(Received for publication, October 20, 1995; and in revised form, March 6, 1996 )
From the
The structure of a fibrin gel depends on the nature of the fibrinogen activation products produced by thrombin and the physical condition under which assembly occurs. Two different structures of the intermediate fibrin protofibril have been proposed, the production of which requires different extents of fibrinopeptide A (FpA) cleavage from fibrinogen. The fibrin activation intermediates must be stable since time is required for the intermediates to diffuse to growing protofibrils. The classic Hall-Slayter model requires cleavage of both FpAs to form a desAA intermediate. The Hunziker model requires cleavage of only one FpA to form an AdesA intermediate. Electrophoretic quasi elastic light scattering has been used to show the time-dependent production of the relevant fibrinogen activation intermediates that includes desAA but not AdesA.
Since the first description of fibrin structure by Ferry and
Morrison(1) , controversy has existed over the exact mechanism
of fibrin assembly. The issues include the order of release of the
fibrinogen activation peptides, fibrinopeptide A (FpA) ()and
fibrinopeptide B (FpB), the importance of the extent of removal of
fibrinopeptide A, the manner in which the fibrin monomers are organized
into protofibrils, and the manner of bundling of the protofibrils into
fibrin fibers. The complexity of the three-step assembly process
including fibrinogen activation, protofibril formation, and fiber
bundling lends itself to the diversity of fibrin structure originally
noted by Ferry(2) . The final fibrin structure depends on many
factors such as the rate of monomer production, fibrin monomer
concentration, the number of polymerization sites present on the fibrin
monomer, pH, ionic strength, solution viscosity, the presence of other
charged molecules, and volume exclusion
effects(1, 3, 4, 5, 6, 7, 8) .
FpA is released before FpB, and the release of FpA is sufficient to
initiate fibrin assembly(9, 10, 11) .
Different models for fibrin assembly have been proposed. Support for
the classical Hall-Slayter model (12) for fibrin assembly in
which fully activated fibrin monomers with both FpAs cleaved (desAA)
are added in an overlapping half-staggered manner to growing
protofibrils has been derived from assembly kinetics, light scattering
studies, and electron
micrographs(13, 14, 15) . A recent challenge
to the Hall-Slayter model by Hunziker et al.(16) refutes the overlapping monomer sequence in the
protofibril. Protofibril assembly in the Hunziker model requires the
existence of a fibrin monomer with only one FpA removed (AdesA) in
sufficient concentrations and with a sufficient lifetime to assemble a
non-overlapping protofibril. The fundamental difference in these two
models arises from differences in the protofibril structure, which
depends on the predominance and stability of AdesA compared with desAA
fibrin monomers. Report for the existence of the AdesA monomer is based
on gel exclusion chromatography and electron
microscopy(16, 17, 18, 19) . Other
investigators using similar methods and peptide sequencing experiments
do not find the AdesA fibrin monomer in either sufficient quantity or
lifetime to be a significant factor in fibrin
assembly(20, 21, 22) .
It is the purpose of this report to apply a methodology that permits direct observation and evaluation in real time of the transient intermediates that form during the activation of fibrinogen. Electrophoretic quasi elastic light scattering (ELS) can resolve structural differences between fibrin monomers because of differences in the columbic charge on the different activation intermediates. Quasi elastic light scattering (QLS) without electrophoresis reports on changes in the diffusion coefficients resulting from differences in the mass of the molecule. Thus, ELS should be better suited to study activation products of fibrinogen activation than QLS, since the fibrinopeptide cleavage changes the charge substantially but does not reduce the mass appreciably.
