|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2064-2074
No Effect of Clot Age or Thrombolysis on Argatroban's Inhibition of
Thrombin
By
Roy R. Hantgan,
W. Gray Jerome, and
Marcie J. Hursting
From the Departments of Biochemistry and Pathology, Wake Forest
University School of Medicine, Winston-Salem, NC; and Texas
Biotechnology Corp, Houston, TX.
 |
ABSTRACT |
The purpose of this study was to establish the effects of clot age
and thrombolysis, with either streptokinase or tissue-type plasminogen
activator (tPA), on argatroban's ability to inhibit thrombin. The antithrombotic activity of argatroban has been quantified in fibrin clot permeation and fibrin clot perfusion systems as a
function of clot age and composition. Analysis of the argatroban dose-response data with a competitive inhibition model has yielded IC50 values in the low micromolar range. Results obtained
in a plasma clot permeation system have also shown that argatroban is a
potent inhibitor of clot-bound thrombin, independent of either clot age
or the presence of hemostatically active platelets. Treatment of aged
plasma clots with either streptokinase or alteplase, at therapeutic
levels, increased the available thrombin activity, yet argatroban still
inhibited this clot-associated thrombin with IC50 values in
the low micromolar range. Scanning electron microscopy/morphometric analyses demonstrated that permeation with argatroban had no
significant effects on clot structure. We conclude that argatroban is
an effective inhibitor of thrombin bound to aged fibrin clots, in
purified systems and in plasma clots, as well as in clots that have
been treated with the thrombolytic agents streptokinase and alteplase.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ARGATROBAN IS A direct thrombin inhibitor
under clinical development as adjunctive therapy to thrombolytic agents
in acute myocardial infarction (AMI). Recent clinical trials have shown argatroban to be especially effective when administered in conjunction with a thrombolytic agent within 6 hours of the onset of AMI
symptoms.1,2 Biochemical studies have shown that argatroban
[((2R,4R)-4-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl-2-piperidine-carboxylic acid monohydrate] is a potent and selective thrombin inhibitor, one
that displays a Ki of 19 nmol/L for thrombin, compared with values of 5 µmol/L for trypsin, 210 µmol/L for factor Xa, and 800 µmol/L for plasmin.3,4 Argatroban (in the absence of any
cofactor) effectively inhibits the ability of thrombin to cleave
fibrinogen, factor XIII, and protein C and to initiate platelet
aggregation.4 Structural studies have shown that argatroban binds tightly to thrombin by inserting the dual hydrophobic
substituents on its arginine backbone into deep clefts on thrombin that
are near to, but distinct from its active site.5,6 Thus,
steric hindrance blocks thrombin's physiological substrates from
access to its catalytic pocket.6
Argatroban's ability to inhibit thrombin that is bound to fibrin and
plasma clots7 as well as to prevent fibrin-bound thrombin from aggregating platelets8 has led investigators to
explore its applications for treatment of cardiovascular
disease.9-12 Studies in experimental models of thrombotic
disease have shown argatroban to reduce cyclic blood flow variations in
a canine model of coronary artery stenosis,13 as well as to
accelerate thrombolysis mediated by urokinase plasminogen activator
(uPA)14 and recombinant tissue-type plasminogen activator
(rtPA)14,15 in vitro and in rabbit occluded
artery models.
Studies in human volunteers have shown argatroban to be well-tolerated
at doses that prolong the activated partial thromboplastin time (aPTT)
up to 2.6-fold.9-11 The therapeutic applications of argatroban have been extensively studied in Japan,9 and
early clinical experience in America has demonstrated the potential of
argatroban to provide an effective anticoagulant for patients with
heparin-induced thrombocytopenia (HIT) and heparin-induced thrombocytopenia and thrombosis syndrome (HITTS).16,17
Argatroban has also been shown to enhance rtPA-mediated coronary
reperfusion as a treatment for acute myocardial infarction when
administered within 6 hours of symptom onset, as documented in the MINT
study.1 In fact, argatroban provided special benefit, compared with heparin, for patients presenting at 6 hours.1 However, with streptokinase, argatroban tends to accelerate coronary reperfusion more readily in patients treated within 3, versus 6, hours
of AMI symptom onset.2 This raises the possibility that
argatroban's ability to inhibit clot-bound thrombin may decrease with
clot age and/or be sensitive to possible temporally related changes imparted on the clot by streptokinase.
These issues were investigated in this study with new in vitro models
of the aged human thrombus. The goals of this study were to determine
if the ability of argatroban to inhibit clot-bound thrombin changes
with clot age and to determine if the ability of argatroban to inhibit
clot-bound thrombin changes after fibrinolysis mediated by either
streptokinase or alteplase. Because clinical trials have demonstrated
that events occurring between 3 and 6 hours after the onset of symptoms
of an acute myocardial infarction can have significant effects on
argatroban's efficacy,1,2 the experimental systems
presented here have been designed to simulate this therapeutic
window. Neither clot age nor clot lysis has been previously addressed
in this context, because data in the literature only describe
argatroban inhibition of thrombin bound to intact clots at a single
time point, ie, 30 minutes.7 Results will be presented here
that demonstrate that argatroban is an effective inhibitor of thrombin
bound to fibrin and plasma clots aged as long as 6 hours and that
argatroban's antithrombin activity is preserved after lysis of these
aged plasma clots with either streptokinase or alteplase. Further,
scanning electron microscopy data will be presented that show that
permeation of intact and partially lysed plasma clots with argatroban
does not cause significant changes in clot structure.
| |
MATERIALS AND METHODS |
Materials
Argatroban (NOVASTAN) was provided by Texas Biotechnology Corp
(Houston, TX). Highly purified human  -thrombin (specific activity, 2,720 NIH units/mg) was a gift (to R.R.H.) from Dr J. Fenton (New York
State Department of Health, Wadsworth Center for Laboratories and
Research, Albany, NY). Highly purified human fibrinogen
(plasminogen-free and factor XIII-free) and streptokinase (4,000 IU/mg)
were purchased from American Diagnostica (Greenwich, CT). Alteplase
(Activase) was purchased from Genentech (South San Francisco, CA).
Chromogenic substrate S-2238 was purchased from Chromogenix AB
(Molndal, Sweden). Aprotinin, bovine serum albumin (Fraction V), and
cytochalasin B were purchased from Sigma Chemical Co (St Louis,
MO).
Plasma Isolation Procedures
Blood was drawn by venipuncture from healthy, adult volunteer donors
(who had taken no aspirin in the preceding 2 weeks) into 1/10 vol of
sodium citrate (110 mmol/L) anticoagulant. The procedures used were
fully examined and approved by the Clinical Research Practices
Committee of the Bowman Gray School of Medicine. Platelet-rich plasma
(PRP) and platelet-poor plasma (PPP) were isolated by differential centrifugation, and platelet counts were determined with a Coulter Model Z instrument (Coulter, Hialeah, FL), as previously
described.18
Thrombin Activity Assays
Solution assay.
