normal
fibrinogen (3, 14), we monitored fibrinopeptide release in the presence of GPRP, a known inhibitor of fibrin polymerization (26). In agreement
with previous reports (3, 14), we found that the release of FpA from
normal fibrinogen was unchanged, but the release of FpB was markedly
slower in the presence of GPRP (Fig. 5).
In contrast, with A' fibrinogen, neither FpA release nor FpA'
release was changed in the presence of GPRP. Comparing the specificity constants from the averaged data (Table I, part C), we saw that with
normal fibrinogen, the release of FpB was 3-fold slower (compare (1.9 ± 0.4) × 106
M1 s1
with (4.6 ± 1.4) × 106
M1 s1)
in the presence of GPRP, whereas FpA was unchanged (compare (13.5 ± 1.4) × 106
M1 s1
with (14.7 ± 4.5) × 106
M1 s1).
In contrast, with A' fibrinogen, the release of FpA' was similar in
the presence of GPRP (compare (4.7 ± 1.3) × 106
M1 s1
with (6.9 ± 2.1) × 106
M1 s1),
as was the release of FpA (compare (7.2 ± 3.5) × 106 M1
s1 with (8.1 ± 2.4) × 106 M1
s1). We conclude that unlike the release of
FpB, the release of FpA' from the N terminus of the chain was not
influenced by polymerization.
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Fig. 5.
Thrombin-catalyzed fibrinopeptide release
from normal and A' fibrinogens in the presence
of 1 mM Gly-Pro-Arg-Pro. Shown are representative
curves of normal (solid lines) and A' (dashed
lines) fibrinopeptide release initiated with 0.002 units/ml
thrombin and 0.3 µM fibrinogen in 20 mM HEPES
(pH 7.4), 150 mM NaCl, and 1 mM GPRP. All FpA
and FpA' release curves were fit to a first-order rate equation,
whereas FpB release curves were fit to an equation describing two
consecutive first-order equations. and , FpA and FpB release,
respectively, from normal fibrinogen; and , FpA and FpA'
release, respectively, from A' fibrinogen.
|
|
Thrombin-catalyzed Fibrin Polymerization--
To determine whether
the altered kinetics of fibrinopeptide release were associated with
altered polymerization, we monitored the polymerization of normal and
A' fibrinogens. The polymerization of A' fibrinogen as measured
by turbidity at 350 nm was similar to the polymerization of normal
fibrinogen, as shown in Fig. 6. We
measured the polymerization parameters lag time,
Vmax, and final turbidity for both proteins and
found no significant differences between these parameters for A' and
normal fibrinogens (Table II).
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Fig. 6.
Thrombin-catalyzed polymerization of normal
and A' fibrinogens. Shown are average
curves of normal (solid lines) and A' (dashed
lines) polymerization with 1.2 µM fibrinogen and 0.4 units/ml thrombin (upper curves; n = 12) and
with 0.3 µM fibrinogen and 0.1 unit/ml thrombin
(lower curves; n = 8) in 20 mM
HEPES (pH 7.4), 150 mM NaCl, and 1 mM
CaCl2.
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|
View this table:
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Table II
Polymerization parameters of normal and A' fibrinogens
Lag time was measured as the time elapsed until an increase in
turbidity was seen, and Vmax was calculated as the
slope of the steepest part of the polymerization curve. Final
absorbance at 350 nm was determined after polymerization for 1 h.
These values represent the mean ± S.D.
|
|
Scanning Electron Microscopy Results--
We examined the final
fibrin clot structures for both normal and A' fibrinogens by
scanning electron microscopy. The two clots were similar in appearance
(Fig. 7), and measurement of the fiber
diameters showed that there was not a significant difference between
the diameters of A' fibers (136 ± 26 nm) and normal fibrin fibers (148 ± 27 nm; p = 0.16). This finding was
consistent with the polymerization curves for normal and A'
fibrinogens, which had similar final A350
values, indicative of similar fiber diameters (27).
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Fig. 7.
