Journal of the American College of Cardiology
`© 2000 by the American College of Cardiology
`Published by Elsevier Science Inc.
`
`Vol. 36, No. 3, 2000
`ISSN 0735-1097/00/$20.00
`PII S0735-1097(00)00817-2
`Platelet Inhibitors
`
`Antiplatelet Effects of Clopidogrel Compared
`With Aspirin After Myocardial Infarction:
`Enhanced Inhibitory Effects of Combination Therapy
`Khatereh Moshfegh, PHD, Maurice Redondo, MD,* Friedgard Julmy, Walter A. Wuillemin, MD, PHD,*
`Mathias U. Gebauer, MD, Andre´ Haeberli, PHD, Beat J. Meyer, MD
`Bern, Switzerland
`
`RESULTS
`
`METHODS
`
`OBJECTIVES We sought to compare the inhibitory effects of the combination of two doses of aspirin plus
`clopidogrel with either drug alone on platelet aggregation and activation.
`BACKGROUND Enhanced platelet inhibitory effects of clopidogrel by aspirin on platelet aggregation and
`activation are suggested by experimental studies but have not been shown in humans.
`The effects of clopidogrel 75 mg or aspirin 100 (300) mg on platelet aggregation and
`activation by flow cytometry after stimulation with various agonists were determined in 30
`patients with a past history of myocardial infarction.
`Clopidogrel alone or in combination with aspirin markedly inhibited adenosine diphosphate
`(ADP)-mediated platelet aggregation compared with monotherapy with aspirin (24.6 6 3.3%
`or 26.6 6 2.7% vs. 44.7 6 2.9%; p , 0.001). Combined treatment significantly inhibited
`collagen-induced aggregation compared with aspirin and clopidogrel (16.4 6 2.4%, 36.5 6
`4.2% and 59.3 6 5.1%, respectively; p , 0.001) and resulted in considerable inhibition of
`aggregation induced by thrombin receptor agonist peptide (TRAP, p , 0.03). Clopidogrel
`with or without aspirin significantly suppressed expression of platelet activation markers CD
`62p, CD 63 and PAC-1 after stimulation with ADP or thrombin (p , 0.001). In addition,
`the combined treatment was more effective than either agent alone after activation with low
`dose thrombin (p , 0.05). Both doses of aspirin equally potentiated the platelet inhibitory
`effects of clopidogrel.
`CONCLUSIONS In this prospective clinical ex vivo platelet study, clopidogrel was more effective than aspirin
`in inhibiting ADP-mediated platelet aggregation and activation. Clopidogrel in combination
`with aspirin showed synergistic inhibitory effects after stimulation with collagen and
`thrombin compared with monotherapies. Thus, this dual antiplatelet treatment strategy
`deserves further evaluation in clinical trials for secondary prevention of acute myocardial
`infarction or unstable angina. (J Am Coll Cardiol 2000;36:699–705) © 2000 by the
`American College of Cardiology
`
`Antiplatelet therapy reduces recurrent ischemic events in
`patients with cardiovascular disease (1). Aspirin, although
`most widely used, cost-effective and safe, is a weak platelet
`inhibitor affecting only the cyclooxygenase pathway. De-
`spite long-term aspirin use, a large number of patients
`continue to have recurrent events associated with bad
`outcomes (2). Recurrent events in such patients may reflect
`natural progression of atherosclerosis or they take place
`through mechanisms that overcome the aspirin-induced
`inhibition of platelet function. This may occur by exposure
`of platelets to high local concentrations of strong agonists,
`such as collagen or thrombin, or by high levels of platelet
`released adenosine diphosphate (ADP) or thromboxane A2
`from nonplatelet sources (3).
`
`From the Department of Cardiology and Thrombosis Research and the *Depart-
`ment of Hematology, University Hospital Bern, Bern, Switzerland. This study was
`supported by grants 32-47077.96 and 32-56705.99 of the Swiss National Foundation
`of Science. This study was presented, in part, at the 21st Congress of the European
`Society of Cardiology, Barcelona, Spain, August 1999.