Thrombin was diluted to concentrations in the range of 0 to 3 NIH
units/mL in either 0.13 mol/L NaCl, 0.05 mol/L Tris, pH 8.3, or 0.13 mol/L NaCl, 0.01 mol/L HEPES, pH 7.4, and aliquots of 100 µL were
transferred to flat-bottom microtiter plates (Falcon 3915; Becton
Dickinson and Co, Lincoln Park, NJ). Next, a 100-µL aliquot of
chromogenic substrate S-223819 (560 µmol/L in water) was
added to each well and the plates were incubated for 30 minutes at
23°C. The reaction was quenched by the addition of 50 µL of 20%
acetic acid and the absorbance at 405 nm was measured in a Vmax
microtiter plate reader (Molecular Devices, Palo Alto, CA). The assay
was found to be linear over the range of 0.03 to 0.90 NIH units/mL
thrombin (correlation coefficient = 0.95); the activity at pH 7.4 was
81% of that at pH 8.3. These observations formed the basis for
development of a chromogenic thrombin assay in fibrin clots, as
described next.
Fibrin clot-based thrombin assay (clot permeation system).
Fibrin clots were formed by the addition of thrombin (to 0.1 to 1 NIH
units/mL) to fibrinogen (1.0 mg/mL) in HEPES-buffered saline, pH 7.4 (HBS). Immediately after mixing, triplicate aliquots (50 µL each) of
the polymerizing fibrin solution were transferred to wells of a
microtiter plate and incubated for 30 minutes at 23°C. Selected
fibrin clot wells were washed by the addition of 100 µL of HBS
followed by careful removal of excess buffer, whereas other wells were
untreated. Chromogenic substrate S-2238 (100 µL at 560 µmol/L) was
added to each well and the plate was transferred to a Vmax microtiter
plate reader, operated in the kinetic mode at 405 nm (1 point per
minute for 30 minutes).
The rate of color development, expressed as milli-optical density (OD)
per minute, was found to be linear up to an absorbance of 0.2. Furthermore, this index of thrombin activity in the washed fibrin clots
was found to be 84% ± 4% of that in the untreated wells, based on
experiments performed at 0.3 NIH units/mL thrombin. Additional
experiments of this type demonstrated a linear relationship between the
initial rate of color development and the concentration of thrombin
present in the fibrin clots, over the range of 0.1 to 1 NIH units/mL
(correlation coefficient = 0.87).
This versatile fibrin clot-based thrombin activity assay was used in
subsequent experiments to characterize the ability of argatroban to
inhibit thrombin bound to aged fibrin clots. In those experiments,
fibrin clots containing 0.5 NIH units/mL thrombin were aged for 0.5, 3, or 6 hours and then a 100-µL aliquot of either HEPES-buffered saline
or argatroban (0 to 300 µmol/L) was added to each fibrin
clot-containing well. After 30 minutes of incubation with the plate on
a rotary mixer, excess liquid (~80 µL) was carefully removed
without disturbing the clot. Next, a 100-µL aliquot of S-2238 (560 µmol/L) was added to each well and the rate of color development (at
405 nm) was measured as described earlier in this section.
In addition, the observation that fibrin clots incubated with S-2238
soon developed a distinct yellow color led us to develop an additional
clot-based thrombin assay performed in optical perfusion chambers.
Fibrin clot-based thrombin assay (clot perfusion system).
Clot-based thrombin assays were also performed in real-time kinetic
mode using optical flow cells (Hellma Cells, Forest Hills, NY; model
178.010-OS, z = 15 mm). Clotting was initiated by addition of thrombin
(to 0.5 U/mL) to fibrinogen (1.0 mg/mL) in HBS, pH 7.4, containing
bovine serum albumin (3 mg/mL) and CaCl2 (2.0 mmol/L), and
then a 200-µL aliquot of polymerizing fibrin solution was transferred
by pipette to fill the optical chamber (80 µL) and its inlet and
outlet ports before the gel point. The absorbance at 405 nm was
recorded as a function of time in an LKB Ultrospec spectrophotometer that was interfaced to a laboratory
microcomputer with a Keithley Model DAS-8PGA 8-channel, 12 bit analog
input board operated in conjunction with the EASYEST LX software
package (Keithley ASYST, Rochester, NY). The increase in absorbance at 405 nm versus time provided a direct index of the clot formation process, which was half-complete in approximately 5 minutes and reached
a plateau by approximately 60 minutes.
After an incubation period of 180 minutes at 23°C, the clot was
gently perfused (6 mL/h) with 2 clot volumes of buffer or argatroban (0 to 300 µmol/L). Thirty minutes after starting the first perfusion
step, the clot was perfused with 1.5 clot volumes of S-2238 (560 µmol/L) with continuous recording of the absorbance at 405 nm. In
control experiments (ie, perfusion of the clots with buffer), the
initial rate of increase in A405 was found to be linear up
to 1.5 absorbance units and to exhibit a slope of 39 ± 7 mOD/min.
This system was used to monitor the effects of argatroban on fibrin
clot structure, as well as to determine dose-response curves for
argatroban inhibition of fibrin clot-bound thrombin. However, the
increased absorbance (>3) at 405 nm measured with plasma clots
precluded their examination in this system. Therefore, a plasma-clot
based permeation assay was also developed, as described next.
Plasma clot-based thrombin assay (clot permeation system).
Plasma clots were formed by addition of thrombin (to 0.5 U/mL) and
calcium chloride (to 20 mmol/L) to citrated human plasma, and then
triplicate 50-µL aliquots were transferred to the wells of a
microtiter plate before the gel point. When the assay was performed
with PRP, cytochalasin B was added (to 100 µmol/L) and the PRP was
preincubated for 5 minutes before the addition of thrombin and calcium.
This additional step was taken to minimize platelet-mediated clot
retraction,20 thus insuring a uniform clot for subsequent
stages of the thrombin assay.
Microtiter plates containing both PRP and PPP clots were incubated for
3 or 6 hours in sealed, moist chambers at 23°C. Next, 100-µL
aliquots of argatroban (0 to 300 µmol/L) or HBS were added to the
plasma clot wells, and the plates were incubated on a rotary mixer for
30 minutes. Excess liquid (~80 µL) was removed and 100-µL aliquots of S-2238 (560 µmol/L) were added to each well. The rate of
color development was measured at 405 nm with the microtiter plate
reader operated in the automix/kinetic mode, and the initial rates
( A405, <0.2) were determined with SOFTMAX software
provided by Molecular Devices, Inc.
Lysed plasma-clot thrombin assays (clot permeation system).
Variations on the plasma-clot-based system were developed to
investigate both the time course of clot lysis by streptokinase and
alteplase and the effects of thrombolysis on the activity of clot-bound
thrombin.
Clot Lysis Kinetics
The time course of plasma clot formation and lysis were measured by
adding 100-µL aliquots of polymerizing plasma (containing 0.5 U/mL
thrombin and 20 mmol/L calcium chloride) to the wells of a microtiter
plate and then measuring changes in the absorbance (turbidity) at 405 nm versus time with the Vmax device operated in the kinetic mode (but
with no mixing) over a 3-hour period. After this incubation, 100-µL
aliquots of either buffer, streptokinase (250 or 500 U/mL), or
alteplase (10 or 20 µg/mL) in HBS were layered on each plasma clot.
Upon returning the microtiter plate to the reader, the subsequent
changes in A405 were monitored for an additional 120 minutes. The extent of clot lysis was determined from the decrease in
turbidity that accompanied the lytic process.
