© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Disaggregation of In Vitro Preformed Platelet-Rich Clots by Abciximab Increases Fibrin Exposure and Promotes Fibrinolysis
Presented in part at the 71st Scientific Sessions of the American Heart Association, Dallas, Tex, November 8–11, 1998.
From the Department of Cardiology (J.P.C., D.T.) and the Hematology Research Center (C.L.), Pitié-Salpêtrière Hospital, Paris; Research Laboratory Sainte Marie (J.P.C., J.S.), Hotel Dieu Hospital, Paris; UPS 937 (Z.M.), CNRS, Paul Brousse Hospital, Villejuif; and Hemostasis Laboratory (C.S.), Lariboisière Hospital, Paris, France.
Correspondence to J.P. Collet, MD, PhD, Department of Cardiology, Centre Hospitalier Universitaire Pitié-Salpêtrière, 47, Boulevard de l’Hôpital, 75013 Paris, France. E-mail jean-philippe.collet{at}psl.ap-hop-paris.fr
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Abstract |
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Abstract—The glycoprotein IIb/IIIa receptor inhibitor abciximab has been shown to facilitate the rate and the extent of pharmacological thrombolysis with recombinant tissue plasminogen activator (rtPA) in patients with acute myocardial infarction. However, the underlying mechanisms remain not fully determined. We sought to demonstrate that this facilitating effect of abciximab could be related to its potential to modify the clot architecture and the clot physical properties. Compared with fibrin-rich clots, platelets dramatically modified the in vitro properties of the fibrin network, leading to a significant increase of the permeability (Ks) and the viscoelasticity (G') indexes but also leading to the appearance of platelet aggregates (surface area [S.ag]). These modifications resulted in a 2.6-fold decrease of the fibrinolysis rate when rtPA (1 nmol/L) was added before the initiation of clotting. Adding aspirin (100 µg/mL) or abciximab (0.068 µmol/L) before the clotting of platelet-rich clots (PRCs) lowered Ks by 50% and 70%, respectively (P<0.01), G' by 41% and 66%, respectively (P<0.01), and S.ag by 32% and 61%, respectively (P<0.01). As a consequence, the lysis speed was increased by 21% with aspirin (P<0.01) and 45% with abciximab (P<0.01). However, unlike aspirin, permeation of preformed PRCs with abciximab (0.068 µmol/L) decreased G' (37%, P<0.01), Ks (35%, P<0.001) and S.ag (25%, P=NS) and resulted in a 27% (P<0.01) increase of the lysis speed when abciximab and rtPA (0.2 µmol/L) were simultaneously permeated. This effect was found to be time dependent and was observed only with early permeation, starting within the first 10 minutes of clotting. These changes in the physical properties of the PRC architecture suggest that fibrin is removed from the platelet-fibrin aggregates and reexposed into the surrounding fibrin network, increasing rtPA access to fibrin and therefore the fibrinolysis rate. The superiority of abciximab over aspirin in accelerating fibrinolysis of forming and preformed PRCs is related to its ability to modulate the interactions of fibrinogen and fibrin with platelets. These findings provide new mechanistic information on reperfusion therapy.
Key Words: platelets • fibrin • fibrinolysis • glycoprotein IIb/IIIa inhibitors
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Introduction |
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Direct inhibition of platelet interactions with fibrin(ogen) by abciximab (Reopro) can restore coronary flow,1 as shown in randomized trials combining abciximab with either primary angioplasty or thrombolysis in acute myocardial infarction.2 3 4 5 However, the comprehensive mechanisms remain to be fully understood. In the present study, we sought to demonstrate that this effect of abciximab is related to its potential to modify platelet-fibrin(ogen) interactions, leading to changes of clot architecture and clot physical properties and facilitating pharmacological thrombolysis.
For the first step in our experiments, differences between the physical properties of fibrin-rich clots (FRCs) and platelet-rich clots (PRC) were studied to delineate the mechanical impact of platelets on the fibrin network properties and their consequences for the fibrinolysis rate. The second step in our experiments evaluated the consequences of the inhibition of fibrinogen interactions with platelets on the fibrin network properties by adding aspirin or abciximab before clotting. In the third set of experiments, preestablished PRCs were permeated with aspirin or abciximab to evaluate further their ability to modulate the interaction between platelets and fibrin. The impact of these pharmacological changes on the fibrinolysis rate was assessed at each step by either loading recombinant tissue plasminogen activator (rtPA) before the initiation of clotting or by permeating preestablished FRCs and PRCs with rtPA.
