Download In Vivo Thrombin Generation and Activity During and After

In Vivo Thrombin Generation and Activity During and
After Intravenous Infusion of Heparin or Recombinant
Hirudin in Patients With Unstable Angina Pectoris
Piera Angelica Merlini, Diego Ardissino, Robert D. Rosenberg, Elisabetta Colombi, Pietro Agricola,
Luigi Oltrona, Filippo Ottani, Marcello Galvani, Kenneth A. Bauer, Bianca Bottasso,
Federico Bertocchi, Pier Mannuccio Mannucci
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Abstract—In patients with unstable angina, intravenous heparin reduces thrombin activity but does not influence thrombin
generation. Recombinant hirudin, a direct thrombin inhibitor, may be more effective in inhibiting both thrombin
generation and activity. We measured the plasma levels of prothrombin fragment 1⫹2 (a marker of thrombin generation)
and fibrinopeptide A (a marker of thrombin activity) in 67 patients with unstable angina enrolled in the GUSTO (Global
Use of Strategies to Open Occluded Coronary Arteries) IIb trial who were receiving either recombinant hirudin (31
patients) or heparin (36 patients). Blood samples were obtained at baseline (before any treatment), after 3 to 5 days of
study drug infusion (immediately before discontinuation), and 1 month later. In the patients receiving recombinant
hirudin, the prothrombin fragment 1⫹2 levels measured immediately before drug discontinuation were significantly
lower than at baseline (P⫽0.0014), whereas they had not changed in the patients receiving heparin; at this time point,
the difference between patients receiving hirudin and those receiving heparin was statistically significant (P⫽0.032).
One month later, the prothrombin fragment 1⫹2 levels in both groups were similarly persistently high and did not differ
from baseline. Fibrinopeptide A plasma levels at the end of infusion were significantly lower than at baseline in both
treatment groups (P⫽0.0005 for hirudin and P⫽0.042 for heparin) and remained lower after 1 month (P⫽0.0001 for
both hirudin and heparin). The fibrinopeptide A plasma levels were not different between patients treated with hirudin
versus heparin at baseline, at the end of infusion, and after 1 month. Thus, in patients with unstable angina, in vivo
thrombin generation and activity are reduced during intravenous infusion of recombinant hirudin. However, the
inhibition of thrombin generation is not sustained, and after 1 month, the majority of patients have biochemical signs
of increased thrombin generation. (Arterioscler Thromb Vasc Biol. 2000;20:2162-2166.)
Key Words: hirudin 䡲 thrombin generation 䡲 unstable angina
I
vation of platelets and factors V, VIII, and X is thought to be
critical for the generation of critical concentrations necessary
for effective hemostasis and thrombosis,5,13 increased thrombin generation may partially contribute to the persistent
thrombotic risk during and after heparin treatment.14
Recombinant hirudin is a 65–amino acid thrombin inhibitor that forms an irreversible complex by directly binding to
thrombin. Unlike heparin, recombinant hirudin does not need
antithrombin as a cofactor, and hirudin also inhibits clotbound thrombin. Such blocking of all thrombin activity
should lead to the interruption of thrombin self-generation by
inhibiting the positive feedback of the multiple mechanisms
involved. The aim of this study was to compare the effects of
heparin and recombinant hirudin on in vivo thrombin gener-
ntravenous heparin has become a recommended therapy
for patients with unstable angina because it leads to an
improved clinical outcome.1 Heparin’s binding to antithrombin produces a conformational change in antithrombin that
considerably accelerates the ability of the latter to inactivate
coagulation enzymes.2,3 This is particularly true in the case of
thrombin, which plays a critical role in the amplification of
the coagulation cascade by activating factor V, factor VIII,4 – 6
and platelets.7,8 However, although the levels of fibrinopeptide A (a sensitive marker of thrombin activity) decrease in
heparin-treated patients with unstable angina,9 –11 the plasma
levels of prothrombin fragment 1⫹2 (a sensitive marker of
thrombin generation) are not reduced.12 Because the selfamplification of thrombin through the above-mentioned acti-
Received December 27, 1999; revision accepted February 18, 2000.