Experiments to examine the release of only fibrinopeptide A were carried out using Atroxin, an enzyme derived from Bothrops atrox (Sigma). In these experiments, Atroxin was added at a final concentration of 1.25 ng/ml, in place of thrombin, which gave equivalent fibrin gelation times. Fibrinopeptide B removal was examined in a similar experiment by adding an enzyme purified from Agkistrodon controtrix venom (Sigma) at 2 µg/ml. Although this enzyme removes predominately fibrinopeptide B, 30% fibrinopeptide A is also cleaved (9, 16) .
where is the time increment, I
is the
intensity of the reference beam (local oscillator), and <I
> is the average intensity of the scattered
light. K is the scattering vector defined by where
is the scattering angle, n is the refractive index,
is the wavelength of the incident
light, v
is the velocity of the scattering particle,
and D is the diffusion coefficient. The important quantity in
this expression is K
v
, the Doppler shift
of the signal resulting from the particle motion.
The magnitude of the Doppler shift is determined from the power spectrum, which is calculated from the Fourier transform of the autocorrelation spectrum. The Doppler shift can then be related to the electrophoretic mobility by ,
where is the Doppler shift,
is the
frequency of the incident light, and C is velocity of light in
the medium. The electrophoretic mobility is related to the velocity of
the scattering particle by the simple equation, v
= µE, where µ is the electrophoretic
mobility and E is the applied electric field(25) .
Temperature, ionic strength, pH, and conductivity affect the
electrophoretic mobility of the scattering particle and were therefore
carefully controlled by monitoring the conductivity of each sample.
Joule heating was governed by regulation of the pulse duration and the
pulse frequency of the electric field. Thermal lensing was avoided by
control of the incident laser power. Snell's law corrections were
made for all scattering angles.
Fig. 1shows the changes in the electrophoretic mobility spectrum for fibrinogen at a concentration of 0.05 mg/ml (peak A) at intermediate times during a 90-min activation by thrombin. At 10 min, a new peak (peak B) with a mobility of -0.6 (µ-cm)/(V-s) is observed. At 25 min, the electrophoretic spectrum shows the appearance of a third peak (peak C) with a mobility of -1.0 (µ-cm)/(V-s). Both peaks B and C continue to increase in intensity with time as shown by the spectra at 40 and 60 min. Other contributions to the mobility spectra are seen at higher mobilities. These experiments show activation intermediates at times of 10, 25, 40, and 60 min. Under conditions of very dilute thrombin concentrations, peak B emerges well ahead of peak C.
Figure 1:
Detection of fibrinogen intermediates.
ELS spectra of fibrinogen and fibrinogen activation intermediates
generated by the addition of 0.005 NIH units/ml of human -thrombin
and sampled at 10, 25, 40, and 60 min. Thrombin removes both FpA and
FpB from fibrinogen. Peak A is fibrinogen. Peaks B and C represent activation intermediates of fibrinogen.
Note that the appearance of peak B precedes that of peak C, which
provides further support that FpA is the first fibrinopeptide released
and that desAA fibrin monomer is the stable
intermediate.
In Fig. 2, the effect of thrombin, which removes both FpA and FpB,
is compared with Atroxin, which removes only FpA. Thus, by using
Atroxin, a homogeneous desAA can be produced. The experimental
conditions are identical to those used in Fig. 1. The 25-min
mobility spectrum using thrombin from Fig. 1has been
superimposed and is shown as a dashed line so that assignment
of peak B can be identified as removal of fibrinopeptide A, yielding
desAA. The same fibrinogen mobility (peak A) is observed in
both the thrombin and Atroxin experiments. The difference occurs in the
absence of peak C. Since Atroxin, the solid curve in Fig. 2, only releases fibrinopeptide A and since the morphology
of the single new peak is symmetric, the new mobility shown as peak B
must represent the desAA fibrin intermediate. Some investigators have
postulated the existence of an AdesA fibrin intermediate. The inset
shows a plot of the line width at half height, , for peak B versus the scattering vector, K, and confirms that
the line broadening is due to diffusion (
/2 =
K
) and not sample heterogeneity (
/2
= K
)(24) . Thus, contribution to
peak B from AdesA is highly unlikely. Furthermore, since the mobility
difference between peak A for native fibrinogen and peak B for desAA is
so large, if AdesA was present, it should appear as an easily
identifiable peak between peak A and peak B. Since no peaks are
observed in this region of the mobility spectra, and since we can show
the appearance of peak B ahead of peak C (see the spectrum for the
10-min sample time in Fig. 1), we take this as evidence that
AdesA does not exist as a significant intermediate. If it exists at
all, it must be extremely short lived or at a very low concentration.