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Methods |
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Materials
Abciximab (Reopro) was supplied by Eli Lilly as a 2 mg/mL solution and was dissolved in buffer 1 (0.15 mol/L NaCl and 0.01 mol/L Tris-HCl, pH 7.4). Aspirin (Aspegic) was supplied by Synthelabo (Meudon-La-Forêt) as a 100 mg/mL solution and was dissolved in buffer 1. Murine monoclonal antibodies (anti-CD9 human platelet receptor [Syb]) were provided by Dr Boucheix (CNRS, Villejuif, France). Goat monoclonal anti-mouse antibodies from Amersham Pharmacia were available as 1 mg/mL solution in buffer 1. Human thrombin (Enzyme Research Laboratories Inc) was stored at 1000 IU/mL. rtPA was purchased from Boehringer-Ingelheim. FITC and streptavidin-tricolor were from Sigma, and CaCl2 (0.456 mol/L solution) was from Assistance Publique-Hôpitaux de Paris.
Preparation of Fibrin-Rich and
Platelet-Rich Thrombi
Blood was from 8 healthy informed volunteers who had
no known bleeding disorder and who had not ingested antiplatelet
medication for at least 10 days. It was anticoagulated with trisodium
citrate (1 vol of 0.13 mol/L citrate for 9 vol blood) and
centrifuged at 800g for
15 minutes to provide platelet-rich plasma (PRP).
Recentrifugation of PRP at
10 000g for 15 additional
minutes provided platelet-poor plasma (PPP), which was then
filtered (0.22-µm filters). PPP was then mixed with the PRP of the
same donor to adjust for the final platelet count. Addition of
CaCl2 (20 mmol/L) and thrombin (0.125
IU/mL) to 0.12 mL of either PPP or PRP led to the formation of FRCs and
PRCs.
Mechanical Properties of Clots
Permeation Experiments
The permeability index
(Ks) of
plasma FRCs and PRCs was measured by the permeation
technique.6 Briefly, clots
were formed in thin glass microchambers (250 µm) and were permeated
with buffer 1 at different gradients of pressure (Figure
I, available
online at http://atvb.ahajournals.org). The calculated
Ks index
(in centimeters squared) provides information on the fibrin network
architecture (shape and size of the pores) and represents the
surface of the gel allowing
flow.6 The thrombus
permeability index equation is as follows:
Ks=(Q · L · 
)/(A · 
P · t),
where Q is the volume of liquid (in milliliters) having the viscosity
(10-2 poise), flowing through the
fibrin gel with length L (2.2 cm) and cross section A (0.03
cm2) in a given time t (in seconds) under a
differential pressure P (ranging from 4000 to 10 000
dyne/cm2).
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Viscoelastic Experiments
FRCs and PRCs were formed between two 12-mm-diameter
glass coverslips in the torsion pendulum device shown in Figure
II
(available online at http://atvb.ahajournals.org) by using the same
clotting conditions as for the permeation
experiments.7 Clots had a
constant width of 1 mm. After the initiation of clotting, a
momentary impulse was carefully applied to the torsion pendulum arm
(air pressure), causing free oscillations of this arm with
strains <3%. The frequency of these free oscillations and
the rate at which they are damped are functions of the elastic and
viscous properties of the clots and are independent of the amplitude of
the initial displacement of the
arm.7 The rigidity index (G',
in dynes per centimeter squared), which reflects the viscoelastic
properties of the clot, was calculated from the recordings of
these oscillations on a chart recorder.
Two different sets of experiment were conducted: (1) FRCs
and PRCs that were formed with buffer or antiplatelet agents were
processed for
Ks and
G' measurements. (2) Preformed FRCs and PRCs were carefully permeated
with buffer or antiplatelet agents at a constant pressure gradient
of 3000 dyne/cm2 before being processed for
Ks and
G' measurements. A small perfusion chamber was added to the torsion
pendulum to allow perfusion of the clots at a constant gradient of
pressure while the viscoelastic properties were constantly monitored
(Figure II).