From the Division of Cardiology (P.A.M., L.O.), Ospedale Niguarda Ca’ Granda, Milan; the Division of Cardiology (D.A.), Ospedale Civile di Parma,
Parma IRCCS; and Policlinico San Matteo and University of Pavia (E.C., P.A., F.B.), Pavia, Italy; the Charles A. Dana Research Institute and
Harvard-Thorndike Laboratory, Department of Medicine (R.D.R.), Beth Israel Hospital and Harvard Medical School, Boston, and the Department of
Biology (K.A.B.), Massachusetts Institute of Technology, Cambridge, Mass; the Division of Cardiology (F.O.), Ospedale di Ravenna, Ravenna; Division
of Cardiology (M.G.), Ospedale Gian Battista Morgagni, Forli’; and the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre and the Department
of Internal Medicine (B.B., P.M.M.), IRCCS Ospedale Maggiore, University of Milan, Milan, Italy.
Correspondence to Piera Merlini, MD, Division of Cardiology, Ospedale Niguarda, Piazza Ospedale Maggiore 3, 20162 Milano, Italy. E-mail
[email protected]
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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Merlini et al
Coagulation Activation During Hirudin Treatment
ation and activity during drug infusion and after 1 month in a
cohort of patients with unstable angina enrolled in the
GUSTO IIb trial.
Methods
Study Population
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The study population consisted of patients with unstable angina or
non–Q-wave myocardial infarction enrolled in the GUSTO IIb trial
at the Division of Cardiology, Ca’ Granda Niguarda Hospital
(Milan), the Division of Cardiology IRCCS Policlinico San Matteo
(Pavia), Ospedale GB Morgagni (Forli), and Ospedale Civile
(Ravenna), Italy. All of the patients had to have reported symptoms
of cardiac ischemia at rest within 12 hours of their admission and to
have shown electrocardiographic signs of acute myocardial ischemia
at presentation: ie, transient ST-segment elevation or depression
⬎0.5 mm or a persistent, definite T-wave inversion ⬎1 mm.15
Patients were excluded from the GUSTO IIb study if they were
taking warfarin at the time of enrollment or if they had active
bleeding, a history of stroke, a contraindication to heparin therapy,
renal insufficiency, or systolic blood pressure ⬎110 mm Hg. Of the
eligible patients, those receiving heparin or thrombolytic therapy at
the time of enrollment or who had a severely limited venous access
were excluded from the hemostatic evaluation.
Study Protocol
Each patient enrolled in the GUSTO IIb study received either
intravenous heparin or desulfato recombinant hirudin (Desirudin,
Ciba-Geigy) for a minimum of 3 and a maximum of 5 days according
to the study protocol, with the dose being adjusted to maintain an
activated partial thromboplastin time between 60 and 85 seconds; all
of the patients received aspirin (165 to 325 mg) before the start of the
study drug. Any associated treatment was given at the discretion of
the patients’ individual physicians and was not dictated by the study
protocol. A baseline blood sample was obtained before any treatment
was started, including the study drug, and another sample was
obtained after 3 to 5 days, immediately before study drug discontinuation. At a follow-up visit after 1 month, a further blood sample was
obtained. Long-term treatment was left to the discretion of the
patients’ individual physicians and in all patients included aspirin
and excluded oral anticoagulants. The study was approved by the
Institutional Review Board of the Ca’ Granda Niguarda Hospital
(Milan, Italy), and written informed consent was obtained from all of
the subjects. All of the clinical studies and informed consent
procedures were also approved by the Committee on Clinical
Investigations of the Beth Israel Hospital (Boston, Mass).
Blood Sampling and Handling
Clean venipunctures were performed by specially trained investigators using 19-gauge butterfly infusion sets and a 2-syringe technique.
Inadequate blood samples were prospectively excluded. After the
first 4 mL of blood was discarded, the samples were placed directly
into refrigerated Vacutainers containing an anticoagulant mixture
consisting of a thrombin inhibitor (Phe-Pro-Arg chloromethyl ketone), EDTA, and aprotinin (Byk-Sangtec); the ratio of anticoagulant
to blood was 1:9, vol/vol. The samples were immediately centrifuged
at 2500g, and the plasma was divided into aliquots, snap-frozen, and
stored at ⫺80°C until analyzed.