Thrombin Activity Assays After Clot Lysis
Plasma clots were formed from 50-µL aliquots of PPP and incubated for
3 or 6 hours, as described earlier. Then, 50-µL aliquots of either
HBS, streptokinase (250 U/mL), or alteplase (10 µg/mL) were layered
on each clot and incubated for 30 minutes. Next, 100-µL aliquots of
either buffer or argatroban (0 to 100 µmol/L) were added to each well
and incubated for 30 minutes. Anticipating that thrombin was likely to
be present both in solution and within these partly lysed clots, excess
liquid was not removed for these assays. Rather, substrate S-2238 (560 µmol/L) was added directly to each well and the increase in
A405 was monitored as described above.
Recognizing that these thrombolytic agents can generate plasmin in
plasma, as well as in plasma clots, and that the resultant plasmin can
cleave S-2238 (albeit at a 200-fold slower rate than thrombin19), control wells containing 50-µL aliquots of
plasma were also carried throughout the assay to determine the
magnitude of this effect. Additional controls for plasmin activity were performed by incubating plasma clots with buffer, streptokinase (250 U/mL), or alteplase (10 µg/mL) and then adding one of the following
mixtures to these partly lysed clots: buffer, argatroban (100 µmol/L), aprotinin (100 KIU/mL), or argatroban (100 µmol/L) + aprotinin (100 KIU/mL).
Scanning Electron Microscopy (SEM)
SEM was used to examine fibrin structure within the clot permeation
systems just described. Cylindrical clots were assembled, aged,
permeated, and lysed in wells (6 to 8 mm in diameter × 2 mm deep)
that had been machined in the center of carbon planchettes. Fibrin
clots (purified system) in HBS, pH 7.4, were aged for 1 hour and then
overlaid with either buffer or argatroban (100 µmol/L) for 30 minutes
before processing for microscopy. Plasma clots were aged for 3 hours
and then overlaid with either buffer, argatroban (100 µmol/L),
streptokinase (250 U/mL), or alteplase (10 µg/mL) for 30 minutes.
Selected plasma clots were first treated with thrombolytic agent for 30 minutes, excess liquid was removed, and then the clots were treated
with argatroban (100 µmol/L) for an additional 30 minutes.
After these treatments, the clotted samples were dehydrated through a
graded series of ethanol and dried from CO2 by the
critical-point method.21 The samples were sputter-coated
with 50 Å of gold-palladium and observed at 15 keV in a Phillips 501 scanning electron microscope (Philips Electronic Instruments, Mahwah,
NJ). Measurements of fiber widths were determined from micrographs
obtained at a magnification of 18,300×; measurements were made on
20 to 50 randomly selected fibers for each sample. Statistical
analyses, including the t-test and ANOVA, were performed with
SigmaStat software (Jandel Scientific, San Rafael, CA).
| |
RESULTS |
Effects of Fibrin Clot Aging on Argatroban's Antithrombin Activity
Argatroban has been previously shown to be an effective inhibitor of
thrombin bound to fibrin clots as well as thrombin free in solution,
exhibiting IC50 values in the low micromolar range in both
cases.7 However, because the available data only describe fibrin clots incubated with argatroban after an initial 30-minute clot
stabilization period,7 the question remains: Do
time-dependent changes in clot structure22-25 influence
argatroban's antithrombin activity? This issue was addressed by
incubating fibrin clots for 0.5, 3, or 6 hours before permeation with
argatroban concentrations ranging from 0 to 300 µmol/L, incubating
each clot for 0.5 hours before removing excess inhibitor, and then
detecting the remaining clot-bound thrombin activity with a chromogenic
substrate.
Characterization of a New Assay for Clot-Bound Thrombin
This assay used coarse fibrin clots, ie, those prepared under
near-physiological conditions of pH (7.4) and salt concentration (0.13 mol/L NaCl) to yield thick fibrin strands. SEM showed a highly
interconnected network of fibrin strands, characterized by an average
fiber diameter of 72 ± 12 nm and a distance between branchpoints in
the range 300 to 600 nm. The total depth of each cylindrical clot
(formed in the wells of a microtiter plate) was set at approximately
1.6 mm to maximize permeation with both argatroban and substrate. SEM
yielded a mean fiber diameter of 72 ± 11 nm for clots that
had been permeated with argatroban (100 µmol/L), indicating that no
significant changes in clot architecture had occurred during this
treatment.
Consistent with earlier observations, thrombin incorporated into these
fibrin clots remained tightly bound,7,26 as evidenced by
the recovery of approximately 85% of the amidolytic activity after
permeation of clots with buffered saline and removing excess liquid
before delivery of the chromogenic substrate (Materials and Methods).
Furthermore, the thrombin activity detected in these fibrin clots was
approximately 30% of that observed with the same thrombin
concentration free in solution (Fig 1,
insert), indicating that a substantial fraction of this clot-bound
thrombin was readily accessible to substrate. However, clot-bound
thrombin differed from that in solution in that its activity remained
constant over a 6-hour period at room temperature. In contrast,
thrombin in solution exhibited a time-dependent loss of amidolytic
activity characterized by a half-time of approximately 36 minutes (Fig 1, insert).
View larger version (24K):
[in this window]
[in a new window]
| Fig 1.
Argatroban inhibition of thrombin bound to aged fibrin
clots (clot permeation assay). Fibrin clots (50 µL each) were formed
in the wells of a microtiter plate by addition of thrombin (to 0.5 NIH
units/mL) to fibrinogen (1 mg/mL) in HBS, pH 7.4, and then aged for 0.5 hours ( ), 3 hours ( ), or 6 hours ( ) at 23°C before
permeation with argatroban (0 to 30 µmol/L). After removal of excess
inhibitor, thrombin activity was determined with chromogenic substrate
S-2238, and the results were presented as the initial rate of color
development at 405 nm (expressed as milli-OD per minute determined in a
Vmax Kinetic Microtiter Reader). These data were fit to equation 1 to
determine the IC50, ie, the concentration of argatroban
that reduced the clot-bound thrombin activity to 50% of its maximum
value. Resultant IC50 values and the maximum inhibition are
presented in Table 1. Data shown as were obtained by clotting
fibrinogen in HBS, pH 7.4, containing 2 mmol/L CaCl2 and 3 mg/mL bovine serum albumin, and then incubating for 3 hours before
permeation with argatroban (0 to 300 µmol/L), followed by removal of
excess inhibitor and addition of chromogenic substrate S-2238. The
solid line was calculated from equation 2, using the IC50
(0.8 ± 0.1 µmol/L) and noninhibited fraction (0.11 ± 0.01)
determined by nonlinear regression analysis. This procedure also yields
the uncertainty in each fitted parameter, expressed as its standard
deviation. (Insert) Time-dependent changes in the activity of thrombin,
in solution and bound to fibrin clots. Thrombin (0.5 NIH units/mL) was
incubated either in HBS, pH 7.4 ( ) or in fibrin clots ( ) (1 mg/mL) for the indicated period before the addition of S-2238. Thrombin
activity is expressed as the initial rate parameter in milli-OD per
minute.
|
|
Argatroban Inhibition Profiles With Aged Fibrin Clots
The concentration of argatroban required to inhibit clot-bound thrombin
was initially determined with fibrin clots aged 0.5, 3, or 6 hours and
then permeated with argatroban concentrations ranging from 0 to 300 µmol/L. Dose-response data, ie, the initial rate of the absorbance
increase at 405 nm after the addition of chromogenic substrate, as a
function of argatroban concentration, are presented in Fig 1. The data
were then fitted to the following competitive inhibition
model27 to determine the IC50, ie, the concentration that reduced the thrombin activity to 50% of its maximal
value:
![Activity = Max Activity × IC<SUB>50</SUB>/[Argatrobin] + IC<SUB>50</SUB>](/content/vol92/issue6/fulltext/2064/img001.gif)
|
(1)
|
As
shown by the data and fitted parameters in
Table 1, this procedure yielded an accurate
description of the argatroban dose-response curves for clots aged 0.5, 3, and 6 hours. The resultant IC50 values were not
dependent on clot age and averaged 1.9 ± 0.3 µmol/L over this
interval. Thrombin activity was inhibited by 89% ± 0.4% at 30 µmol/L argatroban, again independent of clot age (Table 1).