Scanning electron micrographs of normal and
A' fibrin clots. Normal
(upper) and A' (lower) fibrin clots were
formed with 1.2 µM fibrinogen and 0.4 units/ml
thrombin in 20 mM HEPES (pH 7.4), 150 mM NaCl,
and 1 mM CaCl2. The clots were fixed, stained,
dehydrated, critical point-dried, and sputter-coated as described
previously (23). Images were viewed on a Cambridge StereoScan S200 at a
17.0-mm working distance, 20-kV accelerating voltage, and
magnification × 16,200. The magnification bar
represents 1 µm.
|
|
| |
DISCUSSION |
We have synthesized a recombinant fibrinogen (A') to determine
what effect the substitution of FpA' on the N terminus of the chain
has on the kinetics of fibrinopeptide release. The release of FpA and
FpA' from A' fibrinogen occurred from the beginning of the reaction
and followed first-order kinetics, indicating that both FpA and FpA'
are equally competitive substrates for thrombin and have cleavage sites
that are accessible to thrombin at the beginning of the reaction. Thus,
the assumption used to characterize FpB release, that FpB release
depends on prior FpA release, does not accurately describe the release
of FpA' from A' fibrinogen. In addition, the similar rate of release
of either FpA' or FpB from the chain indicates that despite the
fibrinopeptide placed on the N terminus of the chain, the rate of
cleavage of the Arg-Gly bond by thrombin remains constant. Taken
together, we conclude that it is thrombin, in its interaction with FpB, that is responsible for the delay in efficient FpB release during fibrin polymerization, i.e. the specificity of thrombin for
FpB, and not its location on the N terminus of the chain, accounts for the kinetics of fibrinopeptide release from fibrinogen.
Additional experiments support the conclusion that FpA' release from
A' fibrinogen is dictated by thrombin specificity and not fibrin
polymerization. We found that FpA' release is qualitatively different
from FpB release from normal fibrinogen, indicating that the release of
FpA' from the chain is not dependent on prior polymerization of
des-A monomers. This conclusion is supported by our studies in the
presence or absence of GPRP, an inhibitor of fibrin polymerization. Our
results showed that FpB release was impaired in the presence of GPRP,
whereas FpA' release was not affected. Together, these results indicate
that FpA' release from A' fibrinogen is independent of fibrin
polymerization. Thus, we have created a fibrinogen in which the delay
in the rate enhancement of fibrinopeptide release from the chain
has been eliminated.
We present the following models of the interaction of thrombin with
fibrinogen, which accommodate our findings with A' and normal
fibrinogens. Initially, thrombin is oriented such that all three
fibrinogen interaction sites (the apolar specificity pocket,
fibrinogen-binding exosite, and active site) are aligned to accommodate
FpA as the preferred substrate. Upon binding to A' fibrinogen,
thrombin is best suited for FpA or FpA' cleavage, regardless of the
placement of the peptide on the or chain; thus, all four
fibrinopeptides are cleaved simultaneously and with similar affinity.
In normal fibrinogen, the thrombin active site is specific for FpA,
cleaving it efficiently to initiate polymerization, whereas thrombin
cleavage of FpB is much less efficient. During polymerization, however,
efficient cleavage of FpB becomes favorable, i.e. FpB
release becomes more efficient upon the appearance of des-A polymers
(2-4, 15). We propose two possible mechanisms, as depicted in Fig.
8, for this shift to efficient cleavage
of FpB. Either 1) upon the release of FpA, thrombin changes its
conformation such that FpB can be accommodated and cleaved efficiently,
or 2) the local conformation of FpB is altered over the course of
polymerization, which correctly orients the
Arg14-Gly15 bond for efficient cleavage within
the active site of thrombin.
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Fig. 8.