Biochemical Determinations
All of the samples were centrally analyzed by investigators who were
unaware of the clinical data. The plasma levels of prothrombin
fragment 1⫹2 were measured by using a double-antibody radioimmunoassay as previously described16; this method has an interassay
coefficient of variation of ⬇8%. Plasma fibrinopeptide A concentrations were determined in duplicate by means of an enzyme
immunoassay in plasma extracted twice with bentonite to remove
fibrinogen (Diagnostica Stago); this technique has an interassay
coefficient of variation of ⬇5%. Because fibrinopeptide A is known
to be susceptible to in vitro sampling artifacts, blood samples with
fibrinopeptide A levels ⬎20 nmol/L were not used for the determi-
TABLE 1.
2163
Baseline Characteristics
Age, y
Males, n (%)
Hirudin
n⫽31
Heparin
n⫽36
64⫾10
65⫾11
25 (80)
Height, cm
169⫾7
Weight, kg
30 (83)
169⫾7
73⫾13
73⫾10
4 (12)
8 (22)
Smokers, n (%)
18 (67)
20 (55)
Hypertension, n (%)
17 (54)
18 (50)
Positive family history, n (%)
Hyperlipidemia, n (%)
9 (29)
15 (41)
Diabetes, n (%)
9 (29)
11 (30)
Electrocardiographic changes
T-wave inversion
ST-segment depression
ST-segment elevation
7 (23)
8 (22)
23 (74)
24 (66)
1 (3)
4 (13)
Values are either mean⫾SD or n (%).
nation of either marker because it was surmised that they were
affected by poor sample acquisition, handling, or storage.17
Statistical Analysis
Because the plasma levels of the coagulation system markers are not
normally distributed, repeated measures were compared by means of
Friedman’s test, and subsequent pairwise comparisons with baseline
were made with Wilcoxon’s signed-rank test. The Mann-Whitney U
test was used to test the difference between groups. The descriptive
statistics include mean values and standard deviations, or median
values and 25th and 75th percentiles. The number of patients who
exhibited plasma concentrations of prothrombin fragment 1⫹2 and
fibrinopeptide A above the upper normal limits was calculated by
determining the 95th percentile of the distribution in the control
group of healthy individuals, which was set at 1.02 nmol/L for
prothrombin fragment 1⫹2 and 2.2 nmol/L for fibrinopeptide A.18
Prevalences were compared by means of the ␹2 test. All of the tests
were 2-tailed, and a probability value ⬍0.05 were regarded as
statistically significant.
Results
Patient Characteristics
Biochemical evaluation was attempted in 78 consecutive
patients, 42 of whom received heparin and 36, recombinant
hirudin. Eleven patients did not undergo complete evaluation
for the following reasons: death within 1 month (4 patients),
inadequate blood sampling (6 patients), and severely limited
venous access (1 patient). A complete set of 3 samples
(baseline, immediately before study drug discontinuation, and
after 1 month) was therefore available for 67 patients, 31 who
were treated with recombinant hirudin and 36 with heparin.
Their demographic and clinical characteristics are shown in
Table 1. The mean duration of recombinant hirudin and
heparin therapy was respectively, 82⫾11 and 77⫾9 hours
(P⫽NS). The mean activated partial thromboplastin time at
baseline was 23⫾5 seconds for patients receiving hirudin and
24⫾7 seconds for those receiving heparin (P⫽NS), whereas
the activated partial thromboplastin time immediately before
drug discontinuation was 74⫾22 seconds and 69⫾27 seconds, respectively (P⫽NS).
In Vivo Thrombin Generation and Activity in Patients
Receiving Recombinant Hirudin
The median and 25th and 75th percentiles of the plasma
prothrombin fragment 1⫹2 and fibrinopeptide A levels at
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Arterioscler Thromb Vasc Biol.