Additional experiments of this type demonstrated that forming and aging
fibrin clots (for 3 hours) in the presence of millimolar Ca2+ had no significant effect on the IC50
parameters, which averaged 2.8 ± 1.6 µmol/L over the range of 1 to 5 mmol/L CaCl2. There was a moderate reduction in the
maximal inhibition, which averaged 77% ± 4% in the presence of
millimolar Ca2+ (Table 1). However, IC50 values
of 1.2 ± 0.5 µmol/L and maximum inhibition values of 90% ± 1% were obtained when albumin, alone and in the presence of 2 mmol/L
Ca2+, was present (Table 1).
This point was explored in more detail with clots formed and aged
for 3 hours in the presence of 2 mmol/L Ca2+ and 3 mg/mL
albumin. Here, the argatroban concentration was extended to 300 µmol/L and the data were treated with a modified form of equation 1, ie, one that allows for the possibility of incomplete thrombin
inhibition in the presence of saturating concentrations of
argatroban28:
![Activity = Max Activity × (IC<SUB>50</SUB> + Noninhibited Fraction × [Argatroban])/[Argatroban] + IC<SUB>50</SUB>](/content/vol92/issue6/fulltext/2064/img002.gif)
|
(2)
|
As
shown by the correspondence between the experimental data () and
solid line in Fig 1, this equation provided an accurate description of
the argatroban dose-response data. Nonlinear regression analysis
yielded an IC50 value of 0.8 ± 0.1 µmol/L and a
noninhibited fraction of 0.11 ± 0.01, corresponding to 89%
inhibition of fibrin clot-bound thrombin at saturating argatroban
concentrations. Analysis of the complete data set presented in Table 1
yielded a mean IC50 of 2.0 ± 1.0 µmol/L and 85% ± 6% maximum inhibition of fibrin clot-bound thrombin by
argatroban.
Argatroban Inhibition of Fibrin Clot Bound-Thrombin in a
Perfusion Model
The ability of argatroban to inhibit thrombin bound to fibrin was also
examined in a perfusion model, a system that was designed to maximize
delivery of the thrombin inhibitor to the interstices of an aged fibrin
clot. As shown in Fig 2 (insert), fibrin
clots were formed and aged (in the presence of 2 mmol/L
Ca2+ and 3 mg/mL albumin) in an optical flow cell contained
within a spectrophotometer, with continuous monitoring of the optical density.25 These data have been normalized by the
A405 due to clotted fibrin (averaged over the interval 160 to 175 minutes, approximately 1 absorbance unit) to obtain a plot of
relative intensity versus time. After step 1, the clotting and
180-minute aging period, each clot was gently perfused with either
buffer or argatroban (0 to 300 µmol/L) during step 2, denoted by the solid line. A consideration of the complete data set indicated that the
fibrin clots were quite stable, in that perfusion with 2 clot volumes
of either buffer or argatroban changed the turbidity by less than 5%,
ie, a nonsignificant change (P = .25, n = 9). Next, each aged
clot was perfused with 1.5 clot volumes of substrate S-2238 during step
3. A representative control experiment and a perfusion with 100 µmol/L argatroban are shown in Fig 2, insert.
View larger version (25K):
[in this window]
[in a new window]
| Fig 2.
Argatroban inhibition of thrombin bound to aged fibrin
clots (clot perfusion assay). (Insert) Time-course of clot perfusion
assay. Clots were formed by the addition of thrombin (0.5 NIH units/mL)
to fibrinogen (1 mg/mL) in HBS, pH 7.4, containing CaCl2 (2 mmol/L) and bovine serum albumin (3 mg/mL). The solution was then
(before the gel point) transferred to an absorbance flow-cell (path
length, 1 cm) in a spectrophotometer. Relative intensity, ie, the
absorbance at 405 nm expressed as a fraction of the clotted fibrin
signal, is presented as a function of time (1). After 180 minutes of
incubation at 23°C, clots were perfused with either HBS, pH 7.4 (upper trace), or argatroban (100 µmol/L in buffer; lower trace) for
the period indicated by the bracket (2). Thirty minutes after the start
of that perfusion, each clot was perfused with chromogenic substrate
S-2238 (560 µmol/L in water), as indicated by the bracket (3). After
this perfusion (during which the absorbance increases by ~10%), the
initial rate of color development was determined from the digitally
stored data. Argatroban dose-response data. () Thrombin activity
(initial rate of color development at 405 nm) obtained as a function of
argatroban concentration in the perfusate. The solid line was
determined with the IC50 value of 8.0 ± 2.4 µmol/L
obtained by fitting the data to equation 1.
|
|
The initial rate of absorbance increase (after completion of the
substrate perfusion step) was determined by linear regression to obtain
a thrombin activity parameter for each experiment. Figure 2 depicts the
resultant dose-response curve, ie, thrombin activity versus
[argatroban] in the perfusate. Because near-complete inhibition was
obtained at argatroban concentrations at or above 100 µmol/L, these
data were fit to equation 1 to obtain an IC50 = 8.0 ± 2.4 µmol/L. This parameter describes argatroban inhibition of
thrombin bound to 3-hour aged fibrin clots (in the presence of 2 mmol/L Ca2+ and 3 mg/mL albumin).
Both the maximum thrombin activity obtained in this perfusion model (39 ± 7 mOD/min) and the IC50 value (8.0 ± 2.4 µmol/L) are greater than the corresponding parameters obtained in the microtiter plate/clot permeation model described earlier. These observations suggest that more clot-bound thrombin is accessible in the
perfusion model; hence, higher argatroban concentrations are required
to achieve a comparable degree of inhibition.
Argatroban Inhibition of Thrombin Bound to Plasma Clots: Effects of
Clot Age
The ability of argatroban to inhibit thrombin bound to aged plasma
clots was also investigated using the clot permeation system described
earlier. SEM examination of 3-hour aged PPP clots showed a thin surface
coat and an interior network of fibrin strands (Fig 3A). At higher magnification (Fig 3B),
individual fibrin strands can be visualized as thick cables (200 to 300 nm, Fig 4) that extend over distances of
several microns. The open architecture of these plasma clots, which are
characterized by distances between fiber branchpoints ranging from 300 to 1,000 nm, is consistent with earlier observations that plasma clots
are rather freely permeable to water and low molecular weight
solutes.29-31
View larger version (180K):
[in this window]
[in a new window]
View larger version (168K):
[in this window]
[in a new window]
| Fig 3.