Clot Morphological Properties
Confocal Scanning Laser Microscopy
Experiments
Clots used for permeation experiments were then
permeated with a 2 mmol/L FITC solution dissolved in 0.01 mol/L
Tris, 0.1 mol/L NaCl, and 1 mmol/L EDTA (pH 8) buffer for 30
minutes. The excess of dye was eliminated by extensive washing with
buffer 1.8 FITC binds to
fibrin through nonspecific interactions with the COOH residues. Labeled
specimens were scanned with an ACAS 570 interactive laser cytometer
(Meridian Instruments) equipped with confocal optics. Twenty-five
optical sections were collected at intervals of 1.0 µm in the z-axis
and were combined into 1 image, generating a 3D reconstructed image of
the fibrin
network.6
Platelet Localization Within the Fibrin
Network
Platelets within the fibrin matrix were
specifically localized by using the anti-CD9 antibodies coupled to
biotin (25 µg/mL) that were incubated for 5 minutes at 37°C with
the PRP before the initiation of clotting. After clotting occurred,
anti-CD9 antibodies were revealed by permeation of the clot with
streptavidin-tricolor; fibrin was labeled with FITC as described above.
After washing, platelet-rich areas containing fibrin-FITC and
tricolor platelets appeared yellow; fibrin-FITC areas without
platelets appeared green.
Image Analysis
Quantitative analysis of the average area of
platelet aggregates (S.ag, in micrometers squared) was
performed with the Visilog software (version 5.01, Noesis). Because
aggregates and the surrounding fibrin agglomerated within the
reconstructed images from confocal scanning laser microscopy, an
optical algorithm based on "the watershed line" transformation
technique was required to separate them before being
measured.9 10
Lysis Experiments
Fibrinolysis was assessed by
monitoring of the viscoelastic index G' every 4 minutes, and the
fibrinolysis speed corresponded to the time taken for
G' to decrease to 50% of its maximal
value.11 Clots were formed in
the exact same conditions as for viscoelastic measurements, and 2
different assays were developed: (1) In the static lysis assay, rtPA
was added with buffer or with antiplatelet agents before the
initiation of clotting. rtPA concentration (1 or 2 nmol/L) was adjusted
so that lysis started after the first 10 minutes of initiation of
clotting.11 (2) In the
dynamic lysis assay, preformed PRC was carefully permeated with rtPA
and buffer or antiplatelet agents at a final concentration of 0.2
µmol/L, which is the in vivo predicted concentration from computer
simulation of
thrombolysis.12
The different rtPA concentrations used in these 2 assays reflect the
difference of their design: in the static assay, rtPA is already in the
fibrin fibers, whereas in the dynamic lysis assay, rtPA needs to be
delivered to the fibrin network under pressure-driven
permeation.13
Comparison of the Efficacy of Aspirin and
Abciximab in Remodeling the Anatomy of the Clot
To simulate in vitro the dynamic changes in the
structure and the lysis speed of an occlusive coronary
thrombosis after it is exposed to antiplatelet agents in vivo,
occlusive and fully hydrated FRCs and PRCs were used to evaluate the
potential of aspirin and abciximab to either prevent fibrinogen
interactions with platelets or to remove platelets from fibrin.
Aspirin and abciximab were either added before clotting initiation or
carefully infused through preformed clots. Permeation started 5 and 40
minutes after clotting initiation for a 20-minute time period at a
constant gradient of pressure of 3000
dyne/cm2. Fibrinolysis was
assessed by loading rtPA in combination with either aspirin or
abciximab before the initiation of clotting (static lysis assay) or by
permeating rtPA (0.2 µmol/L) in combination with aspirin or abciximab
(dynamic lysis assay). Control clots were formed and/or permeated with
buffer 1. G' and
Ks were
measured before, during, and after permeation. Clots used for
permeation experiments (without rtPA) were then processed for
microscopy.