`Manuscript received October 21, 1999; revised manuscript received January 21,
`2000, accepted April 11, 2000.
`
`Recent clinical trials with intravenous platelet glycopro-
`tein IIb/IIIa (GP IIb/IIIa) receptor blockers have demon-
`strated that aspirin monotherapy has been improved upon
`(4). Further evidence for a combined antiplatelet treatment
`strategy comes from studies with aspirin and ticlopidine.
`They have been shown to lower the risk of subacute stent
`thrombosis and to be safer when compared with conven-
`tional anticoagulant therapy (5–8) and aspirin alone (6). In
`addition, data with clopidogrel, a new thienopyridine de-
`rivative lacking the serious hematological side effects of
`ticlopidine, suggest that its combination with aspirin may be
`an alternative treatment after coronary stent placement (9).
`In patients with cardiovascular disease, clopidogrel alone
`was slightly more effective than aspirin in reducing the
`combined risk of ischemic stroke, myocardial
`infarction
`(MI) or vascular death (10). Although the effects of simul-
`taneous administration of clopidogrel and aspirin on platelet
`aggregation and function in patients after MI has not been
`studied, experimental data suggest synergy between both
`drugs (11–13).
`In our study we compared the inhibitory effects of the
`combination of aspirin and clopidogrel with either drug
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`JACC Vol. 36, No. 3, 2000
`September 2000:699–705
`
`Moshfegh et al.
`Combined Antiplatelet Therapy After MI
`
`701
`
`ter addition of agonists (15). Diluted PRP was aliquoted
`into polypropylene tubes containing the agonists at the
`following final concentrations: ADP 0.1 mmol/L to 100
`mmol/L (Sigma), human and equine fibrillar collagen type I
`25 mg/mL to 75 mg/mL (Horm, Nycomed) and thrombin
`0.05 U/mL to 0.5 U/mL (Diagnotec). To inhibit fibrin
`polymerization, the thrombin aliquots contained additional
`2 mmol/L (final concentration) of the peptide H-Gly-Pro-
`Arg-Pro-OHzAcOH (18)
`(GPRP, Pefabloc Pefa-6003
`Pentapharm). Controls were effected in the absence of
`agonists. After incubation for 15 min, the platelets were
`fixed by adding paraformaldehyde 1% in phosphate buffered
`saline (PBS; ratio 1:1) and incubated with mAbs CD62p,
`CD63 and 7H2 at a final concentration of 10 mg/mL for
`30 min. Controls were effected in the absence of primary
`antibody using PBS. Bound antibody was measured using
`fluorescein isothiocyanate (FITC)-conjugated F(ab9)2 frag-
`ments of rabbit anti-mouse immunoglobulins (Dako) at a
`final concentration of 1.5 mg/mL after incubation for
`another 30 min.
`Whole blood was used for PAC-1 platelet labeling,
`diluted 1:20 with modified Tyrode’s buffer and aliquoted
`into polypropylene tubes containing PAC-1-FITC anti-
`body (20 mg/mL; Becton Dickinson) and agonists at the
`predetermined final concentrations (ADP 0.01 to 10
`mmol/L, collagen type I 25 to 75 mg/mL and thrombin 0.01
`to 0.1 U/mL). The samples were incubated without stirring
`for 15 min, platelets were fixed by adding cold paraformal-
`dehyde 1% (ratio 1:5) and analyzed within 2 h onFACScan
`(Becton-Dickinson). Fluorescence histograms were ob-
`tained for 10,000 cells gated per sample, data being analyzed
`using Cell Quest software (Becton-Dickinson). Antibody
`binding was expressed as the percentage of platelets positive
`for antibody. The gate for activated platelets was identified
`by mAb 7H2 and was set so as to include ,1% of the events
`seen when identical platelet samples were incubated with
`the control murine immunoglobulin (IgM or IgG) used at
`the same concentration as the murine mAb. Percentages of
`activation of unstimulated platelets in PRP and whole blood
`were as follows (given as mean 6 SEM): CD 62p, 1.0 6
`0.1%; CD 63, 1.7 6 0.6% in PRP and PAC-1, 2.3 6 0.4%
`in whole blood, respectively. These values are within our
`normal ranges from blood samples obtained from normal
`subjects.