SEM of aged plasma clots. Plasma clots (50 µL) were
formed by addition of thrombin (to 0.5 NIH units/mL) and
CaCl2 (to 20 mmol/L) to citrated human plasma and then
transferred to cylindrical wells milled in carbon planchettes. After 3 hours of incubation, clots were overlaid with HBS, pH 7.4. After a
30-minute permeation period, excess liquid was removed and each sample
was dehydrated, critical point-dried, and sputter-coated as described
in Materials and Methods. Samples were examined in a Philips Model 515 scanning electron microscope, and micrographs were taken at
magnifications of 1,850× (A; bar = 10 µm) and
18,300× (B; bar = 1 µm).
|
|
View larger version (20K):
[in this window]
[in a new window]
| Fig 4.
Fiber diameters determined by SEM. Each bar denotes the
mean fiber width (in nanometers) and its standard deviation for PRP and
PPP plasma clots aged 3 hours and then treated as follows (30-minute
permeation period with each effector): HBS (pH 7.4); Arg, argatroban
(100 µmol/L); SK, streptokinase (250 IU/mL); Alt, alteplase (10 µg/mL); SK/Arg (250 IU/mL streptokinase, then 100 µmol/L
argatroban); Alt/Arg (10 µg/mL alteplase, then 100 µmol/L
argatroban). Measurements were made on 20 to 50 randomly selected
fibrin strands, as visualized in high-magnification micrographs such as
those presented in Fig 3B.
|
|
The maximum thrombin activity determined in the permeation model with
plasma clots was 12 ± 3 mOD/min, independent of either clot age or
the presence/absence of platelets (Table
2). This level of thrombin activity is threefold to fourfold greater
than that observed with fibrin clots assembled from purified components (Table 1), consistent with the increased generation of thrombin in
clotted plasma.
Data obtained as a function of argatroban concentration were fitted to
equation 2 by nonlinear regression to determine the IC50
and maximum inhibition. Representative data are depicted in
Fig 5A and B and the results of analyzing
the complete data set are presented in Table 2. Statistical analyses
(t-test) indicated that the mean IC50 values
determined for argatroban inhibition of thrombin bound to clots formed
from PRP aged 3 hours and 6 hours, ie, 2.7 ± 1.0 µmol/L (n = 9)
and 3.4 ± 3.0 µmol/L (n = 7), respectively, were not
significantly different (P = .6). The mean IC50
parameters determined for PPP clots aged 3 and 6 hours, ie, 6.1 ± 3.0 µmol/L (n = 9) and 2.9 ± 1.3 µmol/L (n = 7),
respectively, were statistically different (P = .02); however,
the biological significance of this change is uncertain. Taken
together, these data indicate argatroban was an effective inhibitor of
thrombin bound to clotted PRP and PPP after aging periods of either 3 or 6 hours. In fact, analysis of the complete data (Table 2) set yielded a mean IC50 value of 3.8 ± 2.6 µmol/L and a
maximum inhibition of 83% ± 9%.
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
Argatroban inhibition of thrombin bound to aged plasma
clots (clot permeation assay). Plasma clots (50 µL each) were formed
by the addition of thrombin (to 0.5 NIH units/mL) and CaCl2
(to 20 mmol/L) to citrated human plasma (PRP, solid symbols; PPP, open
symbols) in the wells of a microtiter plate and then incubated for
either 3 hours (upper panel) or 6 hours (lower panel) before permeation
with argatroban (0 to 300 µmol/L) in HBS, pH 7.4. After the removal
of excess inhibitor, thrombin activity was determined with chromogenic
substrate S-2238 and the results were expressed as the initial rate of
color development at 405 nm in units of milli-OD per minute (in a Vmax
Kinetic Microtiter Plate Reader). Data obtained as a function of
argatroban concentration were analyzed with equation 2 to determine the
parameters shown in the inserts, IC50, and percentage of
maximum inhibition (defined as 100 * [1  noninhibited fraction]).
The solid lines were calculated from the resultant parameters obtained
with PRP clots at each clot age. Table 2 presents the complete set of
inhibition parameters obtained in these assays.
|
|
SEM examination showed that the highly interconnected network of these
plasma clots remained intact after treatment with argatroban. Morphometric analyses demonstrated the mean fiber diameter was 190 ± 80 nm for PPP clots permeated with buffer and 200 ± 70 nm for
clots permeated with 100 µmol/L argatroban (Fig 4). PRP clots exhibited a somewhat larger diameter (280 ± 50 nm) after permeation with buffer and a similar value (280 ± 65 nm) after permeation with
100 µmol/L argatroban (Fig 4).
Argatroban Inhibition of Thrombin Bound to Plasma Clots: Effects of
Thrombolysis
The ability of argatroban to inhibit thrombin, both that bound to aged
plasma clots and that released by the fibrinolytic activity of
streptokinase and alteplase, was also determined in the plasma clot
permeation model. The concentrations of thrombolytic agent, ie, 250 IU/mL streptokinase and 10 µg/mL alteplase, were designed to
replicate plasma levels typically achieved during thrombolytic
therapy.32-34 Both clot formation in plasma and the extent
of clot lysis were monitored by changes in optical density at 405 nm.
The effects of thrombolytic agents and argatroban on clot structure
were also examined by SEM.
As evidenced by data such as that shown in
Fig 6A, insert, streptokinase lysis of
plasma clots aged 3 hours decreased the A405 by 11% ± 3% in 30 minutes; the turbidity reached a plateau soon after. In
contrast, as shown in Fig 7A, insert,
alteplase decreased the A405 by 6% ± 1% in the first
30 minutes, but the turbidity continued to decrease in an approximately
linear manner over the next 90 minutes. SEM showed that the fibrin
network that remained after exposure to a thrombolytic agent was
comparable to that present before lysis. Morphometric analyses
indicated a modest reduction in fiber thickness after treatment with
streptokinase, but no significant change after exposure to alteplase
(Fig 4).
View larger version (19K):
[in this window]
[in a new window]
| Fig 6.
Argatroban inhibition of thrombin present in aged plasma
clots treated with streptokinase. Plasma clots (50 µL each) were
formed from PRP and aged for 3 hours (as described in the legend to Fig
5). Then, 50 µL of either streptokinase (250 IU/mL in HBS, pH 7.4) or
buffer was layered on each aged plasma clot and incubated for 30 minutes to induce lysis. Control plasma samples (ie, no thrombin added)
were also incubated for 3 hours, and then streptokinase was added to
their wells. In each case, argatroban (0 to 65 µmol/L) was added to
the wells and incubated for an additional 30 minutes before the
addition of substrate S-2238 and determination of thrombin activity (as
described in the legend to Fig 5). Note that excess liquid was not
removed at any step in this assay to recover both clot-bound thrombin
and that released into the supernatant during clot lysis. Argatroban
dose-response data obtained with streptokinase-treated plasma clots
() and with intact plasma clots () were analyzed with equation 2 by nonlinear regression to obtain the IC50 and maximum
inhibition parameters (cited in Table 3 and used to calculate the solid
lines). Data obtained with plasma samples treated with streptokinase
are presented as and provide a measure of the amidolytic activity
due to plasmin generation in plasma, as described in the text. (Insert)
Time course of streptokinase lysis of aged plasma clots. Clots formed
from PPP (100 µL) were aged for 3 hours and then overlaid with an
equal volume of either buffer (control trace) or streptokinase (SK
trace; 250 IU/mL in HBS, pH 7.4). The change in optical density at 405 nm was recorded as a function of time (Vmax Kinetic Microtiter Plate
Reader). The extent clot lysis of was determined from the difference in
optical density between the control and SK-treated data, at selected
time points as described in the text.
|
|
View larger version (23K):
[in this window]
[in a new window]
| Fig 7.