These observations are consistent with the observation of Janmey et
al.(14, 15, 27) that the second FpA is
removed 16 times faster than the first FpA, suggesting that the
possibility of a stable AdesA intermediate is low.
Figure 2:
Identification of peak B as the desAA
intermediate. ELS spectra of fibrinogen (0.05 mg/ml) incubated with
1.25 ng/ml Atroxin (solid line), which removes FpA. Note that
only one new peak is observed. Thus, Peak B is identified as desAA by
the superimposition of the 25-min spectra from Fig. 1(dashed line). The inset shows a plot of
the line width at half height, , versus the scattering
vector, K, and confirms that the line broadening is due to
diffusion (
/2 = K
) and not sample
heterogeneity (
/2 = K
)(24) . If AdesA was present, it
would be seen as a specific mobility between peaks A and B. No mobility
is observed in the region between peaks A and
B.
To identify the
species responsible for the mobility represented by peak C produced by
the thrombin activation of fibrinogen shown in Fig. 1, the
following experiments were performed. The experiments shown in Fig. 3are identical to those shown in Fig. 1and Fig. 2except that fibrinogen is activated by an enzyme from the
venom of the Southern copperhead, Agkistrodon controtrix,
which cleaves FpB at a much faster rate than FpA. Again, the 25-min
spectrum from Fig. 1, shown as a dashed line, has been
superimposed so that the identification of FpB removal can be
established. As expected, no desAA peak (peak B in Fig. 1) is observed because the FpB is removed first. A new
peak, peak C`, with a slightly slower mobility than the peak C from Fig. 1, is produced. The activation product from A. controtrix should produce both desBB and desAAdesBB.
Additional help to resolve the assignment of peak C was obtained from
the production of a homogeneous desAAdesBB generated by first treating
fibrinogen with Atroxin followed by A. controtrix venom, which had identical mobility with peak C from Fig. 1(data not shown). Linewidth analysis on peak C generated
by both venoms, i.e. desAAdesBB, and from thrombin (Fig. 1) both have a K dependence,
confirming a single species identified as desAAdesBB. Linewidth
analysis of the slower mobility peak C` from Fig. 3shows a K dependence, indicating a mixture of species (see Fig. 3, inset). The conclusion from these experiments
is that peak C represents desAAdesBB and is supported by the fact that
we do not expect to find desBB in thrombin activation. These results
also suggest that desAAdesBB has a faster mobility than desBB. Finally,
peak C in Fig. 1is definitely not AdesA, since it would be
highly unlikely that removal of one FpA would produce a faster mobility
than removal of both FpAs and since no peak C is seen in Atroxin
activation. When both desBB and desAAdesBB are present, a slower peak
C` is seen that results from contamination by the slower moving desBB.
Figure 3:
Effect of
fibrinopeptide B removal on peak C. ELS spectra of fibrinogen (0.05
mg/ml) incubated with 2.0 µg/ml A. controtrix venom (ACV) (solid line). Although ACV removes both FpA and FpB, FpB is
removed at a much greater rate than FpA. Linewidth analysis of peak C
from Fig. 1shows a K dependence and
indicates a single species, desAAdesBB. When superimposed on Fig. 1, peak C generated by both Atroxin and ACV, also
desAAdesBB, has an identical mobility to peak C in Fig. 1(data
not shown). In contrast, peak C` generated with ACV alone (shown in
this figure) has a slightly slower mobility. The inset shows
the linewidth of peak C` is dependent on K, which indicates
more than one species is present in peak C`. ACV is known to produce
desBB and desAAdesBB. Thus, the slightly slower moving peak C`
generated with ACV alone results from the presence of desBB in peak C`
and indicates that desBB has a slower mobility than
desAAdesBB.
