|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4721-4729
Structural Studies of Fibrinolysis by Electron Microscopy
By
Yuri Veklich,
Charles W. Francis,
Janice White, and
John W. Weisel
From the Department of Cell and Developmental Biology, University of
Pennsylvania School of Medicine, Philadelphia, PA; and the Vascular
Medicine Unit, Department of Medicine, University of Rochester School
of Medicine and Dentistry, Rochester, NY.
 |
ABSTRACT |
Fibrin is degraded by the fibrinolytic system in which a plasminogen
activator converts plasminogen to plasmin, a serine protease that
cleaves specific bonds in fibrin leading to solubilization. To
elucidate further the biophysical processes involved in conversion of
insoluble fibers to soluble fragments, fibrin was treated with either
plasmin or the combination of plasminogen and plasminogen activator,
and morphologic changes were observed using scanning electron
microscopy. These changes were correlated with biochemical analysis and
with characterization of released, soluble fragments by transmission
electron microscopy. Initial changes in the fibrin matrix included
creation of many free fiber ends and gaps in the continuity of fibers.
With more extensive digestion, free fiber segments associated
laterally, resulting in formation of thick fiber bundles. Supernatants
of digesting clots, containing soluble derivatives, were negatively
contrasted and examined by transmission electron microscopy. Large,
complex fragments containing portions of multiple fibers were observed,
as were pieces of individual fibers and smaller fragments previously
identified. Some large fragments had sharply defined ends, indicating
that they had been cleaved perpendicularly to the fiber direction.
Other fibers showed splayed ends or a lacy meshwork of surrounding
protofibrils. Longer times generated more small fragments whose
molecular composition could be inferred from their appearance. These
results indicate that fibrinolytic degradation results in larger pieces
than previously identified and that plasmin digestion proceeds locally
by transverse cutting across fibers rather than by progressive cleavage
uniformly around the fiber.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
FIBRIN PROVIDES a temporary hemostatic
plug and physiologic mechanisms insure its efficient removal. Most
important is the fibrinolytic system that generates the active
protease, plasmin, through conversion of fibrin-bound plasminogen by
plasminogen activators, including tissue-type plasminogen activator
(t-PA) and urokinase-type plasminogen activator.1-4 Plasmin
then cleaves fibrin at specific sites generating soluble fragments,
many of which have been characterized.5-7 Both the rate of
fibrinolysis and the structures of soluble derivatives are determined
in part by the fibrin structure itself. Fibrin is produced from
fibrinogen through the action of thrombin, which cleaves
fibrinopeptides to produce fibrin monomers that then aggregate in a
half-staggered manner to produce two-stranded protofibrils. These can
aggregate laterally to yield small fibers that then associate and
branch to form the typical fibrin matrix.
The activation of plasminogen by t-PA is accelerated by the conversion
of fibrinogen to fibrin, and this property is determined in part by the
structure of the fibrin formed. For example, clots made of thin fibers
have a decreased rate of conversion of plasminogen to plasmin by t-PA,
and they are lysed more slowly than fibrin composed of thick
fibers.8-10 Under other conditions, however, thin fibers
are lysed more rapidly.11 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display a lower rate of fibrinolysis and decreased plasminogen binding.12-14 The molecular basis for the slower rates of
fibrinolysis within fibers remains unclear. The sequential polypeptide
chain cleavages during plasmic degradation of fibrin have been
characterized and have provided the basis for a molecular model of
fibrin degradation,5,6,15,16 but less is known about the
physical changes in the fibrin matrix that precede solubilization.
Although the molecular details of plasminogen and t-PA binding to
fibrin remain unclear, some information provides an indication of the
possible arrangement of the complex. Specific amino acid sequences that
bind t-PA have been identified,17 and several fibrin
sequences or fragments have been proposed as sites for plasminogen
binding or enhancement of its activation.18-23 An electron microscope study identified the pocket at the end-to-end junction between two fibrin molecules in the protofibril as a plasminogen binding site.24 Soluble degradation products of fibrin have been characterized by both sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis and transmission electron
microscopy, providing the basis for a model of their
structure.5,25 Changes in the polypeptide chain composition
of the fibrin network occurring before solubilization have been
characterized electrophoretically,6 but information is not
available regarding the physical changes in fibrin during plasmin
exposure. In this report, we characterize structural changes occurring
in polymerized fibrin during fibrinolysis, combining visualization by
transmission and scanning electron microscopy with electrophoretic
characterization of the soluble and solid phases of fibrin clots during
the course of plasmic degradation.
| |
MATERIALS AND METHODS |
Preparation of fibrin digests.