Argatroban inhibition of thrombin present in aged plasma
clots treated with alteplase. Plasma clots were formed and aged for 3 hours and then incubated with alteplase (10 µg/mL) before permeation
with argatroban and determination of thrombin activity. The
experimental procedures described in the legend to Fig 6 were followed,
except that alteplase was used to induce lysis rather than
streptokinase. Data obtained with alteplase-lysed plasma clots ()
and intact plasma clots () were analyzed with equation 2 to obtain
the solid lines; the resultant inhibition parameters are cited in Table
3. Data obtained with alteplase-treated plasma () provide a measure
of amidolytic activity due to plasmin, as described in the text.
(Insert) Time course of alteplase lysis of aged plasma clots.
Experimental procedures and data analyses described in the legend to
Fig 4 were followed, except that clots were overlaid with alteplase (10 µg/mL) to induce lysis.
|
|
In the argatroban inhibition experiments, plasma clots were aged for 3 or 6 hours, lysed with either streptokinase or alteplase for 30 minutes, and permeated with argatroban (0 to 100 µmol/L) and then the
residual thrombin activity was determined with chromogenic substrate
S-2238. Representative argatroban dose-response curves obtained with 3- and 6-hour aged plasma clots, lysed with either streptokinase or
alteplase, are presented in Figs 6 and 7, respectively. Each argatroban
inhibition experiment also included clotted plasma controls, to which
buffer was delivered in place of thrombolytic agent. Morphometric
analyses of SEM data showed that no significant changes in fiber
thickness had taken place during the argatroban permeation procedures
(Fig 4).
Argatroban dose-response curves were analyzed by using equation 2 to
determine the IC50 and maximum inhibition of thrombin activity for both intact and partially lysed aged plasma clots. Representative curve fits are shown in Figs 6 and 7, and the complete parameter set is presented in Table 3. It
is noted that IC50 values less than 1 µmol/L resulted in
all cases, indicating that the ability of argatroban to inhibit
clot-bound thrombin was not diminished by clot age or by the action of
thrombolytic agents on these aged plasma clots. In fact, the mean
IC50 values obtained for the following conditions were
indistinguishable: intact clots, 0.67 ± 0.12 µmol/L (n = 8);
streptokinase-lysed clots, 0.67 ± 0.26 µmol/L (n = 5, P = .9 cf controls); alteplase-lysed clots, 0.73 ± 0.10 (n = 4, P = .4 cf controls). This pattern of effective argatroban
inhibition was obtained even under conditions in which streptokinase
lysis increased the available thrombin activity 1.6-fold and alteplase
by 2.7-fold (parameters derived from data in Table 3).
One point that deserves attention is the maximum inhibition determined
with saturating argatroban concentrations (cited in Table 3). With
control plasma clots, this parameter was 94%, indicative of nearly
complete inhibition. In contrast, with streptokinase-lysed clots, the
maximum inhibition appeared limited to approximately 70%. However, as
shown in Fig 6 and Table 3, addition of streptokinase to plasma yielded
an activity comparable to the residual 30% noninhibited activity.
Additional experiments showed that permeating aged/streptokinase-lysed plasma clots with both excess argatroban (60 µmol/L) and the plasmin inhibitor aprotinin (70 KIU/mL) reduced this residual activity to
approximately 8% of controls.
A similar situation was obtained with alteplase in which 72%
inhibition was obtained with excess argatroban (Fig 7). In this case,
plasmin generated in plasma accounts for approximately 10% of the
residual activity (Table 3). However, because fibrin is well-known to
increase the catalytic efficiency of tissue-type plasminogen
activator,35 it is likely that the increased plasmin generated in clotted plasma contributed to the residual activity. Consistent with this postulate, permeating aged/alteplase-lysed plasma
clots with aprotinin alone reduced the total activity by 25%, and the
combination of aprotinin and alteplase reduced it by 79%.
| |
DISCUSSION |
In summary, data presented here have shown argatroban to be an
effective inhibitor of thrombin bound to aged fibrin clots and aged
plasma clots, as well as thrombin released from aged plasma clots by
thrombolysis. By addressing both clot age and thrombolysis as key
experimental variables, this study has expanded the description of
argatroban as a potent inhibitor of clot-bound thrombin.7
First, let us examine the clot age issue in more detail, starting with
the observation that, in a clot permeation model, argatroban exhibited
similar IC50 parameters for inhibition of thrombin bound to
fibrin clots aged 0.5, 3, or 6 hours (2.0 ± 1.0 µmol/L) and that
argatroban inhibited 85% ± 6% of the thrombin activity present in
these aged fibrin clots (Table 1). This study has also shown that
argatroban inhibits virtually 100% of the clot-bound thrombin when
delivered to aged fibrin clots by a pressure-driven perfusion process
(Fig 2).
Additional permeation experiments demonstrated that the
IC50 values for argatroban inhibition of thrombin bound to
plasma clots (Table 2) were independent of clot age, averaging 4.4 ± 2.8 µmol/L and 3.6 ± 2.3 µmol/L, for clots aged 3 and 6 hours, respectively (P = .2). Because the IC50
parameter is sensitive to the total thrombin
concentration,36 the threefold to fourfold increased
thrombin activity generated in plasma can probably explain why nearly
twofold higher IC50 values resulted with plasma clots, compared with fibrin clots. However, argatroban concentrations greater
than 30 µmol/L blocked nearly all this plasma clot-bound thrombin, in
that 86% ± 5% of the thrombin activity in 3-hour aged plasma
clots and 81% ± 12% of that in 6-hour aged plasma clots was
inhibited by a brief argatroban permeation. Some of the residual
argatroban-insensitive amidolytic activity may be due to other serine
proteases that are known to cleave the chromogenic substrate S-2238.
However, this particular substrate was chosen because it displays the
highest available selectivity for thrombin.19
Based on the observations reported here that argatroban displayed
similar inhibition profiles with fibrin clots and in clotted plasma, it
appears that moderate changes in clot structure do not have a large
influence on argatroban's antithrombotic effects. SEM and morphometric
analyses have shown some interesting differences between the purified
system clots and clotted plasma used in this study. In particular, the
fibrin clots were composed of smaller fibrin strands (72 ± 11 nm),
compared with 280 ± 50 nm for PRP clots and 190 ± 80 nm for PPP
clots. However, all of these clots exhibited an open architecture with
distances between network branchpoints (fiber cross-over connections)
in the range of 300 to 1,000 nm. These observations can explain why
both fibrin and plasma clots were readily permeated with low molecular
weight molecules such as argatroban and S-2238, whose sizes are small compared with these fiber dimensions. SEM also demonstrated that permeation of both fibrin and plasma clots did not cause any
significant alterations in clot morphology (as evidenced by the panel
of micrographs in Fig 3 and the morphometric data in Fig 4).