September 2000
TABLE 2. Median Plasma Values (25th to 75th Percentiles) of Prothrombin Fragment 1ⴙ2
and Fibrinopeptide A at Baseline, Before Drug Discontinuation, and After 1 Month in Patients
Receiving Either Heparin or Hirudin
Baseline
Before Drug
Discontinuation
After 1 Month
Hirudin (n⫽31)
1.20 (0.94–1.47)
0.95 (0.76–1.15)‡
1.34 (0.98–1.78)§
0.0011
Heparin (n⫽36)
1.07 (0.84–1.36)
1.16 (0.91–1.39)
1.26 (0.95–1.74)㛳
0.024
NS
0.032
NS
Biochemical Marker/Treatment
P*
Prothrombin fragment 1⫹2, nmol
P†
Fibrinopeptide A, nmol/L
Hirudin (n⫽31)
2.3 (1.2–2.6)
0.9 (0.7–1.1)¶
1.0 (0.6–1.2)#
0.0001
Heparin (n⫽36)
2.3 (1.15–4.2)
1.02 (0.8–1.8)**
1.05 (0.55–1.3)‡
0.0048
P†
NS
NS
NS
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*P refers to comparison within the treatment group.
†P refers to comparison at each time point between the 2 treatment groups.
‡P⫽0.0014 vs baseline.
§P⫽0.0001 vs before drug discontinuation.
㛳P⫽0.040 vs baseline.
¶P⫽0.0005 vs baseline.
#P⫽0.0001 vs baseline.
**P⫽0.042 vs baseline.
different time points for patients treated with recombinant
hirudin are given in Table 2. There was a significant decrease
of plasma prothrombin fragment 1⫹2 during recombinant
hirudin infusion (P⫽0.0014). After 1 month, the levels were
similar to those found at baseline (P⫽NS) but were significantly higher than those found before drug discontinuation
(P⫽0.0001). The prevalence of abnormal levels immediately
before drug discontinuation was significantly lower than at
baseline: 22 patients (71%) versus 14 (45%, P⫽0.039);
however, after 1 month, the prevalence of abnormal values
(21 patients, 70%) was similar to that observed at baseline.
Median fibrinopeptide A levels decreased significantly
after a 72-hour infusion of recombinant hirudin (P⫽0.0005)
and were still significantly lower than baseline after 1 month
(P⫽0.0001). There was also a significant decrease in the
prevalence of abnormal levels at the same time points: 17
patients (57%) at baseline, 4 (13%) immediately before drug
discontinuation, and 1 (3%) after 1 month (P⫽0.0001).
In Vivo Thrombin Generation and Activity in Patients
Receiving Heparin
The median and 25th and 75th percentile values of the plasma
prothrombin fragment 1⫹2 and fibrinopeptide A levels at
different time points for the patients treated with heparin are
given in Table 2. There was no difference between plasma
levels of prothrombin fragment 1⫹2 observed at baseline and
those found before heparin discontinuation. After 1 month,
plasma prothrombin fragment 1⫹2 levels were significantly
increased compared with baseline (P⫽0.040). There was no
difference in the prevalence of abnormal prothrombin fragment 1⫹2 levels at the different time points: 18 patients
(50%) had abnormal plasma prothrombin fragment 1⫹2
levels at baseline, 21 (60%) at the time of drug discontinuation, and 26 (72%) after 1 month.
In comparison with baseline, there was a significant decrease
of plasma fibrinopeptide A levels at the end of the infusion
(P⫽0.042) and after 1 month (P⫽0.0001). At the same time
points, there was also a significant decrease in the prevalence of
abnormal plasma fibrinopeptide A levels, which were found in
19 patients (54%) at baseline, 6 (17%) immediately before drug
discontinuation, and 3 (9%) after 1 month (P⫽0.0001).
Comparison of Heparin and Recombinant Hirudin
With Respect to Their Effects on In Vivo
Thrombin Generation and Activity
The baseline plasma prothrombin fragment 1⫹2 and fibrinopeptide A levels were similar in the 2 treatment groups. The
plasma prothrombin fragment 1⫹2 levels decreased during
treatment in the patients receiving recombinant hirudin but
did not change in those receiving heparin; thus, the postinfusion median plasma prothrombin fragment 1⫹2 levels were
significantly lower in the former (P⫽0.032). However, after
1 month, the levels of plasma prothrombin fragment 1⫹2 had
increased to similar levels in both groups and were not
different from baseline. Plasma fibrinopeptide A decreased to
within normal limits in both groups and remained low after 1
month, without any difference between heparin- and hirudintreated patients.
Discussion
The antithrombotic action of heparin is dependent on antithrombin; in vivo studies have shown that the plasma levels of
fibrinopeptide A (a marker of thrombin activity) dramatically
decrease after heparin treatment.8 –10 However, heparin cannot
inactivate clot-bound thrombin, which retains its catalytic activity against fibrinogen, factor V, and factor VIII and activates the
expression of cell binding sites on platelets for the assembly of
the tenase and prothrombinase vitamin K– dependent complexes
on the platelet membranes.4 – 8,19 –24 Furthermore, heparin is
inactivated by platelet factor 4.25 All of these reasons may
account for the fact that thrombin generation in vivo does not
change during heparin treatment,12 and this high thrombin
generation may underlie the persistent thrombotic risk observed
after heparin discontinuation.14
Merlini et al
Coagulation Activation During Hirudin Treatment
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Recombinant hirudin interacts directly with thrombin,
without depending on antithrombin, and inactivates both
thrombus-bound and soluble thrombin in vitro20,26; thus, it
should be more effective in vivo in blocking thrombin
generation through the obstruction of its feedback-amplifying
mechanisms. In experimental animal models, recombinant
hirudin has been shown to be much more effective than
high-dose heparin and aspirin in reducing platelet deposition
and thrombosis after catheter-induced vascular injury,27 but it
is not known whether direct thrombin inhibition leads to a
decrease in thrombin generation in a clinical setting.
This study shows that in patients with unstable angina, a 3to 5-day infusion of recombinant hirudin leads to a decrease
in thrombin generation that is not observed in the patients
receiving heparin. In patients with acute coronary syndromes,
higher plasma prothrombin fragment 1⫹2 levels are associated with an increased risk of in-hospital events12,28; thus, it
can be surmised that the early reduction in cardiac events
observed in patients receiving recombinant hirudin15,29 may
be related to its ability to decrease thrombin generation. This
finding is different from the results of other studies, which
revealed no effect of recombinant hirudin on thrombin
generation despite decreased thrombin activity.30 –33 This
discrepancy could be due to the longer treatment used in our
study or to the fact that our study population showed signs of
increased thrombin generation at baseline, thereby indicating
that coronary thrombosis was likely to be the main pathogenetic mechanism. It is interesting to note that in a recent
pooled analysis of all large trials of hirudin versus heparin in
acute coronary syndromes, the greater risk reduction (28% at
72 hours) was observed in patients not receiving thrombolytic
therapy,29 which is similar to the population represented in
the present study.
One month after treatment, thrombin generation returned
high levels in both heparin- and hirudin-treated patients. It is
tempting to speculate that the attenuation of the short-term
favorable clinical effect of recombinant hirudin over heparin
observed in the GUSTO IIb and OASIS-2 (Organization to
Assess Strategies for Ischemic Syndrome-2) trial,15,29 with
the occurrence of a significant number of additional ischemic
events after drug discontinuation, may be due to its inability
to persistently inhibit thrombin generation. Recent data have
shown that the healing process of culprit lesions in acute
coronary syndromes takes a long time, and angioscopic signs
of persistent plaque instability and thrombosis can be observed even in the absence of symptoms.34 These findings are
in keeping with the result of previous studies showing that the
hemostatic mechanism, including platelets, remains activated
up to 6 months after an episode of unstable angina or
myocardial infarction.18,35 Continuous thrombin generation
may be due to the unhealed wall injury with exposure of
tissue factor, the chronic activity of the underlying disease,
the persistent platelet activation, and their interplay, which
suggest that prolonged inhibition of the hemostatic mechanism may be needed to include the period of healing of the
culprit lesion and to allow the coagulation system to return to
baseline levels of responsiveness, thereby reducing the risk of
recurrent ischemic events.
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In Vivo Thrombin Generation and Activity During and After Intravenous Infusion of
Heparin or Recombinant Hirudin in Patients With Unstable Angina Pectoris
Piera Angelica Merlini, Diego Ardissino, Robert D. Rosenberg, Elisabetta Colombi, Pietro
Agricola, Luigi Oltrona, Filippo Ottani, Marcello Galvani, Kenneth A. Bauer, Bianca Bottasso,
Federico Bertocchi and Pier Mannuccio Mannucci
Arterioscler Thromb Vasc Biol. 2000;20:2162-2166
doi: 10.1161/01.ATV.20.9.2162
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