`Statistical analysis. Continuous variables were expressed
`as mean 6 SEM. For comparisons between two treatment
`regimens in the same patient group, we used the Wilcoxon
`signed rank test. For comparison of the effects of different
`agonist concentrations within treatment groups, the Fried-
`mann test was used. A value of p , 0.05 was considered to
`indicate a significant difference.
`
`RESULTS
`
`Platelet aggregation studies. As shown in Figure 1A, ex
`vivo platelet aggregation was induced by addition of ADP 3,
`
`Figure 1. (A) Dose-dependent ADP-induced platelet aggregation (per-
`centage of light transmission in platelet-rich plasma) in 30 patients treated
`with aspirin 100 mg/day, clopidogrel 75 mg/day and aspirin 100 mg/day
`plus clopidogrel 75 mg/day. Results represent mean 6 SEM. *p , 0.05
`versus aspirin (Wilcoxon signed rank test). (B) Effects of different aspirin
`doses on ADP-induced platelet aggregation (percentage of light transmis-
`sion in platelet-rich plasma) in 12 patients with aspirin 100 mg/day, aspirin
`300 mg/day, aspirin 100 mg/day plus clopidogrel 75 mg/day and aspirin
`300 mg/day plus clopidogrel 75 mg/day. Results represent mean 6 SEM.
`*p , 0.05 versus aspirin (Wilcoxon signed rank test). ADP 5 adenosine
`diphosphate.
`
`6 and 20 mmol/L in a dose dependent manner (p , 0.001).
`Adenosine diphosphate-induced aggregation was signifi-
`cantly inhibited in patients treated with clopidogrel 75 mg
`(p , 0.001) or clopidogrel 75 mg plus aspirin 100 mg (p ,
`0.001) compared with aspirin 100 mg alone to a similar
`extent at each ADP concentration. Regarding the dose of
`aspirin, Figure 1B illustrates no difference in ADP-induced
`aggregation in patients treated with aspirin 100 mg com-
`pared with aspirin 300 mg and their combination with
`clopidogrel 75 mg, respectively.
`In contrast, mean values of dose-dependent platelet
`aggregation induced by collagen 1.5, 4.0 and 40 mg/mL
`were significantly higher in patients treated with clopidogrel
`75 mg alone compared with aspirin 100 mg alone (p , 0.01,
`Fig. 2). However, the combination of two drugs strongly
`potentiated the antiaggregating effects of the monotherapies
`at all agonist concentrations. The ED50 for collagen-
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`702
`
`Moshfegh etal.
`Combined Antiplatelet Therapy After MI
`
`JACC Vol. 36, No. 3, 2000
`September 2000:699–705
`
`Figure 2. Dose-dependent collagen-induced platelet aggregation (percent-
`age of light transmission in platelet-rich plasma) in 30 patients treated with
`aspirin 100 mg/day, clopidogrel 75 mg/day and aspirin 100 mg/day plus
`clopidogrel 75 mg/day. Results represent mean 6 SEM. *p , 0.05 versus
`aspirin; #p , 0.05 versus clopidogrel (Wilcoxon signed rank test).