Both the permeation and perfusion models described here can have direct
physiological/therapeutic relevance. For example, the clot permeation
model may reflect the difficulty inherent in delivery of argatroban to
a partly occluded thrombus, in which the inhibitor must penetrate the
clot from the blood that flows nearby, driven by a combination of
convection and diffusion.37,38 A mathematical solution to
the appropriate diffusion equations indicates that a small molecule
such as argatroban should be able to effectively penetrate a fibrin
clot and that diffusion alone can increase the argatroban concentration
at the mid-depth of a clot to approximately 75% of that free in
solution in a 30-minute period.39,40 On the other hand, the
clot perfusion model can mimic the more direct delivery of argatroban
when the pressure building up behind a fully occluded thrombus forces
plasma directly, but slowly, through the interstices of the
thrombus.38,41
This study has also shown argatroban to be an effective inhibitor of
thrombin present in aged plasma clots treated with either streptokinase
or alteplase. This point has been explored in detail with a variation
on the plasma clot permeation system, one in which aged clots were
incubated with therapeutic concentrations of streptokinase or alteplase
to induce lysis, before permeation with argatroban and determination of
the clot-associated thrombin activity. As shown in Table 3, the partial
clot lysis induced by both thrombolytic agents increased the total
available thrombin activity, yet argatroban still inhibited this
thrombin with IC50 values less than 1 µmol/L. In fact, there were no significant differences
(P > .5) between the IC50 values obtained in this plasma clot lysis/permeation assay between argatroban inhibition of
thrombin bound to aged, intact plasma clots, and thrombin bound to and
released from plasma clots lysed with streptokinase or alteplase. Clot
age was also not a factor in these argatroban inhibition profiles,
because similar IC50 values resulted with clots aged 3 or 6 hours before treatment with thrombolytic agent (P > .1 for
SK; P > .4 for alteplase).
The observations presented here, namely that argatroban effectively
penetrates aged clots and inhibits the procoagulant activity of
clot-bound thrombin, as well as thrombin released from these aged clots
by treatment with thrombolytic agents, may be relevant to its emerging
clinical applications in the treatment of ischemic heart disease.
Results from the recently completed MINT study have shown argatroban to
be effective as an adjunctive therapy to alteplase in the treatment of
acute myocardial infarction, especially in patients who receive
thrombolytic therapy some 6 hours after symptom onset.1
Evidence for a close correlation between argatroban's clinical
efficacy and its inhibitory activity in a permeation and lysis model of
an aged human thrombus comes from a comparison of the plasma levels
achieved therapeutically, approximately 1 µmol/L (MINT1
and AMI2 trials) to its approximately 1 µmol/L
IC50 determined in this in vitro study (Table 3). In
conclusion, our results indicate that argatroban's ability to inhibit
clot-bound thrombin is not influenced by either clot age or possible
temporally related changes in clot structure imparted by streptokinase
or alteplase.
| |
FOOTNOTES |
Submitted January 27, 1998;
accepted May 6, 1998.
Supported by a grant (to R.R.H.) from the Texas Biotechnology Corp.
Address reprint requests to Roy R. Hantgan, PhD, Department of
Biochemistry, Bowman Gray School of Medicine, Wake Forest Universtiy, Medical Center Blvd, Winston-Salem, NC 27157-1016.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
REFERENCES |
1. Jang IK, The MINT Investigators: A randomized study of argatroban
vs heparin as adjunctive therapy to tissue plasminogen activator in
acute myocardial infarction: MINT (Myocardial Infarction with Novastan
and TPA) study. Circulation 96:I-331, 1997 (abstr)
2. Theroux P: A randomized, double-blind, study of two doses of
Novastan® (brand of argatroban) versus placebo as adjunctive
therapy to streptokinase in acute myocardial infarction. The Argatroban
Myocardial Infarction (AMI) Study. J Am Coll Card 29:viii, 1997 (suppl
A)
3.
Kikumoto R,
Tamao Y,
Tezuka T,
Tonomura S,
Hara H,
Ninomiya K,
Hijikata A,
Okamoto S:
Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methy-1,2,3,4-tetrahydro-8-quinolinyl) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid.
Biochemistry
23:85,
1984[Medline]
[Order article via Infotrieve]
4.
Okamoto S,
Hijikata-Okunomiya A:
Synthetic selective inhibitors of thrombin.
Methods Enzymol
222:328,
1993[Medline]
[Order article via Infotrieve]
5.
Banner DW,
Hadvary P:
Inhibitor binding to thrombin: X-ray crystallographic studies.
Adv Exp Med Biol
340:27,
1993[Medline]
[Order article via Infotrieve]
6.
Bauer M,
Brandstetter H,
Turk D,
Sturzebecher J,
Bode W:
Crystallographic determination of thrombin complexes with small synthetic inhibitors as a starting point for the receptor-based design of antithrombotics.
Semin Thromb Hemost
19:352,
1993[Medline]
[Order article via Infotrieve]
7.
Berry CN,
Girardot C,
Lecoffre C,
Lunven C:
Effects of the synthetic thrombin inhibitor argatroban on fibrin- or clot-incorporated thrombin: Comparison with heparin and recombinant hirudin.
Thromb Haemost
72:381,
1994[Medline]
[Order article via Infotrieve]
8.
Lunven C,
Gauffeny C,
Lecoffre C,
O'Brien DP,
Roome NO,
Berry CN:
Inhibition by argatroban, a specific thrombin inhibitor, of platelet activation by fibrin clot-associated thrombin.
Thromb Haemost
75:154,
1996[Medline]
[Order article via Infotrieve]
9.
Kleiman NS:
Clinical trials of conjunctive anticoagulant strategies in thrombolysis.
Coronary Artery Dis
7:508,
1996[Medline]
[Order article via Infotrieve]
10.
Schwarz RP Jr,
Becker J-CP,
Brooks RL,
Hursting MJ,
Joffrion JL,
Knappenberger GD,
Kogan TP,
Kogan PW,
McKinney AA:
The preclinical and clinical pharmacology of Novastan (argatroban): A small-molecule, direct thrombin inhibitor.
Clin Appl Thromb Hemost
3:1,
1997
11. (suppl 4)
Verstraete M:
Modulating platelet function with selective thrombin inhibitors.
Haemost
26:70,
1996
12.
Clarke RJ,
Mayo G,
FitzGerald GA,
Fitzgerald DJ:
Combined administration of aspirin and a specific thrombin inhibitor in man [see comments].
Circulation
83:1510,
1991[Abstract/Free Full Text]
13.
Eidt JF,
Allison P,
Noble S,
Ashton J,
Golino P,
McNatt J,
Buja LM,
Willerson JT:
Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury.
J Clin Invest
84:18,
1989
14.
Tamao Y,
Yamamoto T,
Kikumoto R,
Hara H,
Itoh J,
Hirata T,
Mineo K,
Okamoto S:
Effect of a selective thrombin inhibitor MCI-9038 on fibrinolysis in vitro and in vivo.
Thromb Haemost
56:28,
1986[Medline]
[Order article via Infotrieve]
15.
Jang IK,
Gold HK,
Leinbach RC,
Fallon JT,
Collen D:
In vivo thrombin inhibition enhances and sustains arterial recanalization with recombinant tissue-type plasminogen activator.
Circ Res
67:1552,
1990[Abstract/Free Full Text]
16.
Lewis BE,
Walenga JM,
Wallis DE:
Anticoagulation with Novastan (argatroban) in patients with heparin-induced thrombocytopenia and heparin-induced thrombocytopenia and thrombosis syndrome.
Semin Thromb Hemost
23:197,
1997[Medline]
[Order article via Infotrieve]
17.
Lewis BE,
Iaffaldano R,
McKiernan TL,
Rao L,
Donkin J,
Wallenga JM:
Report of successful use of argatroban as an alternative anticoagulant during coronary stent implantation in a patient with heparin-induced thrombocytopenia and thrombosis syndrome.
Catheterization Cardiovasc Diag
38:206,
1996
18.