Schematic representation of the
possible mechanisms for the enhanced cleavage of FpB during fibrin
polymerization. A, initially, the conformation of
thrombin preferentially accommodates FpA (arrow) and
initiates cleavage. FpB maintains a conformation that is different from
FpA and does not fit into the thrombin active site. Upon cleavage of
FpA, the conformation of thrombin changes (represented by a
conversion from an arrow to a circle) during
polymerization such that it then accommodates FpB (circle)
and initiates efficient cleavage. B, initially, the
conformation of thrombin preferentially accommodates FpA
(arrow). FpB (skewed arrow) is unavailable for
cleavage by thrombin because its conformation is not compatible with
the thrombin active site. Upon cleavage of FpA, the conformation of FpB
changes (arrow) and is capable of being cleaved by
thrombin.
|
|
Although our results do not give us reason to favor one possibility
over the other, previous studies lend credence to both. Studies on
thrombin binding to exosite-binding fragments of hirudin, heparin
cofactor II, and the thrombin receptor have shown that exosite
interaction can allosterically modify the active site of thrombin
(28-32). Because thrombin binds to fibrinogen in the exosite, it is
possible that conformational changes that occur in fibrinogen during
polymerization can indirectly affect the thrombin active site.
Alternatively, conformational changes in the fibrinogen molecule have
been proposed (2, 3, 14, 23, 33-35) and measured (36) during fibrin
polymerization, and these changes could reposition the E domain such
that the scissile bond in FpB is properly oriented for efficient
cleavage by thrombin. Whether it is conformational changes in
fibrinopeptide B, thrombin, or both, these studies suggest that either
model is possible and reemphasize that we can only conclude from our studies that it is the specificity of thrombin for the fibrinopeptides that dictates the rate and timing of their release.
Production of A' fibrinogen also allowed us to evaluate the effect
of early exposure of the B site on fibrin polymerization. If
early exposure of the B site did affect polymerization, we would expect
a polymerization curve similar to that for fibrin monomer
polymerization, when the A and B sites are exposed from the start of
the reaction. Our results did not follow this pattern, thus suggesting
that exposure of the B site alone does not directly influence
polymerization. Rather, the participation of the B site in
polymerization likely depends on certain polymerization events. Previous studies (33, 37) suggest that this event is the
polymerization of des-A monomers to form protofibrils of a critical length.
In summary, by making this fibrinogen with essentially four
fibrinopeptides A, we have synthesized a model substrate
that eliminated the delay in fibrinopeptide release from the chain. Thus, the normal delay in fibrinopeptide B release likely arises from a
specific interaction between thrombin and FpB. We therefore conclude
that the kinetics of fibrinopeptide release are dictated by the
affinity of thrombin for its substrates. In addition, our work suggests
that early exposure of the B site does not affect the polymerization process.
| |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Li Fang Ping
and Kasim McLain for excellent technical assistance in protein
purification and production. We also gratefully acknowledge John
Weisel, Chandrasekaran Nagaswami, and Yuri Veklich for
teaching us the electron microscopic techniques and Victoria Madden and
Bob Bagnell for technical assistance in this endeavor. We thank Frank
Church for the generous donation of human -thrombin.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01-HL31048 (to S. T. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Present address: Dept. of Human Genetics, Glaxo Wellcome, 5 Moore Dr., Research Triangle Park, NC 27709.
To whom correspondence should be addressed: Dept. of Pathology
and Laboratory Medicine, CB 7525, 605 Brinkhous-Bullitt Bldg., University of North Carolina, Chapel Hill, NC 27599-7525. Tel.: 919-966-2617; Fax: 919-966-6718; E-mail: stl@med.unc.edu.
Published, JBC Papers in Press, June 2, 2000, DOI 10.1074/jbc.M004142200
| |
ABBREVIATIONS |
The abbreviations used are:
FpA, fibrinopeptide
A;
FpB, fibrinopeptide B;
FpA', fibrinopeptide A' (FpA with a G14V mutation);
A', FpA' substituted on the N terminus of the chain;
A', FpA' substituted on the N terminus of the chain;
bp, base pair(s);
HPLC, high performance liquid chromatography;
GPRP, Gly-Pro-Arg-Pro acetate salt peptide.
| |
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ABSTRACT
INTRODUCTION