`induced platelet aggregation slightly decreased from 5.5 6
`1.5 mg/mL in the aspirin 100 mg treated group to 2.5 6
`0.4 mg/mL in the clopidogrel 75 mg group (p , 0.05) and
`significantly increased to 22.4 6 4.0 mg/mL in patients
`receiving the combined treatment (p , 0.001). Again, no
`differences
`in collagen-induced platelet aggregation
`(4 mg/mL) was seen between aspirin 100 mg and 300 mg
`alone (31.7 6 7.7% vs. 33.0 6 5.1%) or their combination
`with clopidogrel 75 mg, respectively (14.5 6 4.5% vs.
`18.2 6 4.3%).
`The thrombin receptor agonist peptide (TRAP) induced
`aggregation was not significantly different at the lowest
`agonist concentration (8 mmol/L) between all groups but
`tended to be lower in the group with combined treatment at
`the 10 mmol/L TRAP dose. At the 15 mmol/L TRAP dose
`where half-maximal aggregations were surpassed, the com-
`bination of aspirin 100 mg and clopidogrel 75 mg signifi-
`cantly reduced platelet aggregation compared with aspirin
`100 mg or clopidogrel 75 mg alone (Fig. 3). No difference
`in TRAP-induced platelet aggregation (15 mmol/L) was
`seen between aspirin 100 mg and 300 mg alone (61.2 6
`3.8% vs. 58.0 6 3.9%, respectively) or their combination
`with clopidogrel 75 mg (54.2 6 3.4% vs. 53.0 6 3.7%,
`respectively).
`Aspirin alone or in combination with clopidogrel fully
`inhibited arachidonic acid-induced aggregation in all pa-
`tients (3.6 6 0.6%), whereas clopidogrel alone did not
`inhibit arachidonic induced aggregation (74 6 3.2%, p ,
`0.001).
`Activation markers after platelet stimulation. The ex-
`pression and induction of CD62p indicating release reaction
`of alpha-granules, CD63 indicating lysosome secretion and
`PAC-1 directed against activation-dependent epitopes on
`GPIIb/IIIa were measured as markers of platelet activation.
`Interestingly, to achieve similar levels of activated platelets
`after agonist stimulation, lower ADP and thrombin con-
`
`Figure 3. Dose-dependent TRAP-induced platelet aggregation (percent-
`age of light transmission in platelet-rich plasma) in 30 patients treated with
`aspirin 100 mg/day, clopidogrel 75 mg/day and aspirin 100 mg/day plus
`clopidogrel 75 mg/day. Results represent mean 6 SEM. *p , 0.05 versus
`aspirin; #p , 0.05 versus clopidogrel (Wilcoxon signed rank test).
`TRAP 5 thrombin receptor agonist peptide.
`
`centrations were required for PAC-1 compared with plate-
`let P-selectin or CD63 mAbs.
`As shown in Figure 4, A to C, clopidogrel alone or
`combined with aspirin had a significant effect on the
`percentage of platelets positive for the three mAbs when
`compared with aspirin alone after stimulation with ADP.
`The combined therapy compared with clopidogrel alone
`equally suppressed the activation markers. These effects
`were seen independently of the ADP concentration used.
`When the groups treated with aspirin 100 mg and aspirin
`300 mg were compared, the results were concordant for all
`three antibodies showing no difference in platelet activation
`except for a slightly lower P-selectin expression with aspirin
`300 mg at the highest ADP concentration (100 mmol/L,
`Fig. 5). There were no differences in levels of activation
`epitopes for all three mAbs between the two doses of aspirin
`when they were combined with clopidogrel.
`Platelet activation after thrombin stimulation is illus-
`trated in Figure 4, D to F. Clopidogrel alone or in
`combination with aspirin compared with aspirin alone
`considerably suppressed the levels of platelet activation as
`measured by the three mAbs. In addition, the combination
`therapy compared with clopidogrel or aspirin alone signif-
`icantly decreased platelet P-selectin expression and degran-
`ulation at low concentrations of thrombin, and a trend
`towards a reduction persisted at high thrombin concentra-
`tions. These effects of combined therapy on platelet activa-
`tion were not seen with PAC-1 where full extent of
`activation was achieved at intermediate to high concentra-
`tions of thrombin. No differences in platelet activation levels
`between the two aspirin groups were detected with CD 63,
`CD 62p and PAC-1, except for CD 62p and CD 63
`expression at the highest dose of thrombin representing
`extensive platelet activation (80.8 6 1.3% vs. 72.1 6 2.4%
`and 51.4 6 3.7% vs. 41.6 6 3.3% for aspirin 100 mg vs.
`aspirin 300 mg [p , 0.05], respectively). However, activa-
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`JACC Vol. 36, No. 3, 2000
`September 2000:699–705
`
`Moshfegh et al.
`Combined Antiplatelet Therapy After MI
`
`703
`
`Figure 4. Changes in the expression of platelet activation epitopes after administration of aspirin (100 mg/day, black bars), clopidogrel (75 mg/day, white
`bars) and aspirin (100 mg/day) plus clopidogrel (75 mg/day, hatched bars). In figures A to C, platelets were stimulated with adenosine diphosphate (ADP;
`0.01, 0.1, 1, 10, 100 mmol/L) and in D to F with thrombin (0.01, 0.05, 0.1, 0.5 U/mL). After stimulation, activated platelets were detected by flow
`cytometry with monoclonal antibodies directed against CD62p (P-selectin), which recognizes alpha-granule release, CD63 recognizing lysosome secretion
`and PAC-1 detecting activated GP IIb-IIIa complex. Results (n 5 30) are expressed as the percentage of the total platelet population binding each
`monoclonal antibody and represent mean 6 SEM. *p , 0.05 versus aspirin; #p , 0.05 versus clopidogrel; §p 5 0.054; ¶p 5 0.065 versus clopidogrel
`(Wilcoxon signed rank test).
`
`tion levels for the three mAbs were identical for both aspirin
`groups when combined with clopidogrel.
`The expression of activation-dependent epitopes mea-
`sured with the three mAbs were always lower than 20% after
`activation with human or equine collagen type I with
`concentrations of 25, 50, 75 mg/mL. Although a trend
`towards lower levels of platelet activation was detected in
`
`Figure 5. CD62p (P-selectin) positive platelets after stimulation with
`ADP in 12 patients receiving two different doses of aspirin: aspirin
`100 mg/day, aspirin 300 mg/day, aspirin 100 mg/day plus clopidogrel
`75 mg/day and aspirin 300 mg/day plus clopidogrel 75 mg/day. Results
`represent mean 6 SEM. *p , 0.05 aspirin 100 mg versus 300 mg
`(Wilcoxon signed rank test). ADP 5 adenosine diphosphate.
`
`patients treated with clopidogrel alone or with the combi-
`nation of clopidogrel with aspirin, the dose response curves
`were too flat to elicit significant differences between groups
`(data not shown). With our experimental conditions used,
`analysis of flow cytometric histograms showed no interfer-
`ence with microaggregate formation, suggesting that the
`collagen types used in our study were not effective enough to
`induce platelet activation. Alberio and Dale (19) recently
`demonstrated that collagen-induced P-selectin expression
`and upregulation of surface GP IIb/IIIa varies significantly
`depending on the type of collagen used.
`Regarding the safety of clopidogrel, two patients devel-
`oped a skin rash, which was rapidly reversible after discon-
`tinuation of the drug. Platelet count, hemoglobin concen-
`tration and leucocyte count
`remained unchanged
`throughout the study. There were no bleeding complica-
`tions noted with each of the treatments.
`
`DISCUSSION
`
`This study in postmyocardial infarction patients demon-
`strates that clopidogrel alone or in combination with aspirin
`markedly inhibited ADP-mediated platelet aggregation in
`platelet-rich plasma compared with monotherapy with as-
`pirin. Moreover, clopidogrel in combination with aspirin
`significantly reduced collagen-induced platelet aggregation
`compared with both monotherapies, suggesting a synergistic
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`Moshfegh etal.
`Combined Antiplatelet Therapy After MI
`
`JACC Vol. 36, No. 3, 2000
`September 2000:699–705
`
`platelet inhibitory effect. In addition, the combined treat-
`ment resulted in a mild inhibition of aggregation induced by
`stimulation of platelet thrombin receptor and was more
`effective in inhibiting platelet activation by thrombin. Both
`doses of aspirin equally potentiated the platelet inhibitory
`effects of clopidogrel.
`Preliminary data indicate that full inhibition of platelet
`activation is present after 2 to 6 h following oral adminis-
`tration of a loading dose of 300 mg or 525 mg (20,21). The
`maximal inhibition at 2 h isequivalent to that achieved by
`a daily dose of 75 mg clopidogrel for seven days (21).
`Results from a dose-finding study demonstrated that the
`inhibition of platelet aggregation by clopidogrel was
`concentration-dependent at low doses, but no differences
`were seen in patients on daily clopidogrel doses of 50 mg,
`75 mg and 100 mg for 7 or 28 days, indicating sustained and
`plateauing effects during chronic administration at doses
`higher than 50 mg (22). A clopidogrel dose of 75 mg was
`chosen in our study and previously evaluated in the CAP-
`RIE study (10). Although animal models suggest a dose-
`dependent inhibition of platelet-mediated thrombosis by
`clopidogrel, this effect is mostly seen within the first hours
`of intravenous administration (13), whereas it plateaus at
`intermediate doses after oral administration of clopidogrel
`for six days as demonstrated by a model of stent and graft
`thrombosis in baboons (11).
`Mechanisms of combination antiplatelet therapy. Al-
`though the exact mechanism is not known, the synergy
`between clopidogrel and aspirin may be secondary to the
`different platelet activation pathways inhibited by these
`antiplatelet drugs. Our results suggest that simultaneous
`antagonism of thromboxane A2 by aspirin and adenosine
`diphosphate by clopidogrel resulted not only in inhibition of
`arachidonic acid- and ADP-mediated platelet activation but
`also in a reduction of collagen- and thrombin-induced
`platelet activation. Collagen is an important compound of
`the extracellular matrix of the vessel wall that is exposed
`after injury. Collagen-induced platelet aggregation at low
`doses is dependent on ADP release by activated platelets
`(14), whereas at high concentrations it can become ADP-
`independent, overcoming inhibition of aggregation by clo-
`pidogrel monotherapy as shown by our findings.
`Potent synergistic effects of clopidogrel and aspirin re-
`garding the inhibition of collagen-induced aggregation,
`direct release reaction of platelets and platelet thrombus
`formation have also been demonstrated in preclinical trials
`in rabbits (12) and nonhuman primates (11). Similar effects
`were seen in a porcine model of high-shear induced stent
`thrombosis (13). Moreover, in patients after stent implan-
`tation, ticlopidine—a prodrug that inhibits platelet function
`through inhibition of the ADP receptor—was found to have
`synergistic platelet inhibitory effects when combined with
`aspirin (23). Consistent with our results in patients after
`MI, these effects were seen particularly in collagen-induced
`aggregation but not in ADP-mediated aggregation.
`Platelet activation and surface expression of adhesive
`
`glycoproteins play a key role in MI (24) and in thrombotic
`complications after coronary interventions (25,26). Evalua-
`tion of platelet activation by flow cytometry uses monoclo-
`nal antibodies that recognize membrane glycoproteins
`present only on activated platelets. Previous studies showed
`that significant platelet activation as measured by surface
`expression of activated GP IIb/IIIa receptors and P-selectin
`occurred in patients after acute coronary syndromes (27) and
`in patients after stenting receiving anticoagulation therapy
`compared with combined antiplatelet therapy with ticlopi-
`dine and aspirin (26). Our findings in patients after MI
`show that ADP-induced platelet activation measured with
`three monoclonal antibodies is significantly reduced by
`clopidogrel compared with aspirin, but platelet reactivity
`was not reduced further after ingestion of clopidogrel and
`aspirin compared with clopidogrel alone. However, a syn-
`ergistic inhibitory effect on platelet alpha-granule and lyso-
`some degranulation was observed using thrombin as a
`strong agonist at lower concentrations, but the effect was
`overpowered at higher concentrations. Since thrombin plays
`a pivotal role in platelet thrombus formation (28), the
`simultaneous administration of clopidogrel and aspirin may
`be beneficial in blocking the effects of physiologic levels of
`thrombin on platelets in patients with acute coronary
`syndromes.
`Safety. Regarding the safety, reduction in the dose of
`aspirin is desirable because side effects, in particular those in
`the gastrointestinal tract, are dose-related. As a separate part
`of our study, we examined the antiplatelet effects of two
`aspirin doses (100 mg and 300 mg) in combination with
`clopidogrel 75 mg. Chronic administration of both doses of
`aspirin prevents thromboxane production in platelets, and
`available data suggest little if any difference in clinical
`efficacy (1). Our results confirm that platelet aggregation
`and reactivity is not dependent on the maintenance dose of
`aspirin, particularly when it is combined with an ADP-
`receptor antagonist. Both drugs lead to a sustained inhibi-
`tion of platelet aggregation for the life span of the platelet
`(14). Recently, a large clinical trial showed an excellent
`safety profile of clopidogrel (10). Compared with aspirin it
`showed fewer side effects, and the incidence of significant
`neutropenia was 0.1% compared with a reported incidence
`of 1–3% for ticlopidine in clinical trials (29). Preliminary
`results from three nonrandomized trials (9,30,31) and the
`Clopidogrel Aspirin Stent International Cooperative Study
`trial (32) suggest that simultaneous administration of clo-
`pidogrel plus aspirin is safer than ticlopidine plus aspirin
`after coronary stent implantation. Although these studies
`suggest similar efficacy of both regimens in reducing coro-
`nary events after stenting, they were not sufficiently powered
`to answer the question of equivalence.
`Study limitations. There are several
`limitations of this
`study, which was designed as a pilot investigation, and,
`therefore, has a small sample size of patients with chronic
`stable coronary disease. In addition, the study is a compar-
`ative investigation of different treatments but is lacking an
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`JACC Vol. 36, No. 3, 2000
`September 2000:699–705
`
`Moshfegh et al.
`Combined Antiplatelet Therapy After MI
`
`705
`
`untreated control group. Although platelet reactivity was
`measured at steady-state drug administration, treatment
`periods were too short to assess long-term antiplatelet
`effects. Despite its wide use, there are also inherent limita-
`tions of ex vivo platelet aggregation studies, which are crude
`indicators of what is happening in vivo. However, using
`flow cytometric analysis as a second method for the evalu-
`ation of platelet reactivity and inhibitory effects of antiplate-
`let drugs, our findings were consistent with two indepen-
`dent methods.
`Conclusions. In this prospective clinical ex vivo platelet
`study, we found that the combination of two antiplatelet
`agents acting through different pathways had greater inhib-
`itory effects on platelet aggregation and activation than
`either agent alone in patients with chronic stable coronary
`disease. Thus, the concept of dual antiplatelet treatment
`with clopidogrel and aspirin is promising and deserves
`further evaluation in large randomized trials for secondary
`prevention in patients after acute MI or unstable angina.
`
`Reprint requests and correspondence: Dr. Beat J. Meyer, De-
`partment of Cardiology and Thrombosis Research, University
`Hospital, Inselspital, Freiburgstrasse 7, CH-3010 Bern, Switzer-
`land. E-mail: bmeyer@insel.ch.
`
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`Downloaded From: http://content.onlinejacc.org/ on 04/29/2013
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`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`Ex. 1009, p. 7 of 7

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