The experiments shown in Fig. 4are identical to those shown in Fig. 1, except that the concentration of fibrinogen is higher, 2 mg/ml, since it represents the normal human plasma concentration of fibrinogen and is also identical to the fibrinogen concentration used by Smith (17) and Hunziker et al.(16) . Under these experimental conditions, the activation rate is thrombin limited. As shown in Fig. 4, an activation profile is seen similar to that in Fig. 1with no evidence for AdesA.
Figure 4:
Fibrinogen activation at physiologic
concentrations of fibrinogen. ELS spectra of fibrinogen at the normal
physiologic concentration (2 mg/ml) and fibrinogen activation
intermediates generated by addition of 0.005 NIH units/ml human
-thrombin sampled at 15, 40, and 60 min are shown. The
designations of the peaks are the same as for Fig. 1.
Fibrin assembly is initiated by thrombin cleavage of the
N-terminal A-chain, fibrinopeptide A, which exposes one of the two
polymerization site ``A's'' on the
E-domain(28, 29, 30) . Structural information
on the chemical nature of ``A'' polymerization site is
limited, but His
on the B
-chain and contributions
from
-chain are necessary for polymerization to
occur(31, 32, 33, 34) . The
polymerization ``A'' site on the E-domain interacts with the
constitutively present ``a'' site on the D-domain of
an adjacent monomer. In contrast to the ``A'' site, the
critical amino acid sequence in the ``a'' site is better
defined and located on the C-terminal
-chain between amino acid
residues 356 and
411(30, 35, 36, 37) .
Once the fibrin monomer is generated following FpA release, fibrin assembly ensues. The classical mechanism for fibrin assembly as described by the Hall-Slayter model suggests that fibrin monomers polymerize in a half-staggered manner so that the D-domain of one monomer interacts with the centrally located E-domain of the adjacent monomer to form protofibrils 2 monomers thick(12) . In this model, polymerization symmetry permits monomer addition to either end of the growing protofibril (bipolar growth), but only if an ``A'' site faces the ``a'' site, which implies rotational symmetry about the minor hemi-axis (equivalent ends) but not about the major hemi-axis with respect to polymerization sites(38) . An additional critical factor is the structural contribution of the dihedral angle present in fibrinogen that introduces helical structure to the protofibril. However, the exact details of the interaction and packing remain unknown(38) .
Recently, the Hall-Slayter model for fibrin assembly has been challenged(16) . A fundamental feature of the alternative model described by Hunziker is that only one D-domain from each fibrin monomer is involved in the initial polymerization process. The second D-domain is then left free to form branches. For this model to be possible, the second ``A'' polymerization site on the E-domain must not be activated, i.e. the AdesA species must predominate and be stable long enough to diffuse to the surface of the assembling fibrin protofibril. If the second FpA is cleaved, protofibril assembly will proceed in an overlapping bipolar manner as postulated in the Hall-Slayter model. Assembly requires close proximity of monomers and growing fibrin oligomers and sufficient time for both rotational and lateral diffusion to occur so that the correct spatial orientation occurs. Thus, the monomer must be stable long enough for diffusive processes to bring monomers and oligomers together. If the AdesA species is the important fundamental monomeric species, it must not encounter a second thrombin molecule that would result in the cleavage of its second FpA before its assembly into the growing fibrin protofibril. These critical interactions between monomer and growing oligomer are highly important in the determination of fiber assembly kinetics and structure(39) .
Thus, a fundamental issue in the differentiation between the Hall-Slayter and Hunziker models is proof of the existence of the AdesA versus the desAA intermediate as the predominant species during fibrin assembly. The existence of a transient AdesA fibrin monomer is controversial and is dependent on the nature of interaction between thrombin and fibrinogen. For example, the AdesA could be produced through one thrombin bound for each FpA so that removal of each FpA is a temporally independent event. The desAA species could be produced by either two thrombin molecules bound simultaneously to fibrinogen or by the sequential removal of FpAs by one bound thrombin molecule. In favor of the latter model, a 16-fold increased rate of removal for the second fibrinopeptide A has been observed(14, 15) .
The existence of an AdesA intermediate in the early stage of fibrin formation was first proposed by Smith(17) , and his analysis, based on N-terminal amino acid analysis of gel chromatography isolated fibrinogen activation intermediates, reported that the interior of fibrin oligomers was composed of desAA monomers but that the oligomer was capped by AdesA monomers. It is not clear from this model how desAA monomers can be added to the growing oligomer if each end is capped by an AdesA. Based on his analysis, Smith postulated that AdesA was the early predominant species. A similar finding also using chromatography to isolate fibrin intermediates was reported by Alkjaersig and Fletcher (18) . Dietler et al.(19) used light scattering to arrive at a similar conclusion. It should be emphasized that quasi elastic light scattering gives highly accurate diffusion coefficients but only for monodispersed solutions(40) . Sample heterogeneity caused by fibrinogen or fibrin monomer aggregates will produce uncertainty in the result as evidenced by a large second moment in the cumulant analysis(40) . More recently, Hunziker et al.(16) have used electron microcopy to examine fibrin oligomers that appear to contain AdesA intermediates. Electron microscopy studies offer the possibility of analyzing individual monomers and oligomers, but it is not clear if the drying process alters the monomers so that their original solution appearance is altered. Monomer aggregation artifacts may also occur during the drying process. Important species present in solution may not be represented in the observed species.
Other investigators do not find evidence
for the AdesA fibrin monomer. Wilf and Minton (21) used gel
permeation chromatography and only found desAA fibrin monomers. Wilf
and Minton (21) also examined Smith's original proposed
assembly mechanism and found that Smith's predictions did not
agree with either their analysis or with Shainoff's sedimentation
analysis(41) . In addition, only desAA was observed by Janmey et al. (14, 27) in their light scattering
studies. Henschen (22) was not able to detect AdesA
intermediates using a more direct analysis of the FpA and amino acid
sequencing of the central, dimeric fragments derived from the
N-terminal region of all A-chains and
-chains present in the
thrombin digest.
In this report, we have used light scattering to examine fibrinogen activation intermediates. However, we have avoided the problem of sample heterogeneity by adding electrophoresis to quasi elastic light scattering as described by Ware and Flygare(42) . Differences between the molecular weight and hence the diffusion coefficient of fibrinogen and fibrin monomer using standard quasi elastic light scattering may not resolve subtle differences present in fibrinogen activation intermediates. On the other hand, substantial differences may be present in the molecular charge for each activation species, which would be highly sensitive to detection by electrophoresis. We view the results from QLS and ELS as complementary. We have shown that ELS is well suited for the study of fibrinogen activation and protofibril formation. ELS reports the surface charge of a particle and is observed as the electrophoretic mobility. The ELS method can measure the mobilities of a mixture of multiple particles with different structures and charges. In the present case, differences in the mobility of fibrin intermediates depend on small changes in the surface charge in activation intermediates. When the fibrinopeptides are removed and fibrin monomers are produced, only a small change in the molecular weight occurs, but a large difference in the electrophoretic mobility occurs. In fact, the change is so large that the existence of the AdesA species would be easily observed between peaks A and B, which is not seen (Fig. 1). We have also used limiting concentrations of thrombin to enhance detection of the AdesA intermediate, if it is present. In separate experiments, physiologic concentrations of fibrinogen were examined (Fig. 4). Our data strongly support the hypothesis that desAA fibrin monomer is the significant intermediate in fibrin assembly. If AdesA does exist, its lifetime and concentration are insufficient to exert a significant role in fibrin assembly.