Hantgan RR:
A study of the kinetics of ADP-triggered platelet shape change.
Blood
64:896,
1984[Abstract/Free Full Text]
19.
Mattler LE,
Bang NU:
Serine protease specificity for peptide chromogenic substrates.
Thromb Haemost
38:776,
1977[Medline]
[Order article via Infotrieve]
20.
Jen CJ,
McIntire LV:
Platelet microtubules in clot structure formation and contractile force generation: Investigation of a controversy.
Thromb Haemost
56:23,
1986[Medline]
[Order article via Infotrieve]
21.
Lewis JC,
Hantgan RR,
Stevenson SC,
Thornburg T,
Kieffer N,
Guichard J,
Breton-Gorius J:
Fibrinogen and glycoprotein IIb/IIIa localization during platelet adhesion: Localization to the granulomere and at sites of platelet interaction.
Am J Pathol
136:239,
1990[Abstract]
22.
Torbet J:
The thrombin activation pathway modulates the assembly, structure and lysis of human plasma clots in vitro.
Thromb Haemost
73:785,
1995[Medline]
[Order article via Infotrieve]
23.
Hantgan RR,
McDonagh J,
Hermans J:
Fibrin assembly.
Ann NY Acad Sci
408:344,
1983[Medline]
[Order article via Infotrieve]
24.
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]
25.
Carr ME,
Hermans J:
Size and density of fibrin fibers from turbidity.
Macromolecules
11:46,
1978[Medline]
[Order article via Infotrieve]
26.
Mosesson MW:
Thrombin interactions with fibrinogen and fibrin.
Semin Thromb Hemost
19:361,
1993[Medline]
[Order article via Infotrieve]
27.
Hantgan RR:
Localization of the domains of fibrin involved in binding to platelets.
Biochim Biophys Acta
968:36,
1988[Medline]
[Order article via Infotrieve]
28.
Hantgan RR,
Hindriks G,
Taylor RG,
Sixma JJ,
De Groot PG:
Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood.
Blood
76:345,
1990[Abstract/Free Full Text]
29.
Carr M,
Shen LL,
Hermans J:
Mass-length ratio of fibrin fibers from gel permeation and light scattering.
Biopolymers
16:1,
1977[Medline]
[Order article via Infotrieve]
30.
Blomback B,
Carlsson K,
Hessel B,
Liljeborg A,
Procyk R,
Aslund N:
Native fibrin gel networks observed by 3D microscopy, permeation, and turbidity.
Biochim Biophys Acta
997:96,
1989[Medline]
[Order article via Infotrieve]
31.
Nair CH,
Azhar A,
Dhall DP:
Studies on fibrin network structure in human plasma. Part I Methods for clinical application.
Thromb Res
64:455,
1991[Medline]
[Order article via Infotrieve]
32.
Sobel BE:
Pharmacodynamics of activation of plasminogen with t-PA
, in Sobel BE,
Collen D,
Grossbard EB
(eds):
Tissue Plasminogen Activator in Thrombolytic Therapy.
New York, NY, Marcel Dekker
, 1987
, p 25
33.
Tebbe U,
Tanswell P,
Seifried E,
Feuerer W,
Scholz KH,
Herrmann KS:
Single bolus injection of recombinant tissue-type plasminogen activator in acute myocardial infarction.
Am J Cardiol
64:448,
1989[Medline]
[Order article via Infotrieve]
34.
Chesebro JH,
Knatterud G,
Roberts R,
Borer J,
Cohen LS,
Dalen J,
Dodge HT,
Francis CK,
Hillis D,
Ludbrook P,
Markis JE,
Mueller H,
Passamani ER,
Powers ER,
Rao AK,
Robertson T,
Ross A,
Ryan TJ,
Sobel BE,
Willerson J,
Williams DO,
Zaret BL,
Braunwald E:
Thrombolysis in myocardial infarction (TIMI) trial, phase 1: A comparison between intravenous tissue plasminogen activator and intravenous streptokinase.
Circulation
76:142,
1987[Abstract/Free Full Text]
35.
Collen D,
Lijnen HR:
The fibrinolytic system in man.
CRC Crit Rev Oncol Hematatol
4:248,
1985
36.
Segel IH:
Multiple inhibition analysis
, in Davis S
(ed):
Enzyme Kinetics. Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems.
New York, NY, Wiley
, 1975
, p 465
37.
Wu JH,
Siddiquik,
Diamond SL:
Transpost phenomenon and clot dissolving therapy: An experimental investigation of diffusion controlled and permeation enhanced fibrinolysis.
Thromb Haemost
72:105,
1994[Medline]
[Order article via Infotrieve]
38.
Blinc A,
Kennedy SD,
Bryant RG,
Marder VJ,
Francis CW:
Flow through clots determines the rate and pattern of fibrinolysis.
Thromb Haemost
71:230,
1993
39. Cantor CR, Schimmel PR: Size and shape of macromolecules, in
Biophysical Chemistry. Part II: Techniques for the Study
of Biological Structure and Function. San Francisco, CA, Freeman,
1980, p 539
40.
Handt S,
Jerome WG,
Tietze L,
Hantgan RR:
PAI-1 secretion by endothelial cellls increases fibrinolytic resistance of an in vitro fibrin clot: Evidence for a key role of endothelial cells in thrombolytic resistance.
Blood
87:4204,
1996[Abstract/Free Full Text]
41.
Blinc A,
Francis CW:
Transport processes in fibrinolysis and fibrinolytic therapy.
Thromb Haemost
76:481,
1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S.-C. Wu, F. J. Castellino, and S.-L. Wong
A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy
J. Biol. Chem.,
May 9, 2003;
278(20):
18199 - 18206.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Zhang, I. Nagata, H. Kikuchi, J-H. Xue, N. Sakai, H. Sakai, and H. Yanamoto
Broad-Spectrum and Selective Serine Protease Inhibitors Prevent Expression of Platelet-Derived Growth Factor-BB and Cerebral Vasospasm After Subarachnoid Hemorrhage : Vasospasm Caused by Cisternal Injection of Recombinant Platelet-Derived Growth Factor-BB
Stroke,
July 1, 2001;
32(7):
1665 - 1672.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. E. Lewis, D. E. Wallis, S. D. Berkowitz, W. H. Matthai, J. Fareed, J. M. Walenga, J. Bartholomew, R. Sham, R. G. Lerner, Z. R. Zeigler, et al.
Argatroban Anticoagulant Therapy in Patients With Heparin-Induced Thrombocytopenia
Circulation,
April 10, 2001;
103(14):
1838 - 1843.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nakatani, S. Takeshita, H. Tsujimoto, Y. Kawamura, and I. Sekine
Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury
J. Leukoc. Biol.,
February 1, 2001;
69(2):
241 - 247.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. S. Regier, D. G. Greene, S. Sergeant, A. J. Jesaitis, and L. C. McPhail
Phosphorylation of p22phox Is Mediated by Phospholipase D-dependent and -independent Mechanisms. CORRELATION OF NADPH OXIDASE ACTIVITY AND p22phox PHOSPHORYLATION
J. Biol. Chem.,
September 8, 2000;
275(37):
28406 - 28412.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Palicz, T. R. Foubert, A. J. Jesaitis, L. Marodi, and L. C. McPhail
Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components
J. Biol. Chem.,
January 26, 2001;
276(5):
3090 - 3097.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract