`or Their Combination After Stent Implantation
`
`Hans J. Rupprecht, MD; Harald Darius, MD; Ulrike Borkowski, MD; Thomas Voigtla¨nder, MD;
`Bernd Nowak, MD; Sabine Genth, MD; Ju¨rgen Meyer, MD
`
`Background—This study was performed to analyze the influence of either aspirin, ticlopidine, or their combination on platelet
`activation and aggregation parameters after stent implantation.
`Methods and Results—Sixty-one patients with successful implantation of a single Palmaz-Schatz stent in a native coronary
`artery were randomly assigned to either group A (aspirin 300 mg/d⫹ticlopidine 2⫻250 mg/d), group B (ticlopidine
`2⫻250 mg/d), or group C (aspirin 300 mg/d). Platelet activation was evaluated on days 1, 7, and 14 by flow cytometry
`measurement of expression of CD62p (p-selectin) and the binding of fibrinogen to the platelet surface glycoprotein IIb/IIIa
`receptor. Platelet aggregation was induced by addition of ADP or collagen. Differences between treatment groups were
`compared by ANOVA. Between days 1 and 14, we observed a significant decrease in collagen-induced platelet aggregation
`in group A (62.2⫾2.5% versus 36.9⫾3.1%), whereas an increase was seen in group B (58.3⫾2.5% versus 67.7⫾3.2%) and
`no change was seen in group C (P⬍.0001). The ADP-induced aggregation declined significantly in group A (74.7⫾1.4%
`versus 55.3⫾2.6%), whereas a delayed reduction was seen in group B (72.0⫾3.0% versus 52.6⫾4.2%) and no change was
`seen in group C (P⫽.0017). The CD62p expression declined significantly in groups A (68.2⫾2.7% versus 41.3⫾2.7%) and
`B (64.8⫾2.9% versus 39.3⫾3.5%) but not in group C (P⬍.0001). Moreover, the fibrinogen binding decreased significantly
`in group A (61.0⫾4.3% versus 36.3⫾4.2%) and with delay in group B (58.3⫾2.2% versus 39.4⫾3.0%), whereas no
`alterations were seen in group C (P⫽.012).
`Conclusions—Our results demonstrate synergistic and accelerated platelet inhibitory effects of ticlopidine plus aspirin in
`patients after stent implantation compared with a monotherapy with either ticlopidine or aspirin alone. (Circulation.
`1998;97:1046-1052.)
`
`Key Words: stents 䡲 platelet aggregation inhibitors 䡲 aspirin
`
`Combined antiplatelet therapy with ticlopidine and aspirin
`
`subacute stent
`has been shown to lower the risk of
`thrombosis compared with conventional anticoagulant thera-
`py.1–5 This has enabled stenting to become a breakthrough
`technology in interventional cardiology, and the rate of stent
`implantation now amounts to 30% to 50% of all procedures in
`most centers.6,7
`Bearing in mind the inherent risks and side effects of
`ticlopidine and aspirin, especially the risk of neutropenia for
`the former and the risk of gastrointestinal bleeding for the
`latter, the question as to whether aspirin or ticlopidine alone or
`a combination of both might be sufficient
`to counteract
`platelet activation and aggregation after stenting deserves
`further investigation. Therefore we randomly assigned patients
`after stent implantation to a treatment with aspirin alone,
`ticlopidine alone, or a combination of both to compare
`magnitude and temporal changes of markers of platelet activa-
`tion in flow cytometry and aggregation within the first 2 weeks
`after stent implantation.
`
`Patient Selection
`Patients with successful implantation of a single Palmaz-Schatz
`stent in a native coronary artery were selected for the study if
`there was a low risk for subacute stent thrombosis. This
`included a vessel diameter of the stented segment of ⱖ3.0 mm,
`absence of thrombus formation before and after stent place-
`ment, a TIMI grade 3 blood flow, absence of a residual
`dissection, and absence of a residual lesion ⬎20% within or
`adjacent to the stent.
`Patients with bleeding disorders, contraindications to treat-
`ment with aspirin and/or ticlopidine, abnormal blood cell
`count, childbearing potential, acute myocardial
`infarction,
`depressed left ventricular function, renal insufficiency, or an
`indication for oral anticoagulation were excluded from the
`study. Eligible patients adhering to the prespecified criteria
`were randomly assigned to the treatment groups immediately
`after the intervention and after written informed consent was
`obtained. The study was performed according to the Declara-
`tion of Helsinki and the protocol was approved by the local
`ethical committee.
`
`Received October 31, 1997; accepted November 25, 1997.
`From the Department of Medicine II, Johannes Gutenberg University, Mainz, Germany.
`The authors contributed equally to the paper.
`Correspondence to Hans-Ju¨rgen Rupprecht, MD, Department of Medicine II, Johannes Gutenberg-University, Langenbeckstr 1, D-55101 Mainz,
`Germany.
`© 1998 American Heart Association, Inc.
`
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`Rupprecht et al
`
`1047
`
`Intervention and Adjunctive
`Concomitant Therapy
`All patients received 10 000 IU of heparin. The first 5000 U
`was given after arterial puncture and another 5000 U imme-
`diately before guidewire insertion. Palmaz-Schatz stents were
`folded by hand on the angioplasty balloon (Europass, Cordis),
`and implantation was performed with a pressure of 10 to 16
`atm. The arterial sheath was left in the groin for 12 to 24 hours.
`After sheath removal, manual compression of the groin was
`carried out until hemostasis occurred. Thereafter, a pressure
`bandage was applied for at least 12 hours.
`Heparin was administered intravenously for 24 hours, when
`the pressure bandage was removed to maintain a partial
`thromboplastin time of 60 to 90 seconds.
`
`Antiplatelet Therapy
`All patients were pretreated with 100 mg aspirin/d for at least
`1 week before randomization, with the last drug intake at the
`day of percutaneous transluminal coronary angioplasty. Ac-
`cording to the randomization schedule, the patients were
`attributed to one of three antiplatelet regimens, starting imme-
`diately after the procedure for a duration of four weeks: group
`A: aspirin 300 mg/d⫹ticlopidine 2⫻250 mg/d, group B:
`ticlopidine 2⫻250 mg/d, and group C: aspirin 300 mg/d.
`After the initial 4-week treatment period, antiplatelet therapy
`was continued with aspirin 100 mg/d.
`
`Laboratory Investigations
`The following laboratory tests were performed on day 1, day 7,
`and day 14 after stent implantation.
`
`Blood Sampling
`Venous blood was withdrawn immediately after patient ran-
`domization before intake of the first dose of study medication,
`on day 7 and on day 14 of treatment. Twenty milliliters of
`blood was drawn by venipuncture and anticoagulated in acid
`citrate dextrose (15% vol/vol; Biostabil, Biotest). Blood was
`centrifuged for 12 minutes at 180g at room temperature to
`obtain platelet-rich plasma. Aliquots of the platelet-rich plasma
`were used for platelet aggregation studies, for the preparation
`of platelet-poor plasma (centrifugation 1500g, 10 minutes) to
`calibrate the platelet aggregometer, and another aliquot for
`flow cytometric analysis.
`
`Platelet Aggregation Studies
`Platelet aggregation was performed in platelet-rich plasma by
`addition of ADP or collagen and the alterations in light
`transmission were measured photometrically (APACT Aggre-
`gometer) and analyzed automatically. The aggregometer was
`calibrated for the difference in light transmission between
`platelet-poor and platelet-rich plasma, which was set as 100%
`by definition. Alterations in light transmission in response to
`the respective aggregating agent were expressed as percentage
`of light transmission. ADP (Boehringer Mannheim) was added
`in concentrations between 1 to 20 mol/L to facilitate
`construction of a dose-response curve and calculation of ED50
`values, with 1.0, 3.3, and 20.0 mol/L used for each time
`point. Collagen was used as the aggregating agent in at least
`three concentrations of 1.0, 3.3, and 33.3 g/mL plus addi-
`
`tional concentrations
`dose-response relation.
`
`if necessary for the evaluation of a
`
`Flow Cytometric Analysis
`Platelet activation was evaluated by flow cytometry measure-
`ment of
`the surface membrane expression of CD62p
`(p-selectin, GMP140) by use of a monoclonal antibody (clone
`CLBI Cell Systems, dilution 1: 300) and the binding of
`fibrinogen to the platelet glycoprotein (GP)IIb/IIIa receptor.8
`Aliquots of platelet-rich plasma were activated by addition of
`ADP (1.0, 10, 100 mol/L), and reaction was terminated after
`5 minutes by fixation of the cells with paraformaldehyde (1%)
`for 30 minutes.
`For flow cytometric analysis, the platelet gate was identified
`by staining control platelets with an CD61 antibody (clone
`SZ21, Dianova, dilution 1:100), an epitope constitutively
`expressed on the platelet surface. More than 95% of all cells
`measured in the delineated window were CD61 positive in all
`treatment groups at all time points. As an isotype control, a
`mouse anti-human MHC II antibody (OX6) was used.
`
`Platelet Count
`Platelets were counted in venous blood samples taken in
`EDTA by an automatic cell counter (Coulter Instruments).
`
`Statistics
`Continuous data are expressed as mean⫾SEM. ANOVA for
`repeated measures was used for comparisons between different
`time points and between treatment groups as well. A value of
`P⬍.05 was considered to indicate a statistically significant
`difference.
`
`Results
`Platelet Aggregation Studies
`induced by addition of
`Platelet aggregation ex vivo was
`collagen 1.0, 3.3, and 33.3 g/mL. In Fig 1A, the results of
`collagen 3.3 g/mL are depicted. The initial values in groups
`A, B, and C were not significantly different before initiation of
`therapy. After 7 days of therapy, a significant decrease in group
`A from 62.2⫾2.5% to 46.2⫾3.9% was observed. A further
`decline in platelet aggregation response to 36.9⫾3.1% oc-
`curred until the end of the observation period on day 14
`(P⫽.0001). In contrast, in group B a significant increase in
`collagen-induced platelet aggregation from 58.3⫾2.5% to
`70.9⫾3.5% was seen on day 7 when compared with day 1.
`During the second week of
`the observation period,
`the
`collagen-induced platelet aggregation amounted to 67.7⫾3.2%
`(P⫽.005). In group C there was no significant alteration in
`collagen-induced platelet aggregation detectable (P⬍.0001 for
`comparison of treatment groups by ANOVA (Fig 1A). The
`influence of different collagen concentrations on platelet ag-
`gregation in group A is shown in Fig 1B.
`For
`the highest concentration of collagen used (33.3
`g/mL), the inhibitory effects of the platelet antagonists were
`surmounted by the collagen concentration used (Fig 1B). In
`the lower concentrations (1.0 and 3.3 g/mL), a significant
`decrease in platelet aggregation response was seen during the
`observation period.
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`Ex. 1014, p. 2 of 8
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`
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`1048
`
`Comparison of Antiplatelet Effects
`
`Figure 2. Time-dependent alterations in the ED50 for collagen-
`induced platelet aggregation in patients of group A
`(ticlopidine⫹aspirin, dashed line), group B (ticlopidine, black
`line), and group C (aspirin, gray line). Data represent mean⫾1
`SEM. Probability values relate to differences between different
`time points in each treatment group. Comparison of treatment
`groups by ANOVA revealed a value of P⬍.0001.
`
`Platelet Adhesion Molecule Expression
`As a marker of platelet adhesion molecule expression, the
`ADP-induced expression of CD62p was measured. Basal
`CD62p expression in the absence of stimulating agents was
`⬍5% of positive cells in all groups. Activation of platelets with
`ADP (100 mol/L) resulted in an increase in CD62p-positive
`cells, which was not different for the three treatment groups
`(Fig 4A). In group A, CD62p expression declined from
`68.2⫾2.7% to 43.7⫾3.1% on day 7 and to 41.3⫾2.7% on day
`14 (P⫽.0001). In group B the percentage of positive cells
`decreased from 64.8⫾2.9% to 46.7⫾3.8% on day 7 and to
`39.3⫾3.5% on day 14 (P⫽.0001). In contrast, no significant
`changes were noted in group C during the 2-week observation
`period (P⬍.0001 for comparison of
`treatment groups by
`ANOVA). The number of CD62p-positive cells in group A
`decreased significantly, independent of the ADP concentra-
`tions used (Fig 4B).
`Binding of biotin-labeled fibrinogen to the platelet
`GPIIb/IIIa receptors was ⬍5% in all samples measured before
`activation. Stimulation of
`fibrinogen binding by ADP
`(100 mol/L) resulted in a comparable increase in all three
`groups on day 1 (Fig 5A). In group A fibrinogen binding
`decreased significantly from 61.0⫾4.3% on day 1 to
`40.8⫾3.8% on day 7 and to 36.3⫾4.2% on day 14 (P⫽.0001).
`In group B, a moderate reduction of positive cells from
`58.3⫾2.2% on day 1 to 52.5⫾4.2% on day 7 was observed,
`with a further decrease on day 14 to 39.4⫾3.0% (P⫽.0001). In
`contrast, in group C there were no significant time-dependent
`alterations in fibrinogen binding (P⫽.012 for comparison of
`treatment groups by ANOVA). The fibrinogen binding de-
`creased significantly in group A only when an ADP concen-
`tration of 100 mol/L was used (Fig 5B).
`
`Clinical Results
`With regard to clinical or lesion characteristics, there were no
`significant differences between the three groups of patients
`
`Figure 1. A, Time-dependent alterations in collagen-induced
`platelet aggregation (collagen 3.3 g/mL) measured as percent-
`age of light transmittance in groups A (ticlopidine⫹aspirin),
`group B (ticlopidine), and group C (aspirin). Numbers within bars
`represent mean⫾1 SEM. Probability values relate to differences
`between different time points in each treatment group. Compari-
`son of treatment groups by ANOVA revealed a value of
`P⬍.0001. B, Dose-dependent platelet stimulatory effects of col-
`lagen (33.3, 3.3, or 1.0 g/mL) in patients of group A receiving
`ticlopidine and aspirin. Numbers within bars represent mean⫾1
`SEM. Probability values relate to differences between different
`time points in each treatment group.
`
`for collagen-induced platelet aggregation in-
`The ED50
`creased significantly from 1.7⫾0.2 to 3.9⫾0.7 g/mL on day
`7 and 5.4⫾0.9 g/mL on day 14 in group A (P⫽.0005; Fig 2).
`In group B a slight decrease of the ED50 was seen after
`withdrawal of the routine aspirin medication. In contrast, the
`ED50 was unaltered in patients receiving aspirin only (P⬍.0001
`for comparison of treatment groups by ANOVA).
`The ADP-induced aggregation was not significantly differ-
`ent on day 1 between the treatment groups (Fig 3A). Aggre-
`gation declined in group A from 74.7⫾1.4% on day 1 to
`57.0⫾2.6% on day 7 and to 55.3⫾2.6% on day 14 (P⫽.0001).
`In group B only a moderate reduction of the ADP-induced
`platelet aggregation from 72.0⫾3.0% on day 1 to 61.6⫾3.0%
`on day 7 and a further reduction to 52.6⫾4.2% on day 14 was
`measured (P⫽.0002). In group C no significant changes
`compared with the baseline value were noted (P⫽.0017 for
`comparison of treatment groups by ANOVA).
`A significant reduction of platelet aggregation in group A
`was measured irrespective of the ADP concentration used
`(ADP 20, 3.3, or 1.0 mol/L; Fig 3B).
`
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`Ex. 1014, p. 3 of 8
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`Rupprecht et al
`
`1049
`
`Figure 3. A, Time-dependent alterations in ADP-induced plate-
`let aggregation (ADP 20 mol/L) measured as percentage of
`light transmittance in group A (ticlopidine⫹aspirin), group B
`(ticlopidine), and group C (aspirin). Numbers within bars repre-
`sent mean⫾1 SEM. Probability values relate to differences
`between different time points in each treatment group. Compari-
`son of treatment groups by ANOVA revealed a value of
`P⬍.0017. B, Dose-dependent platelet stimulatory effects of ADP
`(20.0, 3.3, or 1.0 mol/L) in patients of group A receiving ticlopi-
`dine and aspirin. Numbers within bars represent mean⫾1 SEM.
`Probability values relate to differences between different time
`points in each treatment group.
`
`study did not
`(Table). The patients randomized for this
`subacute stent
`experience any angina attacks,
`infarctions,
`thromboses, or any other adverse cardiovascular events. One
`major bleeding event with a drop in hemoglobin concentra-
`tion by 4 mg/dL occurred at the groin puncture site of one
`patient in group C; however, a transfusion of red blood cells
`was not required. There was no necessity for repeat interven-
`tions during the follow-up period of 2 weeks or until the
`medication was altered to aspirin 100 mg/d 4 weeks after
`stenting. As a safety measure with regard to the ticlopidine
`administration, white blood cell counts of all patients involved
`in this study were taken 2, 4, and 6 weeks after intervention.
`No clinically relevant alterations in white blood cell counts
`were detected.
`
`Discussion
`Since its introduction by Sigwart et al,9 coronary stent implan-
`tation has been hampered for many years by the occurrence of
`subacute stent thromboses and bleeding complications.6,7,10,11
`
`Figure 4. A, CD62p (p-selectin)-positive platelets after stimula-
`tion with ADP (100 mol/L) for patients in group A
`(ticlopidine⫹aspirin), group B (ticlopidine), and group C (aspirin).
`Numbers within bars represent mean⫾1 SEM. Probability values
`relate to differences between different time points in each treat-
`ment group. Comparison of treatment groups by ANOVA
`revealed a value of P⬍.0001. B, CD62p (p-selectin)-positive
`platelets after stimulation with ADP (100, 10, or 1 mol/L) for
`patients in group A (ticlopidine⫹aspirin). Numbers within bars
`represent mean⫾1 SEM. Probability values relate to differences
`between different time points in each treatment group.
`
`Certain investigators demonstrated a reduction of the afore-
`mentioned complications by means of improved stent deploy-
`ment
`techniques and more adequate postprocedural
`management.3,8,12–22
`It is the merit of a group of French investigators2,5 who first
`demonstrated that a combination of aspirin and ticlopidine,
`another antiplatelet drug that acts by the inhibition of the
`ADP-induced platelet activation, was clearly superior to the
`conventional oral anticoagulation regimen with regard to a
`reduction of subacute stent thrombosis. A randomized single-
`center trial thereafter reported a reduction of major adverse
`cardiac events from 6.2% with oral anticoagulation to 1.5%
`with a combined antiplatelet treatment involving ticlopidine
`and aspirin.1 This correlated with a reduced stent occlusion rate
`of 0.8% compared with 5.4% with anticoagulation. In addi-
`tion, the rate of noncardiac events, comprising mainly hem-
`orrhagic complications, was reduced from 12.3% to 1.2%.1
`Major multicenter trials (STARS, FANTASTIC) have con-
`firmed these results.23,24
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`Ex. 1014, p. 4 of 8
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`
`1050
`
`Comparison of Antiplatelet Effects
`
`ment with aspirin and ticlopidine would be necessary or
`whether a monotherapy would be sufficient to counteract the
`problem of stent thrombosis. Especially in the light of im-
`proved implantation techniques with high-pressure balloon
`inflation and intravascular ultrasound guidance, it was sug-
`gested that the antithrombotic strategy might not be of major
`concern after optimal stent deployment.18 An observational
`trial with ticlopidine alone for 3 to 6 months and subcutaneous
`heparin for 1 week revealed a subacute stent thrombosis rate of
`4.2%, a myocardial infarction rate of 1.2%, and a death rate of
`2%.15 A randomized trial comparing a monotherapy of aspirin
`with a combination of ticlopidine and aspirin was prematurely
`terminated because 3 of 103 patients in the aspirin group died
`compared with none of 123 patients in the ticlopidine⫹aspirin
`group.27 Although the total event rate (3.9% versus 0.8%) did
`not reveal a statistically significant difference,
`the authors
`believed that it was not justified to withhold ticlopidine from
`their patients any longer. Preliminary data from the much
`larger STARS trial suggest a relevant reduction of clinical
`events through the use of a combination of ticlopidine and
`aspirin compared with a monotherapy with aspirin.23
`To determine whether monotherapy with ticlopidine, aspi-
`rin, or a combination of both agents are equivalent or whether
`there might be a meaningful difference, we compared these
`three treatment strategies in 61 randomly assigned patients and
`determined the collagen-induced as well as ADP-induced
`platelet aggregation immediately after stent implantation and 1
`week and 2 weeks after stent implantation. In addition, platelet
`activation was determined by means of the ADP-induced
`CD62p expression and the ADP-induced fibrinogen binding
`to platelets.
`In patients with aspirin monotherapy no change in collagen-
`induced platelet activation occurred during the observation
`period because all patients were already pretreated with aspirin
`before stent implantation. Although the results in the ticlopi-
`dine group on day 7 might still be contaminated by the
`long-lasting effects of aspirin pretreatment (Fig 1A), collagen-
`induced aggregation on day 14 is still higher if compared with
`day 1. Thus the increased aggregation response on day 7
`represents an aspirin withdrawal phenomenon. There is no
`clear evidence for an aspirin rebound phenomenon because
`aggregation responses on days 7 and 14 are not strikingly
`different from each other. Surprisingly, the combination of
`aspirin and ticlopidine resulted in a significant reduction of
`collagen-induced platelet aggregation, suggesting a synergistic
`platelet inhibitory effect of both agents. Accordingly, we found
`no change of the ED50 for collagen-induced platelet aggrega-
`tion in the aspirin group, a slight decrease in the ticlopidine
`group, but a significant increase in the aspirin⫹ticlopidine
`group.
`Monotherapy with aspirin alone did not alter the ADP-
`induced platelet aggregation, whereas monotherapy with ticlo-
`pidine led to a marked decrease in platelet aggregation,
`pointing out that ticlopidine in contrast to aspirin acts by
`inhibition of the ADP-induced platelet activation. This effect
`increased over time during the observation period in the
`ticlopidine group. The combined treatment with aspirin and
`ticlopidine led to a highly significant suppression of ADP-
`induced platelet aggregation, which was already completely
`
`Figure 5. A, Fibrinogen receptor (glycoprotein IIb/IIIa) occupa-
`tion measured as binding of biotin-labeled fibrinogen and its
`time-dependent alterations after stimulation with ADP
`(100 mol/L) for patients in group A (ticlopidine⫹aspirin), group
`B (ticlopidine), and group C (aspirin). Numbers within the bars
`represent mean⫾1 SEM. Probability values relate to differences
`between different time points in each treatment group. Compari-
`son of treatment groups by ANOVA revealed a value of P⫽.012.
`B, Platelet fibrinogen receptor (glycoprotein IIb/IIIa) occupation
`after stimulation with ADP (100, 10, or 1 mol/L) for patients in
`group A (ticlopidine⫹aspirin). Numbers within bars represent
`mean⫾1 SEM. Probability values relate to differences between
`different time points in each treatment group.
`
`Gawaz et al25 recently demonstrated that platelet activation
`after coronary stent implantation can be modified by selection
`of antithrombotic strategies. He found an increase in fibrino-
`gen receptor activity in patients receiving oral anticoagulation
`compared with a decrease in patients receiving ticlopidine.
`Platelet surface expression of CD62p was enhanced in patients
`under oral anticoagulation but not in patients treated with
`ticlopidine. In another study, Neumann et al26 were able to
`demonstrate that platelet fibrinogen receptor expression was an
`independent predictor of subacute stent occlusion, whereas
`prothrombin fragment F1 and F2, sensitive markers of throm-
`bin generation, and fibrinogen,
`the final substrate of
`the
`coagulation cascade, did not show a strong correlation to the
`risk of stent occlusion.
`With regard to potential side effects of either aspirin or
`ticlopidine, which is known to cause leukopenia in ⬇1% of all
`patients and skin rash and diarrhea in a sizeable number of
`patients, the question arose as to whether a combined treat-
`
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`Ex. 1014, p. 5 of 8
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`
`1051
`
`Patient and Lesion Characteristics
`
`Ticlopidine
`ⴙAspirin
`
`Ticlopidine
`
`Aspirin
`
`P
`
`21
`59⫾8
`16 (76%)
`4 (19%)
`10 (48%)
`10 (48%)
`7 (33%)
`4 (19%)
`4 (19%)
`2 (10%)
`8 (38%)
`
`18 (86%)
`17 (81%)
`4 (19%)
`
`䡠 䡠 䡠
`10 (48%)
`7 (33%)
`4 (19%)
`
`20
`59⫾10
`14 (70%)
`4 (20%)
`8 (40%)
`9 (45%)
`8 (40%)
`5 (25%)
`3 (15%)
`1 (5%)
`9 (45%)
`
`16 (80%)
`14 (70%)
`5 (25%)
`1 (5%)
`9 (45%)
`6 (30%)
`5 (25%)
`
`20
`58⫾9
`15 (75%)
`3 (15%)
`9 (45%)
`8 (40%)
`7 (35%)
`4 (20%)
`3 (15%)
`2 (10%)
`8 (40%)
`
`17 (85%)
`16 (80%)
`3 (15%)
`1 (5%)
`10 (50%)
`5 (25%)
`5 (25%)
`
`NS
`NS
`NS
`NS
`NS
`NS
`NS
`NS
`NS
`NS
`
`NS
`NS
`NS
`NS
`NS
`NS
`NS
`
`Patient characteristics
`Patients, n
`Age, y
`Male
`Diabetes
`Hypertension
`Hypercholesterolemia
`Smoking
`Previous MI
`Previous PTCA
`Previous CABG
`Unstable angina
`Lesion characteristics
`De novo lesion
`Single-vessel disease
`Double-vessel disease
`Triple-vessel disease
`LAD
`RCA
`LCx
`Largest balloon size
`NS
`11 (55%)
`9 (45%)
`10 (48%)
`3.0 mm
`NS
`5 (25%)
`8 (40%)
`6 (29%)
`3.5 mm
`NS
`4 (20%)
`3 (15%)
`5 (24%)
`4.0 mm
`72⫾13
`72⫾12
`76⫾15
`NS
`Diameter stenosis before intervention, %
`11⫾7
`12⫾8
`12⫾7
`NS
`Diameter stenosis after intervention, %
`13⫾3
`12⫾3
`13⫾4
`NS
`Maximal inflation pressure, atm
`MI
`indicates myocardial
`infarction; PTCA, percutaneous transluminal coronary angioplasty; CABG,
`coronary artery bypass grafting; LAD, left anterior descending coronary artery; RCA, right coronary artery;
`and LCx, left circumflex artery.
`
`present 1 week after randomization. Thus the combination of
`both antiplatelet agents obviously caused a faster inhibition of
`the ADP-induced platelet aggregation compared with a mono-
`therapy with ticlopidine,
`suggesting a possible role for
`thromboxane A2 for the augmentation of the stimulatory effect
`of ADP.
`The ADP-induced expression of CD62p was not altered
`during follow-up in the aspirin group, but a significant
`reduction of
`the percentage of CD62p-positive cells was
`measured in both the
`ticlopidine
`as well
`as
`the
`aspirin⫹ticlopidine group, with a trend toward a faster effect in
`the latter.
`The ADP-induced binding of fibrinogen to the GPIIb/IIIa
`receptors did not change in the aspirin group but was markedly
`reduced in the two ticlopidine groups. Again, the combined
`treatment revealed a relevant decrease of the ADP-induced
`fibrinogen binding already on day 7, whereas a comparable
`effect was seen in the ticlopidine group on day 14.
`It is remarkable that the expression of the adhesion molecule
`CD62p being necessary for platelet adhesion is reduced to a
`
`comparable magnitude as the aggregation response. This might
`indicate a possible role for ticlopidine and aspirin in inhibition
`of platelet adhesion processes in vivo. There is recent evidence
`in the literature that aspirin might produce an artifact during
`aggregation in platelet-rich plasma that
`is not present
`if
`aggregation is performed in whole blood.28 However, because
`platelet aggregation in platelet-rich plasma is a widely per-
`formed and acknowledged test for the evaluation of platelet
`activity ex vivo, we used this method, for which many other
`clinical
`studies exist. This
`is
`in contrast
`to whole blood
`aggregation, which is rarely used in clinical studies. In addition,
`we performed flow cytometric analysis to circumvent the
`problems with platelet aggregation in platelet-rich plasma and
`to involve a second, independent method for the evaluation of
`platelet activity and the inhibitory effects of platelet-active
`drugs.
`Our results demonstrate a synergistic platelet-inhibitory
`effect of ticlopidine plus aspirin in patients after stent implan-
`tation that might be responsible for the beneficial effects of this
`drug combination in patients after stent implantation. The
`
`Downloaded from
`
`
`
` by guest on January 7, 2015http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1014, p. 6 of 8
`
`
`
`1052
`
`Comparison of Antiplatelet Effects
`
`combined inhibition of ADP and arachidonic acid pathway–
`dependent platelet activation antagonized the two most im-
`portant avenues of platelet stimulation within the coronary
`circulation. Whether clopidrogrel,
`the likely successor of
`ticlopidine, yields equivalent platelet inhibitory effects needs to
`be shown in future trials.29 From the above data it can be
`concluded that the combination of aspirin and ticlopidine is
`clearly superior in terms of platelet aggregation parameters and
`platelet activation markers compared with a monotherapy with
`ticlopidine or aspirin and thus should be the preferred treat-
`ment strategy after stent implantation.
`
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