Aspirin concentrations (50 and 100 µg/mL) correspond to an intravenous administration of 500 and 1000 mg aspirin, which completely inhibits platelet cyclooxygenase activity.14 Abciximab concentrations (0.034 and 0.068 µmol/L) correspond to an intravenous bolus administration of 0.15 and 0.30 mg/kg, which produces near-binding saturation of platelet surface glycoprotein (GP) IIb/IIIa receptors and inhibits thrombosis in vivo.15 This was further ascertained by the use of the rapid platelet functional assay (Accumetrics), which confirmed that 99% of the platelet GP IIb/IIIa receptors were occupied with both abciximab concentrations, whereas 95% and 100% of the platelet receptors remained free with aspirin at 50 and 100 µg/mL, respectively.16 These abciximab concentrations were also shown to inhibit 75% and 100% of the mechanical effect of platelets on fibrin, respectively.17
All experiments were conducted with a range of platelets of 75 000 to 150 000/µL. A dose-related effect of platelets on mechanical properties and on fibrinolysis speed was found. Results with a final platelet count of 75 000/µL are presented here, especially because measurements of permeability constant Ks and morphological index S.ag were more accurate than with 150 000 platelets per microliter.
Statistical Analysis
Statistical analysis was performed with
StatView software. Continuous variables were expressed as
mean±SEM, and group differences were determined by ANOVA. The Student
t test for paired samples was
used to take into account the heterogeneity among the
plasma of the 8 donors. A value of
P<0.05 was considered to
indicate statistical
significance.
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Results |
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Comparison Between FRC and PRC Properties
Intra-assay and interindividual variabilities in permeability and viscoelastic experiments were similar to those previously reported for plasma FRCs.8 The coefficients of variation for Ks and G' were 12.5% and 9.2%, respectively, for a double measurement on a single sample (intra-assay variability, n=8). Measurement of Ks was performed in different chambers. The coefficient of variation of Ks was slightly higher when platelets were added before clotting (16.5%, n=8).
Platelets increased the whole-clot permeability
(Ks) by
3.5-fold and the whole-clot rigidity (G') by 3-fold
(Table).
These platelet-dependent modifications of the physical properties
of the fibrin network were ascertained further by the reverse
correlation (R) between
Ks and
G' in PRCs (R=0.55,
P=0.02) compared with FRCs
(R=-0.64,
P=0.0061;
Figure 1). It is likely that platelets within the PRC
simultaneously strengthen and distort the fibrin network,
resulting in more space within the pores delimited by fibrin fibers.
This was confirmed by confocal microscopic analysis of the same
clots. A typical FRC appeared as a homogeneous structure
made up of straight rodlike elements corresponding to branching and
crossing fibrin fibers organized in a 3D network
(Figure 2A). The round white elements correspond to branching
fibers perpendicular to the plane of the scanning area. PRCs were
rather inhomogeneous and were characterized by aggregates
of platelets, for which the average surface area was 5562±521
µm2 (n=8,
Figure 2B). Fibrin fibers located within and at the edge of
these aggregates were bent as if they were stressed and trapped. The
double fluorescence labeling experiment showed that these
aggregates corresponded to the colocalization of platelets and
fibrin
(Figure 2C). Hence, superposition of fibrin-FITC and
platelet-tricolor within the aggregates appeared as yellow areas,
and FITC-fibrin alone appeared as green areas surrounding the
platelet-fibrin aggregate
(Figure 2C).
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Platelet-mediated fibrin retraction led to a
2.6-fold increase in the lysis time of PRCs compared with FRCs formed
in the same conditions and with use of the static lysis assay (1 nmol/L
rtPA,
Figure 3a). Raising the rtPA concentration up to 2 nmol/L
lowered the average lysis time of PRCs by 32.8±20% (n=16,
P<0.0001), but it still
remained significantly higher than that of FRCs with 1 nmol/L of rtPA
(Figure 3a).
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Effect of Adding Antiplatelet Agents
Before Clotting
Unlike FRCs, mechanical properties of PRCs were greatly
affected by the addition of abciximab or aspirin before the initiation
of clotting
(Table).
In all treated groups, a significant decrease in
Ks and
G' was found. Unlike aspirin, abciximab displayed a dose-dependent
efficacy in reducing G' but not
Ks.