Fibrin clots were prepared in specially adapted tubes to facilitate
removal of the clot after formation and digestion. Polyethylene tubes
(9 × 32 mm) were obtained from Dynalab (Rochester, NY), and the
bottom 2 mm was removed. The tops were covered with teflon tape and
inverted over tight fitting caps. To prepare fibrin, 0.2 mL of
fibrinogen (Kabi Pharmacia, Inc, Franklin, OH) at a concentration of 2 mg/mL and 0.05 mol/L Tris-HCl buffer, pH 7.4, containing 0.10 mol/L
NaCl, 0.018 mol/L calcium chloride, and 0.01% sodium azide was
pipetted into the inverted tubes, and human thrombin (Calbiochem,
LaJolla, CA) was then added to a final concentration of 0.5 U/mL. As
soon as clotting occurred (~30 seconds) the clot was overlaid with
0.2 mL buffer and incubated at 37°C for 60 minutes. For enzymatic
digestion, the buffer was removed, and the clot was rinsed 5 times with
0.2 mL of buffer to which was added t-PA (Genentech, South San
Francisco, CA) or plasmin at 71 CTA units/mg (Bureau of Biologics
Standards, Bethesda, MD) at varying concentrations. After incubation at
37°C, the soluble digestant was removed from the top of the clot
and transferred to a separate tube containing 100 kallikrein inhibitory
units (KIU)/mL of aprotinin (Bayer, Kankakee, IL). The remaining,
partly digested clot was then overlaid with 0.2 mL of buffer containing
200 KIU/mL of aprotinin and allowed to incubate for 1 hour. The buffer
was then removed and replaced with 0.1 mol/L phosphate buffer, pH 7.4, containing 200 KIU/mL of aprotinin and incubated for 16 hours at
25°C. The buffer was again removed and replaced with a new 0.2 mL
aliquot of buffer containing 200 KIU/mL of aprotinin, and this step was
repeated three times over 1 hour. Phosphate buffer was then removed,
and the clot was fixed with 1 mL of 2% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Electrophoresis in 7% SDS polyacrylamide gels was performed as described.26 Gradient gels of 4% to
10% polyacrylamide were prepared as previously described27
and run using a discontinuous buffer system.28
Scanning electron microscopy of digested clots.
Clots were prepared for scanning electron microscope experiments by
fixation, dehydration, critical point drying, and sputter coating with
gold-palladium as described previously.29,30
Specimens were observed and photographed digitally using a Philips XL20 scanning electron microscope (Philips Electron Optics, Eindhoven, The
Netherlands).
Transmission electron microscopy of negatively contrasted specimens.
A drop of the fiber suspension was transferred to a 500 mesh copper
grid coated with a thin carbon film, rinsed with several drops of
buffer, and negatively stained with 2% uranyl
acetate.31 The specimens were observed in a Philips EM 400 electron microscope operating at 80 kV.
| |
RESULTS |
Clot digestion and biochemical characterization.
Fibrin was overlaid with plasmin at concentrations of 0.2, 0.5, and 2.0 U/mL or with buffer only for 0.5, 0.75, 1, 2, and 3.5 hours. Grossly,
all control clots incubated with buffer for different times were
similar in size with a cylindrical shape and an approximate diameter of
6 mm and height of 5 mm. The top surface had a slight concavity of
approximately 0.5 to 0.7 mm. Fibrin clots treated with 0.2 U/mL of
plasmin did not appear different for periods up to 1 hour, but
developed deeper concavities at 2 and 3.5 hours. Clots overlaid with 2 U/mL of plasmin showed a clear decrease in clot thickness, declining to
4.0, 3.0, 2.5, and 0.5 mm at exposure times of 30 minutes, 1 hour, 2 hours, and 3.5 hours, respectively. Fibrin clots incubated with plasmin
at the intermediate concentration of 0.5 U/mL had dimensions between those exposed to the high and lower concentrations.
Fibrin clots were also digested with plasmin produced by activation of
endogenous plasminogen with t-PA. In these experiments, 0.75 µg/mL
t-PA was layered on the clots and digestion was stopped after either 1 or 3 hours; the concentration of endogenous plasminogen in these
preparations was measured to be 0.16 µmol/L. The clots showed similar
changes in overall clot dimensions and electrophoretic appearance of
residual clot and soluble products.
Analysis by SDS polyacrylamide gel electrophoresis showed that fibrin
cross-linking was complete, with no residual monomeric  or  chains remaining (Fig 1A), and the
cross-linked fibrin showed characteristic  , , and polymer
chains as prominent features. Degradation was stopped after 2 hours
(lane 2) or 4 hours (lane 3), and the supernatant containing soluble
derivatives was removed. Electrophoresis of the remaining, insoluble,
partly degraded fibrin showed little change in polypeptide chain
composition, with intact and chains and only some decrease
in the amount of polymer. This appearance was expected, considering
that plasmin action was limited to the buffer-fibrin interface, so that
a large amount of undegraded fibrin remained. Electrophoresis of the
soluble products generated during a typical experiment showed little
change in composition during plasmic degradation for up to 4 hours, at which time 26% of the original fibrin had been solubilized. The prominent derivatives present were fragments E, DD, DY, and YY (Fig
1B).
View larger version (59K):
[in this window]
[in a new window]
View larger version (56K):
[in this window]
[in a new window]
| Fig 1.
