(Received for publication, September 18, 1995; and in revised form, November 30, 1995)
From the
Fibrinogen Caracas II is an abnormal fibrinogen involving the
mutation of A serine 434 to N-glycosylated asparagine.
Some effects of this mutation on the ultrastructure of fibrinogen
Caracas II molecules, fibers, and clots were investigated by electron
microscopy. Electron microscopy of rotary shadowed individual molecules
indicated that most of the
C domains of fibrinogen Caracas II do
not interact with each other or with the central domain, in contrast to
control fibrinogen. Negatively contrasted Caracas II fibers were
thinner and less ordered than control fibers, and many free fiber ends
were observed. Scanning electron microscopy of whole clots revealed the
presence of large pores bounded by local fiber networks made up of thin
fibers. Permeation experiments also indicated that the average pore
diameter was larger than that of control clots. The viscoelastic
properties of the Caracas II clot, as measured by a torsion pendulum,
were similar to those of control clots. Both the normal stiffness and
increased permeability of the Caracas II clots are consistent with the
observation that subjects with this dysfibrinogenemia are asymptomatic.
Fibrinogen is a plasma glycoprotein with a molecular mass of
340,000 daltons. The molecule consists of three pairs of nonidentical
polypeptide chains known as A (64,000 daltons), B
(56,000
daltons), and
(47,000 daltons). These polypeptide chains are
linked by 29 disulfide bonds and form two identical halves to the
fibrinogen molecule, each half being comprised of one set of A
,
B
, and
chains. The chains are folded into several domains to
form an elongated molecule 47.5 nm in length. There is a globular
central domain containing the amino termini of all six chains linked by
disulfide bonds to form a ``disulfide knot.'' Rod-like
regions extend from either side of this central domain and consist of
three-stranded
-helical coiled-coils that terminate in two outer
nodules. Each A
chain extends from the outer domains to eventually
terminate in a globular carboxyl-terminal region known as the
C
domain. The two
C domains have been shown to be bound both to each
other and to the central domain in normal fibrinogen under
physiological conditions(1, 2) .
Thrombin, a
proteolytic enzyme, cleaves two pairs of peptides from the A and
B
chains in the central domain of fibrinogen, called
fibrinopeptides A and B, to produce the fibrin monomer. This peptide
cleavage exposes binding sites in the central domain that interact with
complementary binding sites in the outer domains of other fibrin
monomers in a half-staggered arrangement. Fibrin monomers spontaneously
polymerize initially to form oligomers, followed by longer two-stranded
structures termed protofibrils. Lateral aggregation of protofibrils
eventually yield fibrin fibers that have a characteristic pattern of
striations with a periodicity of 22.5 nm as observed by electron
microscopy. These fibers can also undergo lateral aggregation to form
thicker fiber bundles. Branching of fibers and fiber bundles results in
the formation of a three-dimensional network that is the basic
structural framework of the blood clot.
Studies on the mechanism of
fibrin polymerization have shown that sequential cleavage of
fibrinopeptide A, followed by fibrinopeptide B, is important for the
formation of a normal clot structure(3, 4) . The
release of fibrinopeptide A allows the initial formation of
two-stranded protofibrils. Cleavage of fibrinopeptide B is delayed
until the fibrin molecules have been incorporated into protofibrils, at
which point lateral aggregation to form fibers is enhanced.
Fibrinopeptide B cleavage also releases the C domains from their
intramolecular binding sites on the central domain(2) . It has
been postulated that the
C domains enhance lateral aggregation by
intermolecular binding to other
C domains located on adjacent
protofibrils, hence pulling the protofibrils together(2) .
Several studies have provided evidence to support this proposed role
for the
C domains in lateral aggregation of protofibrils.
Different preparations of fragment X, missing the
C domains, have
impaired lateral aggregation(2, 5, 6) .
Purified fragment X
monomer, which consists of fibrin
monomer lacking both of the
C domains, exhibits a decreased rate
of lateral aggregation when compared with fibrin monomer containing
both
C domains(2) . However, these results also indicated
that although the
C domains accelerate the polymerization of
fibrin and influence the final structure of the clot, they are not
essential for either branching or lateral aggregation. When the
C
domains are blocked by
C fragments (1, 7) or
monoclonal antibodies specific for for the
C domain(8) ,
both the rate and extent of lateral aggregation are decreased. The
latter effect, which results in thinner fibers, suggests that
modification of the
C domains, as opposed to their removal, may
have an additional inhibitory effect on fibrin polymerization.
Several dysfibrinogenemias have been reported that involve mutations
in the C domain, including fibrinogen Caracas II (A
Ser
to N-glycosylated Asn)(9) ,
fibrinogen Dusart (A
Arg
to albumin-linked
Cys)(10) , and fibrinogen Marburg (A
461-611
missing)(11) . All of these mutant fibrinogens appear to be
defective in lateral aggregation. Other substitutions have also been
identified in this region of the A
chain, but their effects on
fibrin polymerization have not been defined (12, 13) .
The Caracas II and Dusart fibrinogens have additional material on the
C domains, carbohydrate and albumin, respectively, and both
produce fibers that are thinner than normal. Extensive studies have
been carried out on the ultrastructure of molecules, fibers, and clots
from fibrinogen Dusart, in addition to an investigation of the
biomechanical properties of the final clot
structure(10, 14, 15, 16) . These
studies have revealed a lack of interactions between the
C domains
of fibrinogen Dusart, the formation of very thin fibers that exhibit
less order than normal, and a clot structure that has very short
distances between branch points resulting in greatly diminished pore
sizes. The viscoelastic and permeation properties of the Dusart clots
show a marked increase in stiffness and a much lower permeation rate,
reflecting the above ultrastructural observations. Clinical symptoms of
subjects who have the Dusart syndrome include a high incidence of
thromboembolism and a resistance to thrombolysis, both of which can be
related to the defective clot structure. In contrast, fibrinogen
Caracas II is a congenital dysfibrinogenemia found in an asymptomatic
subject and was originally discovered through a prolonged thrombin time
observed in a routine coagulation test(17) . The rate of
lateral aggregation of protofibrils, as observed by turbidity
measurements, was lower than normal, and the final clot was
translucent. The fibers formed from this mutant fibrinogen were shown
to be thinner and more disordered than normal, although striations were
still discernible(18) . The defect in the fibrinogen Caracas II
molecule was found to be an N-glycosylated asparagine
substitution for serine at position 434 of the A
chain, with the
majority of the oligosaccharide consisting of a disialylated
biantennary structure(9) .
There is considerable evidence
that the similar carbohydrate moieties normally present on the B
and
chains of fibrinogen molecules have an inhibitory effect on
the polymerization process. Hyperglycosylated fetal fibrinogen
aggregates more slowly than normal with reduced turbidity in the final
clot(19, 20) . Removal of the sialic acid residues
from either fetal (21, 22, 23) or normal
adult (24, 25, 26) fibrinogen results in
faster polymerization, higher final turbidity, and thicker fibers.
Complete deglycosylation of fibrinogen also promotes lateral
aggregation to form thicker fibers, along with a significant reduction
in the degree of branching(27) . Fibrinogen Caracas II not only
has a higher sialic acid content than normal, which would be expected
to have an adverse effect on the extent of lateral aggregation, but
this additional carbohydrate is also located on the
C domains,
which have been directly implicated in the process of lateral
aggregation itself.
The study presented here involves an
ultrastructural investigation of fibrinogen Caracas II molecules,
fibers, and clots to elucidate the effects this mutation has on the
structure and function of fibrinogen at the molecular level.
Viscoelastic and permeation properties of the gel network were also
measured to relate the ultrastructural observations to the overall
physical behavior of the clot. The results provide insight into both
the role of the C domains in clot formation and the basis for the
lack of clinical symptoms exhibited by the fibrinogen Caracas II
subjects.
Fibrinogen was purified from plasma prepared from the blood of the subject and a normal individual as described previously(9) .
Permeation measurements were
made as described previously(34) . Tubes containing the gels
were placed in a holder and connected via plastic tubing to a reservoir
containing the buffer described above. Permeation experiments were
performed at different pressure heads, where the pressure was
determined by the vertical distance between the buffer reservoir and
the tip of the gel; pressure was kept constant during each experiment.
A dye, bromphenol blue, was applied to the clot after each experiment
to detect leaks between the gel and the walls of the tube or defects in
the clot itself, which were grounds for discarding the results from any
such gel. Such defects could be detected as a nonuniform progression of
dye through the gel. Average rates of flow were determined by multiple
measurements of the volume of liquid eluting from the gel in a given
period of time. Accurate measurements of the small volumes involved
were made by weighing the eluate on an analytical balance. Flow rate
(in ml/h) plotted against the pressure head (in dyne/cm)
gave a straight line for any one clot. In these experiments, flow rates
at different pressure heads were determined in random order to avoid
any potential effects of compression of the gel, although such effects
were not generally observed. In most experiments, the results were
highly reproducible, with standard deviations of about ±5%.
Occasionally, the formation of a defect in a clot (later detected by
the dye) caused a striking increase in flow rate during the course of
multiple experiments; the results of these experiments were discarded.
The permeation coefficient or Darcy constant, which represents the surface of the gel allowing flow through a network and thus provides information on the pore structure, was calculated from the flow measurements, pressure, and geometric parameters of the clot(35, 36) .
where Q is the volume of liquid (in ml) having the
viscosity (in poise), flowing through a gel with length L (in cm) and cross-sectional area A (in
cm
) in time t (in seconds) under differential
pressure
P (in dyne/cm
). The resulting Darcy
constants (K
) are in units of cm
. In
our experiments, L = 1.3 cm, A = 0.061
cm
,
= 10
poise; Q/t was measured for different values of
P.
Figure 1: Electron microscopy of individual rotary shadowed molecules of fibrinogen Caracas II. A, field of fibrinogen molecules showing examples of both simple trinodular molecules and molecules showing extra nodules (indicated by arrowheads). Bar, 100 nm. B, gallery of fibrinogen molecules showing simple trinodular structures. C, gallery of fibrinogen molecules showing an additional nodule at the center. D, gallery of fibrinogen molecules showing an extra nodule at one end. E, gallery of fibrinogen molecules showing extra nodules at both ends. Bar, 50 nm.
Figure 2: Electron micrographs of negatively contrasted fibrin fibers. A, fiber network formed from fibrinogen Caracas II. Some examples of free fiber ends are indicated by arrowheads. B, fiber network formed from control fibrinogen. Bar, 1 µm. C, control fibrin fiber that exhibits a distinctive band pattern with a 22.5-nm repeat. D, fibrin Caracas II fiber that shows longitudinal striations that almost obscure the 22.5 nm repeat. E, thin fibrin Caracas II fiber that still shows a 22.5 nm repeat. F, thin fibrin Caracas II fiber that lacks a 22.5 nm repeat but still has distinct edges. G, fibrin Caracas II fiber that lacks a 22.5 nm repeat and has frayed edges and readily discernible gaps between the protofibrils. H, fibrin Caracas II fiber that has a similar appearance to that in G but also exhibits protofibrillar branches (indicated by arrowheads). I, fibrin Caracas II fiber that shows a tapered end. J, fibrin Caracas II fiber that shows a frayed end. Bar, 100 nm. K, fibrin Caracas II fiber that provides an example of regional structural variation. Bar, 200 nm.
The substructure of Caracas II fibers (Fig. 2, D-K) exhibited marked regional heterogeneity as they varied in appearance along their lengths to a much greater extent than control fibers (Fig. 2C). In regions where cross-striations were visible, they were not as prominent as normal, although the spacings within the band pattern were essentially normal, as was the periodicity of 22.5 nm (Fig. 2, D and E). Commonly, longitudinal strands were visible in these regions and probably corresponded to individual protofibrils. A decrease in protofibrillar order apparently contributed to the reduction in the clarity of the cross-striations. Striations were observed over a wide range of diameters and usually extended across the complete width of the fibers. Therefore, ordering did not appear to be correlated with fiber width. Some regions exhibited very pronounced longitudinal strands that retained a fairly high degree of parallel order but lacked discernible striations (Fig. 2F). Other regions showed neither transverse nor parallel order and had relatively wide spaces between the protofibrils, and thus displayed an open ``spongy'' or ``frayed'' appearance (Fig. 2, G and H). However, it should be noted that in spite of the regional variation along the fibers from a relatively ordered to a very disordered appearance, there was considerable uniformity in substructure across the width of a fiber regardless of the overall nature of the particular region (Fig. 2K).
Another important difference between Caracas II and control fibers was the frequent occurrence of fiber ends within the dysfibrinogenemic sample. Some fibers retained considerable order toward their termini to form a relatively smooth tapered end (Fig. 2I), whereas others became increasingly disordered and terminated with a very frayed appearance (Fig. 2J).
Figure 3: Scanning electron micrographs of fibrin clots. A, clot formed from control fibrinogen. B, clot formed from fibrinogen Caracas II that shows distinct large pores bounded by smaller secondary networks. C, clot formed from fibrinogen Caracas II that shows thin fibers forming a secondary network. D, clot formed from fibrinogen Caracas II that shows many examples of free fiber ends (indicated by arrowheads). Bar, 5 µm.
However, the most striking feature of these clots was the presence of large pores or open areas bounded by local fiber networks. The largest of the pores had the appearance of caves or tunnels bounded by long, curved bundles of fibers (Fig. 3B). Along the walls of these channels were a mixture of fine fiber meshworks together with smaller pores (Fig. 3C). Free fiber ends were commonly observed, particularly at the boundary between the fiber meshworks and the pores (Fig. 3D). Owing to the extreme heterogeneity of the Caracas II clot structure, it was not possible to obtain meaningful measurements of average fiber bundle diameters or mean distance between branch points.
For a fibrinogen concentration of 0.5 mg/ml, the frequency of free
oscillation was 0.3 radian/sec for both the Caracas II and control
clots. The storage modulus (G`) was 20 dyne/cm
for
the Caracas II clot and 28 dyne/cm
for the control clot,
whereas the loss modulus (G") was 3 dyne/cm
in
both cases. The storage modulus is a measure of clot stiffness, and the
loss modulus is a measure of the energy dissipated by nonelastic,
viscous processes. Thus, the viscoelastic properties of the clots
formed from fibrinogen Caracas II were similar to those of the control
fibers, with the storage modulus for Caracas II being about 70% of the
control value, and there was no discernible difference between the
respective loss moduli.
In a prior study of fibrinogen Caracas II(9) , the
results were consistent with approximately half of the C domains
being abnormal and half being normal. The evidence (Table 1)
suggests that the polypeptide chains are assembled such that half of
the molecules are completely normal and half contain two abnormal
C domains, because it was observed that about 50% of the molecules
of fibrinogen Caracas II displayed normal morphology. A random
distribution of chains would result in only 25% of the molecules having
completely normal
C domains, whereas 25% would be completely
abnormal and 50% would have one abnormal A
chain.
Although the fibers
prepared by the addition of thrombin to fibrinogen Caracas II were
generally thinner, less ordered, and more variable in appearance than
normal, it appears that most molecules in these fibers must still be
arranged in a half-staggered manner because the striation spacings and
general band pattern are not affected by the mutation. There are often
larger spaces between the protofibrils than normal, resulting in fibers
that are more open in structure and hence of lower density (Fig. 2, G and H). This may indicate that the
extra charge and increased bulk of the carbohydrate on the C
domains prevents the protofibrils from packing close together in the
fiber. The modification of the
C domains may also account for the
reduction in the average fiber diameter to about half of the thickness
of control fibers, because the weaker intermolecular interactions
between the
C domains would be expected to inhibit lateral
aggregation of the protofibrils.
The appearance of Caracas II fibers varies considerably along their lengths, from relatively ordered to very disordered (Fig. 2K). It is possible that this variability arises from the presence of differing amounts of abnormal molecules in different regions of the fibers, because it is likely that these molecules will assemble more slowly than the normal molecules in these heterozygous individuals. The presence of many fiber ends is also consistent with molecular segregation, because the abnormal molecules may be impeding further growth by ``capping'' protofibril ends.
The viscoelastic properties of the clots formed from fibrinogen Caracas II were similar to those of the control clots, especially with respect to the storage modulus (G`), which differed from the control by only about 30%, indicating a similar degree of stiffness between the two types of clot. These results are somewhat surprising when the marked difference between the respective clot structures is taken into consideration. For example, a six-fold difference was observed between both the G` and G" values for clots formed from Dusart plasma and control plasma(15) . However, the Caracas II results are consistent with the absence of clinical symptoms observed in the Caracas II subject because there is evidence to suggest that the mechanical properties of clots are related to the propensity of individuals toward thrombotic disease(38, 39) .
Although some of the mechanisms responsible for the elasticity of fibrin clots have yet to be elucidated(40) , it is possible that the thinner fibers, the relative abundance of free fiber ends, and the spaces left by the large pores in the Caracas II clot structure would contribute to a decrease in clot stiffness and thus lower the value of G`. However, this decrease may essentially be offset by an increase in stiffness resulting from the greater degree of branching in the Caracas II clot. The similar values for the loss modulus (G") exhibited by the Caracas II and control clots indicate that energy dissipation by nonelastic, viscous processes is the same in both cases. It has been suggested that the primary contribution to G" arises from slippage between protofibrils(32, 41, 42, 43) . In the case of Caracas II, there is likely to be more slippage because of weaker interprotofibrillar interactions, but this may be offset by fewer opportunities for slippage as a result of the thinner fibers.
Permeation, a direct measure of the bulk transport of fluid through a gel, is related to the clot structure. We have demonstrated by scanning electron microscopy that clots made from fibrinogen Caracas II have very large pores compared with control clots. Such pores would be expected to yield very high flow rates. From the electron micrographs, the average pore diameter of the larger pores in Caracas II clots is roughly 3.3 ± 2.4 µm, whereas that in control clots is about 0.6 ± 0.5 µm, although it should be noted that it is sometimes difficult to identify what would constitute individual pores. Thus, the average cross-sectional area of the larger pores in Caracas II clots, an approximate measure of the flow rate, is 30-fold higher. On the other hand, electron micrographs also reveal a very fine meshwork of fibers surrounding the pores. As a consequence, the measured flow rate for fibrin Caracas II clots is 16.5 times larger than that of the control clots, somewhat lower than expected from the size of the large pores alone.
These results are considerably
different than those of permeation experiments from another
dysfibrinogenemia, fibrinogen Dusart, which also has a single amino
acid substitution in the C domains leading to defective lateral
aggregation(14) . Although the permeation experiments for
fibrin Dusart were carried out with plasma rather than purified
fibrinogen and the protein concentrations were different, the results
may be compared qualitatively. Fibrin Dusart clots, made up of a dense
meshwork of thin fibers with small pores, have extremely low rates of
permeation (K
was 175-fold lower than the
control). In contrast, even though the fibrin Caracas II clots are also
made up of thin fibers, the large pores that are present lead to higher
than normal permeation rates. This observation highlights the
importance of determining the ultrastructure of clot networks, in
addition to permeation values, in order to adequately explain clot
porosity(36, 44) .
Permeation may be relevant for the clinical consequences of these dysfibrinogenemias. The rate of permeation is an important determinant of how rapidly molecules, such as those involved in fibrinolysis, travel through a clot. Permeability may also affect the neutralization of free thrombin. In addition, cellular interactions with fibrin are likely to be affected by the pore sizes in the clot. Thus, the higher than normal permeation in fibrin Caracas II clots is consistent with the observation that subjects who have this dysfibrinogenemia are asymptomatic, whereas subjects with fibrinogen Dusart, having clots with much lower permeability, have severe thrombotic problems. However, in the case of fibrinogen Dusart, it should be noted that possible anomalous interactions of plasminogen or plasminogen activator with the clot may also provide a significant contribution to the observed clinical symptoms(45, 46) .
The converse may be true for Caracas II where the C
domains are not only free from the central nodule at all stages of
polymerization but would be expected to undergo repulsive interactions
owing to the negatively charged sialic acid groups, in addition to the
steric hindrance induced by the carbohydrate moieties. This
interference from the abnormal
C chains is likely to slow down the
initial rate of oligomer formation, which is supported by the
observation of an extended lag period during turbidity
experiments(17) . However, half of the molecules present in
fibrinogen Caracas II have normal
C domains, and these will
probably polymerize at a faster rate than the abnormal molecules,
resulting in an initial selective increase in the concentration of
oligomers containing predominately normal fibrin molecules. As the
oligomers elongate into two-stranded protofibrils, the pool of
available normal molecules will eventually become depleted relative to
mutant molecules, which may result in capping of the ends of
protofibrils with increasing proportions of abnormal fibrin monomers.
The end result may be a heterogeneous pool of protofibrils containing
varying proportions of normal and abnormal molecules.
Near the
completion of the lag period, the protofibrils will begin to undergo
lateral aggregation to form fibers; this process is assisted by the
normal C domains but inhibited by the mutant chains. Turbidity
experiments have demonstrated a decrease in the rate of lateral
aggregation of Caracas II protofibrils compared with controls, as shown
by a lowered rate of optical density increase following the lag period,
reflecting the probable inhibitory effect of the abnormal
C
domains (9, 17) . Again, it is likely that the regions
of protofibrils containing higher populations of normal
C domains
will undergo lateral aggregation at a faster rate than mutant-rich
regions, resulting in the fibers being more ordered during earlier
stages of formation with a tendency toward decreasing order later on as
more of the carbohydrate-rich
C domains become involved. This
process may explain the large number of fiber ends observed for Caracas
II, because eventually the population of defective
C domains may
become so high that their repulsive effects prevent further fiber
growth by either protofibril elongation or lateral aggregation. The
loose protofibril association observed in Caracas II fibers, combined
with the tendency toward fraying, particularly at fiber ends, is likely
to be a result of the repulsion between the abnormal
C domains and
suggests another mechanism that may contribute to the formation of this
unique clot architecture. Loose, frayed ends could increase the
frequency of branching, giving rise to the dense, highly branched
meshwork made up of thin fibers that was observed in the areas adjacent
to the pores.
The final structure of Caracas II clots, therefore, is
probably a result of the effect of the decreased rates of both
protofibril elongation and lateral aggregation, combined with the
effect that the repulsive forces associated with the additional charge
and mass of the carbohydrate bound to the C domains have on the
ultrastructural integrity of the constituent fibers.
These studies demonstrate that clots made up of fibers with similar diameters can differ dramatically in structure and physical properties. It is also evident that clot structure can be related to some clinical symptoms. Structural studies of these dysfibrinogenemias have revealed important aspects of the molecular mechanisms of assembly of fibrin clots.