Unlike aspirin, abciximab (0.068 µmol/L) reversed the relation
between
Ks and
G', but in a nonsignificant manner
(R=-0.28,
P=0.28;
Figure 1). This relation was intermediate between the
positive one of PRCs (R=0.55,
P=0.02) and the negative one of
FRCs (R=-0.64,
P=0.0061). This indicates that
despite a nearly complete inhibition of fibrinogen-platelet
interactions, as shown by the measurement of GP IIb/IIIa receptor
occupancy with the Accumetrics device, abciximab failed to restore a
platelet-free–like fibrin structure. The significantly higher
values of G' and
Ks of
PRCs formed with 0.068 µmol/L of abciximab compared with FRCs further
support this assumption. It seems likely that interactions between
platelets and fibrin differ from interactions between platelets
and fibrinogen.
These mechanical changes are correlated with morphological
changes. Hence, adding abciximab or aspirin before PRC formation
significantly decreased the average area of platelet-fibrin
aggregates (S.ag), with a trend for a dose-dependent effect
(Table
and
Figure 2D and 2E). However, the only significant reduction
between treated groups was found between 50 µg/mL of aspirin and
0.068 µmol/L of abciximab. These morphological changes resulted in a
more homogeneous fibrin architecture with more fibrin
exposed within the fibrin network, which is consistent with the
concomitant reduction of both
Ks and
G'
(Table).
Adding abciximab (0.034 µmol/L) and rtPA (1 nmol/L) before
the initiation of clotting reduced the average lysis time of PRCs by
36.7±11% (P<0.001) compared
with PRCs formed without abciximab. Addition of abciximab had the same
effect as a 2-fold increase in the rtPA concentration (20.2±2.7 and
20.75±3.95 minutes, respectively;
P=0.77;
Figure 3a). Although nonsignificant, there was a trend for a
dose-related efficacy of the profibrinolytic effect of abciximab. The
lysis rate of PRCs formed with 0.068 µmol/L abciximab still remained
slower than the lysis rate of FRCs at a similar concentration of rtPA
(P=0.019); this was
consistent with the remaining difference in the physical
properties between these 2 types of clots
(Figure 3a). Aspirin at 50 µg/mL increased the lysis rate
of PRCs by 21±8% (n=8,
P<0.01), but unlike abciximab,
there was no trend for a dose-related effect (not
shown).
Effects of Permeating Preestablished PRCs With
Antiplatelet Agents
Permeation of preformed FRCs with aspirin or abciximab
did not affect the physical properties of fibrin. Unlike buffer or
aspirin, permeation of PRCs at a constant pressure gradient with 0.068
µmol/L abciximab significantly reduced
Ks and
G', whereas S.ag was decreased in a nonsignificant manner
(Table
and
Figure 2F). Interestingly, these changes were found to be
time-related. Early perfusion, starting within the 10 first minutes of
clotting, was effective, whereas late perfusion, starting 40 minutes
after the initiation of clotting, had no significant effect
(Table).
Permeation of preformed PRCs with rtPA (200 nmol/L) and
aspirin (100 µg/mL) had an effect on the lysis time similar to that
obtained by permeation with rtPA alone. Unlike aspirin, permeation of
PRCs with abciximab (0.068 µmol/L) and rtPA (200 nmol/L) produced a
27% increase of the lysis speed
(P<0.001,
Figure 3b). This accelerating effect of abciximab was not
dose-related but time-related. As for mechanical properties, it was
found with early permeation (starting within the first 10 minutes of
initiation of clotting) but not with late permeation (starting after 40
minutes).
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Discussion |
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The present work was designed to evaluate the effect of platelets and platelet GP IIb/IIIa antagonists on fibrin physical properties and the fibrinolysis rate of native fully hydrated PRCs. It was hypothesized that inhibition of the interactions of platelet-membrane receptor GP IIb/IIIa with either fibrinogen or fibrin could modify the architecture of PRCs, leading to an acceleration of its dissolution. The most important finding of this in vitro study is that unlike aspirin, early permeation of preformed PRCs with abciximab can disaggregate these clots and facilitate fibrinolysis. These results provide new information on the mechanisms of abciximab to achieve full reperfusion of initially occluded coronary arteries.
Impact of Adding Platelets and
Antiplatelet Agents Before Clotting on Physical Properties and
Fibrinolysis Rate of PRCs
Our results provide for the first time direct
correlations between mechanical and morphological properties of fully
hydrated clots. Compared with FRCs, PRCs are stiffer, more porous
(increase of G' and
Ks,
respectively), and more heterogeneous, because of the
presence of platelet-fibrin aggregate-like structures. These
platelet-related changes of the fibrin physical properties account
in part for the fibrinolysis resistance of PRCs. Adding
antiplatelet agents before clotting prevented platelet-related
mechanical (reduction of G' and
Ks) and
morphological changes (reduction of S.ag) of the fibrin network,
leading to a significant increase of the fibrinolysis
rate. Unexpectedly, decreased permeability was found to be associated
with increased lytic speed in the static lysis assay. This is because
fibrin, which was trapped into the platelet-fibrin aggregates, has
been reexposed in the surrounding fibrin network as a consequence of
the inhibition of the interaction between fibrinogen and platelets
by abciximab. Consequently, more fibrin is accessible to rtPA, which is
loaded before the initiation of clotting, leading to a faster
fibrinolysis, but the overall permeability of the clot
is decreased.
The superiority of abciximab over aspirin in this setting
was found to be directly related to a better inhibition of the
fibrinogen-platelet interactions. Although aspirin provided a
complete inhibition of the platelet
cyclooxygenase activity, nearly all of the GP
IIb/IIIa receptors remained functional after platelet activation
with thrombin. The dose-related effect of abciximab that is reported in
the present study further confirms previous in vivo pharmacodynamic
studies showing a dose response of abciximab binding to platelet GP
IIb/IIIa receptors.15
Interestingly, although only few GP IIb/IIIa receptors remained
unoccupied with the highest dose of abciximab (0.068 µmol/L), a
significant difference between the physical properties and the
fibrinolysis rate of PRCs and FRCs was found. This
finding is consistent with recent studies using recombinant
fibrinogens showing that platelet interactions with fibrin may
involve receptors other than GP
IIb/IIIa.18 19
Furthermore, the present study corroborates previous data showing
that at 0.034 to 0.068 µmol/L abciximab, platelet-mediated fibrin
retraction is not completely abolished, although there is a complete
inhibition of platelet aggregation at 
0.034 µmol/L
abciximab.20
Impact of Permeation of Antiplatelet Agents
and/or rtPA on Physical Properties and Fibrinolysis
Rate of Preformed PRCs
To consider rheological, transport, and enzymatic
events, preformed PRCs were permeated at a constant gradient of
pressure with antiplatelet agents and/or rtPA. As expected, PRCs
were found more resistant to fibrinolysis than
were FRCs. These findings are consistent with previous studies
showing a decreased binding of tPA in PRCs as a consequence of
platelet
retraction.21 22
Unlike aspirin, permeation of PRCs with abciximab significantly
decreased
Ks and
G', suggesting that fibrin is removed from preexisting aggregates and
reexposed into the surrounding fibrin network, as confirmed by confocal
microscopy (decrease of S.ag). Interestingly, these abciximab-related
changes of the fibrin network structure were found to be time-related
and were observed only with early permeation (<10 minutes) and led to
a faster fibrinolysis. Therefore, it is likely that the
dissociating capacity of abciximab depends on the age of the PRCs and
that the structure of the PRCs changes over time. This was ascertained
by the continuous increase of G' up to 40 minutes after the initiation
of clotting and relates to the development of the
platelet-contractile force that slowly retracts the fibrin
network.23 Therefore, we
assume that within the first 10 minutes of the initiation of clotting,
the dissociating capacity of abciximab is strong enough to reverse the
interactions between fibrin and activated
platelets,24 whereas
after 40 minutes, tight contacts between fibrin and platelets may
prohibit the big molecule of abciximab (molecular weight of 47 000) to
access the GP IIb/IIIa receptors. The fact that the interactions of
fibrin with platelets may involve receptors other than GP IIb/IIIa,
as shown above, is an important additional explanation for the limited
efficacy of permeation of abciximab in disaggregating
PRCs.
Relations of Our Experimental Design to
Clinical Realities
Our data relate to clinical realities in different
ways. Coronary thrombosis involves simultaneous
deposition and lysis of red (fibrin-rich) and white (platelet-rich)
clot components, causing intermittent vessel occlusion. Although this
dynamic aspect of coronary thrombosis could not be directly
assessed in our experimental design, our data clearly emphasize that
abciximab could affect simultaneously preformed PRCs
(disaggregating potential) and clot extension by the prevention of
platelet-related fibrin remodeling. Moreover, it is likely that the
potential of abciximab to reduce the generation of thrombin may
accentuate the limitation of the clot extension in vivo, although this
has not been directly demonstrated in our
work.25 Hence, fibrin
expression from platelets plays a minor role in our experimental
system because fibrin formation is mainly related to the activation of
fibrinogen molecules by the exogenous thrombin addition. Thrombin that
is generated as a consequence of platelet activation has a
different kinetic profile and usually appears after the activation of
nearly all the fibrinogen molecules.
The time-related efficacy of abciximab in disaggregating
preformed PRCs is consistent with the temporal relationship
detected between the age of the thrombus and the efficacy of abciximab
in reperfusing the occluded coronary artery in
vivo.26 These in vitro data
further support recent results of randomized trials showing that
abciximab with reduced-dose fibrinolytic agents enhances fibrin and
platelet lysis, resulting in rapid and complete reperfusion (within
60 minutes) in a high proportion of patients with myocardial infarction
(nearly 70%).4 5
Moreover, our findings are consistent with those of the same
trials showing that adding low-dose reteplase to abciximab provides
slightly superior rates of culprit artery patency compared with those
with standard-dose reteplase alone (see
Figure 3).
Study Limitations
In terms of clot mechanics, final platelet count is
obviously a critical parameter and therefore a limitation
of our experimental design. We found a correlation between clot
strength and platelet count with a dose-related enhancement of G',
as described earlier: G'=420+p0.76 (where p
is platelet
concentration).27 In
parallel, the whole-clot permeability,
Ks, was
found to increase linearly with platelet concentration. However,
accurate measurements of
Ks were
impaired at >100 000 platelets per microliter because retraction
led to the formation of leaks. For the same reasons, morphological
analyses were not possible. Fibrinolysis
resistance using the static lysis assay was found to be positively
correlated with the platelet concentration, and the effect of
abciximab on physical properties and the fibrinolysis
rate was of a similar magnitude over the whole range of platelet
concentration.
The question arises as to whether our experimental conditions are relevant to an in vivo scenario, especially regarding the retraction phenomena. Red blood cells are an important component of the thrombus, primarily because their incompressibility limits clot retraction to 50%.13 In our system, red blood cells were discarded because the system did not allow optical microscopy to be performed; however, the high surface area of contact of the clots within the microchamber considerably limited the retraction phenomena. Therefore, we assume that a final platelet count of 75 000 per microliter may have a similar impact on the fibrin network as a platelet concentration of 150 000 per microliter in the presence of red blood cells.
Although the present study emphasizes the role of PRC architecture as an important determinant of the fibrinolysis rate, other factors are of major importance. Plasminogen activator inhibitor type 1, which has been shown to be a major determinant of the failure of thrombolytic therapy in vitro and in vivo, may have an important role in our experimental setting.28 29 30 It has been detected only in platelet-fibrin aggregates and with a higher intensity in PRCs formed with buffer compared with abciximab. Further quantitative analyses are needed to evaluate the impact of plasminogen activator inhibitor type 1 in our setting. A structural and dynamic approach to the early stage of PRC formation and fibrinolysis is also an important issue and is also needed to evaluate our findings and to extend previous work on the concept of a heterogeneous architecture of occlusive PRCs with aggregates that resist fibrinolysis.31 32 33
In conclusion, inhibition of platelet-mediated fibrin remodeling and removal of fibrin from preexisting aggregates contribute to the disaggregating potential of abciximab and are potential mechanisms for its additional benefit over aspirin in the setting of thrombolysis in myocardial infarction.
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Acknowledgments |
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This study was supported by a grant from the Association de Cardiologie Ile-de-France, Paris, France. We wish to thank J.W. Weisel, PhD, from the Department of Cell and Developmental Biology, University of Pennsylvania (Philadelpha, Pa), for the fruitful discussion and his expert review of the manuscript.
Received May 12, 2000; accepted July 12, 2000.
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References |
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1. Gold HK, Garabedian HD, Dinsmore RE, Guerrero LJ, Cigarroa JE, Palacios IF, Leinbach CL. Restoration of coronary flow in myocardial infarction by intravenous chimeric 7E3 antibody without plasminogen activators. Circulation. 1997;95:1755–1759.
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