Electrophoretic characterization of plasmic derivatives
of fibrin. (A) SDS polyacrylamide gel electrophoresis of cross-linked
fibrin (lane 1) and cross-linked fibrin after exposure to t-PA (1 µg/mL) for 1 hour (lane 2) or 4 hours (lane 3). After 2 and 4 hours,
9% and 26%, respectively, of the original fibrin clot had been
solubilized. Electrophoresis of reduced protein was performed using 7%
polyacrylamide gels. The locations of the polypeptide chains are
indicated. (B) SDS polyacrylamide gel electrophoresis of digests of
fibrin after incubation with plasmin for 45 minutes, 1 hour, 2 hours,
and 3.5 hours in lanes 1 through 4, respectively. The percentage
degradation of the original fibrin was 4% at 45 minutes, 5% at 1 hour, 11% at 2 hours, and 12% at 3.5 hours. The location of fragment
E and the smallest cross-linked degradation products (DD, DY, and YY)
are indicated. Electrophoresis of nonreduced protein was performed in
4% to 10% gradient gels.
|
|
Scanning electron microscopy of digested clot surfaces.
The appearance of fibrin clots before plasmin treatment or after
incubation with buffer rather than plasmin was consistent with past
observations demonstrating an extensively branched fiber network
(Fig 2D). The mean diameter of fibers
measured from micrographs of these control clots was 98 ± 19 nm. In
some cases, the bottom surface of the digested clots was examined as an
internal control and showed the same appearance as clots never exposed
to plasmin.
View larger version (203K):
[in this window]
[in a new window]
| Fig 2.
Scanning electron micrographs of digested clot surfaces.
Different concentrations of plasmin were applied to the clot surface
and samples were prepared for microscopy at various times. (A) 0.2 U/mL
plasmin, 30 minutes. (B) 0.2 U/mL plasmin, 1 hour. (C) 0.2 U/mL
plasmin, 3.5 hours. (D) Control, no plasmin. (E) 0.5 U/mL plasmin, 30 minutes. (F) 2.0 U/mL plasmin, 3.5 h. Bar equals 2 µm.
|
|
After plasmin exposure, the most obvious feature observed by scanning
electron microscopy was the presence of many fiber ends (Fig 2A),
whereas fiber ends were observed rarely in control clots. Although the
presence of many fiber ends in the early stages of digestion was
greater than in control samples, there were no changes in pore sizes,
fiber diameters, or other properties at the very earliest stages of
digestion. After 30 or 45 minutes of digestion by plasmin at a
concentration of 0.2 U/mL, the fiber bundles were larger in diameter
(130 ± 49 nm). Both long and short cleaved fibers with sharply
truncated ends were seen (Fig 2A).
In fibrin clots exposed for 1 hour to plasmin at a concentration of 0.2 U/mL, long individual fibers with cleaved ends were rare, but the ends
of many shorter fibers were visible over the entire surface, as were
fiber bundle aggregates composed of fibers cleaved at one end but
remaining attached to the clot at the other (Fig 2B). The fiber bundles
appeared to be covered with follicles of varying size representing the
stubs of fibers that had been cleaved and removed. Although individual
fibers making up the bundles could still be seen in some cases, these
fiber bundles were different in appearance from the ones found in
undegraded fibrin clots. The fiber bundles were larger in diameter (222 ± 89 nm) and they appeared flatter with rough and irregular
surfaces that were often fragmented in one or more places. This
fragmentation as well as the bundling of individual fibers into larger
aggregates resulted in the formation of networks with larger pores than
in control clots.
After 3.5 hours of plasmin treatment, fiber aggregates were larger and
more numerous (Fig 2C) and individual cleaved fibers were difficult to
identify. The fine structure of these aggregates also changed, with
fiber bundles spread over a larger area, and their surfaces were more
irregular. Many areas looked amorphous with poor definition of
individual fibers, and there was extensive fragmentation with large
pores evident. The average fiber diameter was 220 ± 78 nm.
Structural changes observed on the fibrin clot surface after treatment
with 0.5, 1.0, or 2 U/mL of plasmin were similar but occurred earlier.
For example, large fragmented fiber aggregates dominated the surface of
clots treated with 0.2 U/mL plasmin after 2 or more hours of treatment,
whereas at higher plasmin concentrations, these fragmented structures
were prominent after only 30 minutes (compare Fig 2C and E). The fibers
were also greater in diameter and had a flatter appearance than those
seen with 0.2 U/mL of plasmin (compare Fig 2C and F). For example, with
1 U/mL plasmin at 30 minutes, the average diameter was 314 ± 106 nm. The individual fibers making up such aggregates were poorly defined
and appeared fused along their lengths. Few individual fibers were
identifiable.
Histograms of fiber bundle diameters for different stages of digestion
provide additional quantitative information about these changes
(Fig 3). With increasing digestion, the
histograms became broader, indicating greater heterogeneity in fiber
diameter. The peaks also shifted to larger diameters as a result of the
presence of thicker fiber bundles formed from the aggregation of fibers transected by plasmin. Clots digested with a combination of t-PA and
plasminogen showed a similar sequence of changes and overall appearance.
View larger version (36K):
[in this window]
[in a new window]
| Fig 3.
Histograms of fiber bundle diameters from scanning
electron micrographs of digested clot surfaces. Fiber bundle diameters
were measured from micrographs similar to those shown in Fig 2. Fiber
bundle diameters are in nanometers. The histograms for each digestion
experiment are arranged in the same way as the corresponding
representative micrographs in Fig 2, for ease of comparison. The same
number of measurements was made for each digestion condition. (A) 0.2 U/mL plasmin, 30 minutes. (B) 0.2 U/mL plasmin, 1 hour. (C) 0.2 U/mL
plasmin, 3.5 hours. (D) Control, no plasmin. (E) 0.5 U/mL plasmin, 30 minutes. (F) 2.0 U/mL plasmin, 3.5 hours.
|
|
Transmission electron microscopy of soluble derivatives.
To characterize the structures of soluble derivatives released from
fibrin and to follow their digestion over time, the specimens were
negatively contrasted and observed by transmission electron microscopy.
A large variety of fragments with marked variability in size and
appearance were observed, but the same structures were observed from
digestion with plasmin or with t-PA and plasminogen. The largest
products appeared to be composed of fragments of multiple fiber bundles
(Fig 4A). The ends of these structures were
often sharply delineated, showing that the fibers had been cut
approximately perpendicular to the fiber axis (Figs 4A and D and
5D). In early digests at low plasmin
concentration, these fiber bundles sometimes retained their overall
shape so that individual fibers were still detectable and the 22.5 nm
repeat was visible, although in most cases the normal fibrin band
pattern was no longer clear. After more extensive digestions, there
were loose amorphous aggregates made up of pieces of smaller size
linked together to make complex lacy networks (Figs 4B and C and 5A
through C). In such aggregates, few individual fibers could be
recognized, although the size of these aggregates and their mesh-like
appearance suggested that they consisted of several highly digested
fibers or protofibrils fused together.
View larger version (137K):
[in this window]
[in a new window]
| Fig 4.
Transmission electron micrographs of negatively
contrasted digestant removed from the clots. Large pieces of clot
observed in the supernatants. (A) This chunk consisting of pieces of
fibers and aggregated smaller structures has been cleaved so that it is
nearly separated from a larger mass at the location indicated by the
arrow. (B) Lacy meshwork of fibers, protofibrils, and other cleaved
pieces. (C) Aggregate consisting of pieces of fibers and smaller
structures. Bar for (A) through (C) equals 0.5 µm. (D) Fiber bundles
with splayed ends. Bar for (D) equals 0.1 µm.
|
|
View larger version (148K):
[in this window]
[in a new window]
| Fig 5.
Transmission electron micrographs of negatively
contrasted digestant removed from the clots. Pieces of fibers,
protofibril aggregates, and smaller fragments observed in the
supernatants. Arrows indicate areas that are darker as a result of
removal of protein by digestion. Arrowheads point out brighter areas
where stain was excluded because of plasmin binding. (A) Fiber
surrounded by lacy meshwork of protofibril-like structures. (B) Fiber
surrounded by meshwork made up of protofibrils and other smaller
structures. (C) Fiber splayed into bundles of protofibrils. Bar for (A)
through (C) equals 0.5 µm. (D) Fiber showing sharply cut end (at
right) and another chunk that is largely separated from the rest of the
fiber but still associated with it (asterisk). (E) Digested fiber with
missing pieces and truncated end; note some smaller fragments in the
background. (F) Piece of remaining fiber where individual protofibrils
can be visualized. (G) Very thin fiber remnant with other smaller
fragments. (H) Small fragments present at longer times of digestion or
with higher plasmin concentrations. Some examples of DDE complexes are
circled. Bar for (D) through (H) equals 0.1 µm.
|
|
Individual digested fibrin fibers that were also found in all samples
of soluble digestant displayed more structural detail (Fig 5A through
G). Typically, these fibers had compact lateral packing in the center
but splayed into thin strands laterally and at their ends. The darker
areas corresponding to lower protein density in these regions arise
from the presence of gaps as a consequence of protein removed by
digestion (Fig 5A, D, and F, arrows). Some brighter areas of fibers
were observed, indicating areas where stain was excluded because of a
higher protein content, possibly representing sites of plasmin
binding24 (Fig 5B and C, arrowheads). In some fibers, the
lateral packing along their length was uniform, such that individual
protofibrils with a lateral spacing of about 9 nm were visible (Fig 5B,
E, and F). Areas with cleaved fiber ends were found in every sample of
digested clot, indicating the cross-sectional direction of fibrin
cleavage (Fig 5D, E, and F). In most preparations there were also small
structures that could be identified as complexes arising at the later
stages of plasmin digestion (Fig 5H). Globular regions corresponding to
the nodules present in fibrin(ogen) could be visualized, permitting identification of complexes such as DD/E, DY/YD, and DXD/YY. These smaller fragments predominated at the longest digestion times.
| |
DISCUSSION |
Detailed information regarding the structures of fibrinogen and fibrin
degradation products appearing during the course of plasmic degradation
provided the basis for a model of their structure and origin from the
fibrin fiber.5-7,15,16 The carboxyl terminal ends of the
chains and the amino terminal ends of the chains are first
removed from fibrinogen, yielding fragment X. Then all three
polypeptide chains are cleaved at plasmin-sensitive sites in the
coiled-coil region connecting the lateral and central domains. This
digestion yields from each fibrin monomer a single fragment E
consisting of the central domain and two D fragments, each consisting of lateral domains, as well as two C fragments representing the carboxyl terminal chains. If fibrin is cross-linked by factor XIIIa, the degradation products are different, with cross-linked D-dimers and C multimers produced. Additional noncovalent
interactions result in formation of a variety of complexes of which
DD/E is the smallest.5
Much less is known about physical changes in the residual, insoluble
fibrin matrix during plasmic degradation. One model proposes that a
fibrin clot is digested from the outside-in with products of
degradation released layer-by-layer.6,32 This analysis is
based on characterization of fibrin degradation products released from
clots that had been lyophilized and ground to small pieces before
plasmin exposure. Recent investigations have also suggested that plasma
clot degradation results in sequential, layer-by-layer reduction in
size.33,34 These studies included dynamical changes during
degradation monitored by confocal laser-scanning fluorescence microscopy, but there was not sufficient resolution to observe fine
structural details at the clot surface.
At the level of individual fibers, Gabriel et al8 suggested
an outside-in model in interpreting results of experiments with
digestion of thick and thin fibrin fibers. In other words, it was
assumed that plasmin will first digest the outer surface of the fibers,
producing progressively thinner fibers as digestion proceeds.
Similarly, a model of fibrinolysis developed to predict effects of clot
transport also used an outside-in scheme to keep track of
changes.35
This report presents direct observations of changes during fibrinolysis
at the resolution of individual fibers. An important finding was that
local plasmin action proceeds laterally, resulting in transection of
fibers. With progressive degradation there was an increase in free
fiber ends but little change in fiber diameter. We also observed an
irregular or nodular appearance of fibers suggesting cleavage similar
to the fragmented and flocculent appearance of fibrin during lysis of
platelet-fibrin thrombi.36 At later stages of digestion,
many cut fiber ends were observed, and mean fiber diameters increased
rather than decreased, contrary to our expectations. We interpret this
as lateral aggregation of the cut fibers to form thick bundles, which
could have been expected, because lateral aggregation of fibrin fibers
occurs during polymerization, yielding thick fiber
bundles.37 The average size of the fiber bundles increased
dramatically from 129 ± 49 to 220 ± 78 nm, and such fiber
aggregates became the dominant feature of the surface of digesting
fibrin. These changes were accompanied by a clear increase in the size
of pores between bundles, providing further evidence of lateral
cleavage and fiber aggregation. Fibers cleaved by plasmin have
increased mobility, providing freedom for lateral polymerization.
Our observations are consistent with data obtained by other means. For
example, during lysis of fibrin clots polymerized in the presence of
plasminogen and t-PA, the turbidity increases during polymerization and
then decreases as the clot is lysed. A spike of high turbidity can
sometimes be observed at the initiation of clot degradation, consistent
with the transient presence of very thick fibers at this time. Similar
experiments have monitored clot stiffness during fibrinolysis,
demonstrating a transient phase of hyper-rigidity before clot
dissolution,38 which is also consistent with the transient
generation of thicker fibers.
The results presented suggest an explanation for the observed
resistance to fibrinolysis of fibrin composed of thin in comparison with thick fibers.8-10,12 If plasmin transects fibers at
localized sites, fibrin composed of thick fibers will be degraded more
rapidly, because there would be fewer fibers to transect. We also
suggest that the progressive fiber aggregation during plasmic
degradation may provide an additional mechanism for local enhancement
and acceleration of the fibrinolytic process, because cleavage proceeds laterally.
Transmission electron microscopy of soluble products also provided new
insight into the initial stages of fibrin solubilization in more
detail. Larger and more heterogeneous soluble products were observed in
this study than expected, reflecting the largest derivatives released
from fibrin before complete solubilization of the matrix. Products that
appeared to represent aggregates of several fibrin fibers were
observed, as well as individual fibers. Smaller degradation products
identical to those observed previously25 were also seen.
Soluble products composed of sections of entire fibers or fibrin
bundles could only arise from degradation due to localized proteolytic
transection of fibers rather than an overall uniform action, which
would result in a progressive decrease in fiber diameter.
The largest soluble derivatives, which had diameters up to 400 nm and
displayed no visible internal structure, corresponded in appearance to
large fiber aggregates observed by scanning electron microscopy of the
clot surface before solubilization. The lack of structural order
suggests extensive internal degradation before release from the fiber
surface. In contrast, portions of individual fibrin fibers in solution
demonstrated more structural detail and appeared similar to fibers seen
in undigested clots. They exhibited sharp, transected ends, indicating
that they were produced by cleavage across the fiber. The band pattern
arising from the specific molecular packing was faint, suggesting some
internal degradation or conformational change.
The most frequent structures observed in the digesting fibrin matrix
appeared as fibers with a compact structure centrally but splayed into
a lace-like mesh at the edges. The splaying of partially digested
fibers may be explained from knowledge of fibrin structure and
assembly. During clot formation, protofibrils of fibrin start
aggregating laterally to form fibers after they reach a critical
length.30,39,40 When this process is reversed by plasmic
degradation, disaggregation of protofibrils may occur if transection
generates a fragmented piece shorter than the critical length needed
for lateral aggregation. This disaggregation may be modified by the
extent of chain cross-linking that stabilizes lateral
aggregation.41
The pattern of degradation observed is consistent with evidence that
plasminogen binds to a fiber in the pouch at the end-to-end junction of
two fibrin molecules in a protofibril.24 In this location,
plasmin is able to reach cleavage sites located on adjacent protofibrils, creating additional carboxyl-terminal lysines as new
sites for plasmin binding as degradation proceeds.42 The results presented herein suggest that plasmin molecules then move across the fibers laterally, cleaving additional sites. In support of
this concept, it should be noted that equivalent plasminogen binding
sites along the length of a protofibril would be located 22.5 nm apart,
whereas those on an adjacent protofibril are likely to be less than 5 to 10 nm away. Movement of plasmin across the fibrin may result from
binding of a second kringle to the next fibrin binding site before
release of the first kringle. Such a crawling of plasmin across the
fiber is consistent with the presence of more than one binding kringle
in each plasmin molecule43 and with its known
conformational changes.44,45 Additional evidence for such a
bridging mechanism is provided by the observation that plasminogen can
precipitate fibrin degradation products, forming complexes with a ratio
of one plasminogen to two fibrin degradation products.46
Also, plasminogen added to polymerizing fibrin enhances lateral
aggregation of protofibrils yielding clots made of thicker
fibers.47 In addition, observations of plasminogen covalently bonded to fibrin with a photoaffinity activated cross-linker show examples of one plasminogen molecule linked to the ends of three
or four fibrin molecules24 that must arise from adjacent protofibrils.
The results presented provide a new degree of detail regarding the
physical process of fibrinolysis. At the level of individual fibers,
the consequences of plasmin activity locally are to fully transect
individual fibers and release a heterogeneous group of large, soluble
products. However, from the perspective of a macroscopic clot, the
process may still be viewed as digestion from the outside-in, as the
action of plasmin is limited to the shell of fibers at the clot
periphery. Its action proceeds centrally with progressive degradation.
The observations also demonstrate that there are large-scale changes in
the three-dimensional integrity and mechanical stability of fibrin
coincident with release of the soluble degradation products. These
changes in the fibrin matrix will enhance permeation of fluid into the
clot, which is the most important mode of enzyme transport.35,48 Therefore, the cross-lateral cleavage of
individual fibrin fibers by plasmin with generation of larger pores may
result in progressive acceleration of fibrinolysis as resistance to
permeability decreases. This will facilitate access of enzyme to deeper
layers of clot and promote fiber degradation.
| |
FOOTNOTES |
Submitted May 27, 1998;
accepted August 12, 1998.
Supported in part by National Institutes of Health Grants No. HL30954
(to J.W.W.) and HL30616 (to C.W.F.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John W. Weisel, PhD, Department of Cell and
Developmental Biology, University of Pennsylvania School of Medicine,
Philadelphia, PA 19104-6058.
| |
REFERENCES |
1.
Wiman B, Collen D:
Molecular mechanism of physiological fibrinolysis.
Nature
272:549, 1978[Medline]
[Order article via Infotrieve]
2.
Collen D, Lijnen HR:
Fibrin-specific fibrinolysis.
Ann NY Acad Sci
667:259, 1992[Medline]
[Order article via Infotrieve]
3.
Collen D, Lijnen HR:
Molecular basis of fibrinolysis, as relevant for thrombolytic therapy.
Thromb Haemost
74:167, 1995[Medline]
[Order article via Infotrieve]
4.
Thorsen S:
The mechanism of plasminogen activation and the variability of the fibrin effector during tissue-type plasminogen activator-mediated fibrinolysis.
Ann NY Acad Sci
667:52, 1992[Medline]
[Order article via Infotrieve]
5.
Francis CW, Marder VJ, Barlow GH:
Plasmic degradation of crosslinked fibrin. Characterization of new macromolecular soluble complexes and a model of their structure.
J Clin Invest
66:1033, 1980
6.
Francis CW, Marder VJ:
A molecular model of plasmic degradation of crosslinked fibrin.
Semin Thromb Haemost
8:25, 1982[Medline]
[Order article via Infotrieve]
7.
Hantgan RR, Francis CW, Marder VJ:
Fibrinogen structure and physiology, in
Colman RW,
Hirsh J,
Marder VJ,
Salzman EW
(eds):
Hemostasis and Thrombosis: Basic Principles and Clinical Practice (ed 3). Philadelphia, PA, Lippincott, 1994, p 277.
8.
Gabriel DA, Muga K, Boothroyd EM:
The effect of fibrin structure on fibrinolysis.
J Biol Chem
267:24259, 1992[Abstract/Free Full Text]
9.
Carr ME, Alving BM:
Effect of fibrin structure on plasmin-mediated dissolution of plasma clots.
Blood Coagul Fibrinolysis
6:567, 1995[Medline]
[Order article via Infotrieve]
10.
Williams S, Fatah K, Ivert T, Blombäck M:
The effect of acetylsalicylic acid on fibrin gel lysis by tissue plasminogen activator.
Blood Coagul Fibrinolysis
6:718, 1995[Medline]
[Order article via Infotrieve]
11.
Kolev K, Tenekedjiev K, Komorowicz E, Machovich R:
Functional evaluation of the structural features of proteases and their substrate in fibrin surface degradation.
J Biol Chem
272:13666, 1997[Abstract/Free Full Text]
12.
Collet JP, Soria J, Mirshahi M, Hirsch M, Dagonnet FB, Caen J, Soria C:
Dusart syndrome: A new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure.
Blood
82:2462, 1993[Abstract/Free Full Text]
13.
Collet J-P, Woodhead JL, Soria J, Soria C, Mirshahi MC, Caen JP, Weisel JW:
Fibrinogen Dusart: Electron microscopy of molecules, fibers and clots and viscoelastic measurements.
Biophys J
70:500, 1996[Medline]
[Order article via Infotrieve]
14.
Wada Y, Lord ST:
A correlation between thrombotic disease and a specific fibrinogen abnormality (A alpha 554 Arg  Cys) in two unrelated kindred, Dusart and Chapel Hill III.
Blood
84:3709, 1994[Abstract/Free Full Text]
15.
Marder VJ, Shulman NR, Carroll WR:
High molecular weight derivatives of human fibrinogen produced by plasmin.
J Biol Chem
244:2111, 1969[Abstract/Free Full Text]
16.
Marder VJ, Budzynski AZ:
Data for defining fibrinogen in its plasmic degradation products.
Thromb Diath Hemorrh
33:199, 1975[Medline]
[Order article via Infotrieve]
17.
Schielen WJG, Adams HPHM, Voskuilen GJ, Tesser GJ, Nieuwenhuizen W:
Structural requirements of position Aalpha-157 in fibrinogen for the fibrin-induced rate enhancement of the activation of plasminogen by tissue-type plasminogen activator.
Biochem J
276:655, 1991
18.
Verheijen JH, Nieuwenhuizen W, Wijngaards G:
Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments.
Thromb Res
27:377, 1982[Medline]
[Order article via Infotrieve]
19.
Verheijen JH, Nieuwenhuizen W, Traas DW, Chang GTG, Hoegee E:
Differences in effects of fibrin(ogen) fragments on the activation of 1-glu-plasminogen and 442-val-plasminogen by tissue-type plasminogen activator.
Thromb Res
32:87, 1983[Medline]
[Order article via Infotrieve]
20.
Varadi A, Patthy L:
Location of plasminogen-binding sites in human fibrin(ogen).
Biochemistry
22:2440, 1983[Medline]
[Order article via Infotrieve]
21.
Varadi A, Patthy L:
Beta(Leu121-Lys122) segment of fibrinogen is in a region essential for plasminogen binding by fibrin fragment E.
Biochemistry
23:2108, 1984[Medline]
[Order article via Infotrieve]
22.
Hasan AAK, Chang WS, Budzynski AZ:
Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator.
Blood
79:2313, 1992[Abstract/Free Full Text]
23.
Lezhen TI, Kudinov SA, Medved LV:
Plasminogen-binding site of the thermostable region of fibrinogen fragment D.
FEBS Lett
197:59, 1986[Medline]
[Order article via Infotrieve]
24.
Weisel JW, Nagaswami C, Korsholm B, Petersen LC, Suenson E:
Interactions of plasminogen and polymerizing fibrin and its derivatives monitored with a photoaffinity cross-linker and electron microscopy.
J Mol Biol
235:1117, 1994[Medline]
[Order article via Infotrieve]
25.
Weisel JW, Francis CW, Nagaswami C, Marder VJ:
Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments.
J Biol Chem
268:26618, 1993[Abstract/Free Full Text]
26.
Weber K, Osborn M:
The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis.
J Biol Chem
244:4406, 1969[Abstract/Free Full Text]
27.
Francis CW, Marder VJ, Martin SE:
Detection of circulating crosslinked fibrin derivatives by a heat extraction-SDS gradient gel electrophoretic technique.
Blood
54:1282, 1979[Abstract/Free Full Text]
28.
Neville DM:
Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system.
J Biol Chem
246:6328, 1971[Abstract/Free Full Text]
29.
Langer BG, Weisel JW, Dinauer PA, Nagaswami C, Bell WR:
Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers.
J Biol Chem
263:15056, 1988[Abstract/Free Full Text]
30.
Weisel JW, Nagaswami C:
Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: Clot structure and assembly are kinetically controlled.
Biophys J
63:111, 1992[Medline]
[Order article via Infotrieve]
31.
Weisel JW:
The electron microscope band pattern of human fibrin: Various stains, lateral order, and carbohydrate localization.
J Ultrastruct Mol Struct Res
96:176, 1986[Medline]
[Order article via Infotrieve]
32.
Marder VJ, Francis CW:
Plasmin degradation of cross-linked fibrin.
Ann NY Acad Sci
408:397, 1982[Medline]
[Order article via Infotrieve]
33.
Sakharov DV, Rijken DC:
Superficial accumulation of plasminogen during plasma clot lysis.
Circulation
92:1883, 1995[Abstract/Free Full Text]
34.
Sakharov DV, Nagelkerke JF, Rijken DC:
Rearrangements of the fibrin network and spatial distribution of fibrinolytic components during plasma clot lysis. Study with confocal microscopy.
J Biol Chem
271:2133, 1996[Abstract/Free Full Text]
35.
Diamond SL, Anand S:
Inner clot diffusion and permeation during fibrinolysis.
Biophys J
65:2622, 1993[Medline]
[Order article via Infotrieve]
36.
Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC, Hantgan RR:
Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: Light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus.
Blood
81:1290, 1993[Abstract/Free Full Text]
37.
Weisel JW:
Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.
Biophys J
50:1079, 1986[Medline]
[Order article via Infotrieve]
38.
Shen LL, McDonagh RP, McDonagh J, Hermans J:
Early events in the plasmin digestion of fibrinogen and fibrin. Effects of plasmin on fibrin polymerization.
J Biol Chem
252:6184, 1977[Free Full Text]
39.
Hantgan R, Fowler W, Erickson H, Hermans J:
Fibrin assembly: A comparison of electron microscopic and light scattering results.
Thromb Haemost
44:119, 1980[Medline]
[Order article via Infotrieve]
40.
Hantgan RR, Hermans J:
Assembly of fibrin. A light scattering study.
J Biol Chem
254:11272, 1979[Abstract/Free Full Text]
41.
Francis CW, Marder VJ:
Rapid formation of large molecular weight alpha-polymers in cross-linked fibrin induced by high factor XIII concentrations.
J Clin Invest
80:1459, 1987
42.
Suenson E, Lützen O, Thorsen S:
Initial plasmin-degradation of fibrin as the basis of a positive feedback mechanism in fibrinolysis.
Eur J Biochem
149:193, 1984[Medline]
[Order article via Infotrieve]
43.
Christensen U, Mølgaard L:
Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change.
Biochem J
285:419, 1992
44.
Marshall JM, Brown AJ, Ponting CP:
Conformational studies of human plasminogen and plasminogen fragments: Evidence for a novel third conformation of plasminogen.
Biochemistry
33:3599, 1994[Medline]
[Order article via Infotrieve]
45.
Ponting CP, Marshall JM, Cederholm-Williams SA:
Plasminogen: A structural review.
Blood Coagul Fibrinolysis
3:605, 1992[Medline]
[Order article via Infotrieve]
46.
Garman AJ, Smith RAG:
The binding of plasminogen to fibrin: Evidence for plasminogen-bridging.
Thromb Res
27:311, 1982[Medline]
[Order article via Infotrieve]
47.
Petersen LC, Suenson E:
Effect of plasminogen and tissue-type plasminogen activator on fibrin gel structure.
Fibrinolysis
5:51, 1991
48.
Blinc A, Planinsic G, Keber D, Jarh O, Lahajnar G, Zidansek A, Demsar F:
Dependence of blood clot lysis on the mode of transport of urokinase into the clot A magnetic resonance imaging study in vitro.
Thromb Haemost
65:549, 1991[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. Vadseth, J. M. Souza, L. Thomson, A. Seagraves, C. Nagaswami, T. Scheiner, J. Torbet, G. Vilaire, J. S. Bennett, J.-C. Murciano, et al.
Pro-thrombotic State Induced by Post-translational Modification of Fibrinogen by Reactive Nitrogen Species
J. Biol. Chem.,
March 5, 2004;
279(10):
8820 - 8826.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-P. Collet, C. Lesty, G. Montalescot, and J. W. Weisel
Dynamic Changes of Fibrin Architecture during Fibrin Formation and Intrinsic Fibrinolysis of Fibrin-rich Clots
J. Biol. Chem.,
June 6, 2003;
278(24):
21331 - 21335.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kolev, K. Tenekedjiev, K. Ajtai, I. Kovalszky, J. Gombas, B. Varadi, and R. Machovich
Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution
Blood,
June 1, 2003;
101(11):
4380 - 4386.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.Ph. Collet, G. Montalescot, C. Lesty, and J.W. Weisel
A Structural and Dynamic Investigation of the Facilitating Effect of Glycoprotein IIb/IIIa Inhibitors in Dissolving Platelet-Rich Clots
Circ. Res.,
March 8, 2002;
90(4):
428 - 434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Collet, D. Park, C. Lesty, J. Soria, C. Soria, G. Montalescot, and J. W. Weisel
Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis Speed : Dynamic and Structural Approaches by Confocal Microscopy
Arterioscler Thromb Vasc Biol,
May 1, 2000;
20(5):
1354 - 1361.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.Ph. Collet, G. Montalescot, C. Lesty, and J.W. Weisel
A Structural and Dynamic Investigation of the Facilitating Effect of Glycoprotein IIb/IIIa Inhibitors in Dissolving Platelet-Rich Clots
Circ. Res.,
March 8, 2002;
90(4):
428 - 434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction