Is a 300 mg Clopidogrel Loading Dose Sufficient to Inhibit
Platelet Function Early After Coronary Stenting? A Platelet
Functio

ABSTRACT: Background. Clopidogrel combined to aspirin reduces the early risk of stent thrombosis and a clopidogrel pre-treatment strategy is associated with a better outcome. However, in clinical practice such pre-treatment strategy is not always feasible and clopidogrel is frequently not administered until the time of intervention. Aim of the study was to compare platelet function profiles in patients undergoing coronary stenting receiving clopidogrel pre-treatment (75 mg x 2 daily at least 48 hours before intervention) compared to that of patients receiving a 300 mg loading dose at intervention time. Methods: A total of 50 patients were included in whom patients’ platelet aggregation (using light transmittance aggregometry) and platelet activation (P-selectin and PAC-1 expression by whole blood flow cytometry) were assessed following ADP stimuli at baseline, and 4 hours and 24 hours following coronary stenting. Results: In the overall study population, 16/50 (32%) patients were pre-treated with clopidogrel and 34/50 (68%) received clopidogrel loading dose at intervention time. Platelet aggregation, as well as P-selectin and PAC-1 expression were significantly lower in clopidogrel pre-treated patients at baseline (p1–2 In particular, in patients undergoing percutaneous coronary interventions (PCI) a standard 300 mg clopidogrel oral loading-dose (LD) is generally administered to achieve a more accelerated inhibition of platelet function^2–4 with the goal to reduce the early risk of stent thrombosis, which most frequently occurs within the first 24 hours.^1–4 In particular, clopidogrel pre-treatment, at least 6 hours before PCI, is associated with a reduced risk of ischemic events at one month follow-up and therefore a clopidogrel pre-treatment strategy is strongly suggested.^5–6 However, such pre-treatment strategy within the requested time frame is frequently not applicable in the real life clinical setting and clopidogrel front-loading is usually given at intervention time, and not before as it ideally should be. The aim of the present study was to compare platelet function profiles in patients undergoing coronary stenting receiving clopidogrel according to a pre-treatment strategy with that of patients receiving a 300 mg clopidogrel loading dose at the time of intervention. Methods Patient population. Fifty patients referred to our interventional cardiology unit for coronary stenting were included. Two groups of patients were identified: 1) patients approaching PCI according to a pre-treatment strategy in whom clopidogrel was initiated at a regimen of 75 mg x 2 daily at least 48 hours before intervention, thus all patients approached intervention having received >= 300 mg of clopidogrel (pre-treatment group); and 2) patients receiving a 300 mg clopidogrel loading dose at intervention time (loading-dose group). All patients were on treatment with aspirin (100–250 mg/daily) for at least 7 days before intervention. Clopidogrel (75 mg/daily) was maintained for one month after intervention. During intervention, unfractionated heparin (100 IU/kg) was administered and activation clotting time values were maintained above 250 seconds. In all patients commercially available metallic stents were used. Exclusion criteria were the use of platelet glycoprotein (GP) IIb/IIIa blockers before, during, or after intervention; prior use of thienopyridines; and/or aspirin intolerance/allergy. This study was approved by the Ethical Committee of the San Carlos University Hospital and all patients gave their informed written consent. Blood Sampling. Blood samples were collected before intervention (baseline sample), 4 hours, and 24 hours following PCI. Blood was drawn from a 6 French arterial sheath for baseline and 4-hour samples and from an antecubital vein using a 21-gauge needle for the 24 hour samples. The initial first milliliters of blood were discarded to avoid spontaneous platelet activation. Assessment of Platelet Aggregation. Blood samples were collected in tubes containing 3.8% trisodium citrate for assessment of platelet aggregation. Platelet aggregation induced by 6 mM ADP (Chrono-Log Corp., Havertown, Penn) as agonist was assessed using platelet rich plasma (PRP) by the turbidimetric method in a 2-channel aggregometer (Chrono-Log 490 Model, Chrono-Log).⁷ PRP was obtained as a supernatant after centrifugation of citrated blood at 800 rpm for 10 minutes. The isolated PRP was kept at 37º C before use. Platelet poor plasma (PPP) was obtained by a second centrifugation of the blood fraction at 2500 rpm for 10 minutes. Light transmission was adjusted to 0% with PRP and to 100% for PPP for each measurement. Platelet aggregation was assessed within 2 hours from blood sampling. Curves were recorded for 5 minutes and analyzed according to international standards.⁷ Platelet aggregation was determined as the maximal percent change in light transmittance from baseline PPP as reference. Assessment of Platelet Activation. Platelet activation was determined by assessing platelet surface expression of activated GP IIb/IIIa and P-selectin following 2 mM ADP (ChronoLog, Havertown, Penn.) stimuli through flow cytometry (EPICS-XL PROFILE II Coulter; Coulter Corp. Miami, FL) as previously described.8,9 Whole blood was drawn into sterile tubes containing 3.8% trisodium citrate and then diluted with HEPES-tyrodes buffer (5 mM HEPES hydroxyethylpiperazineethane-sulfonic acid, 137mM NaCl, 2.7mM NaHCO3, 0.36 mM NaH2PO4, 2 mM CaCl2, 5 mM glucose, 0.2% BSA) to a final volumen of 1:8:1 (blood:HEPES-Tyrodes:citrate) resulting in a 1/10 dilution of whole blood during sampling. Previously HEPES-Tyrodes buffer were filtered through 0.22mm sterile filters to avoid interference from particles. Then, 50 mL of diluted whole blood was stimulated in vitro with 2mm ADP before immunolabelling. GP IIb/IIIa activation was assessed using a PAC-1 fluorescein isothiocyanate (FITC)-conjugated antibody (33 mg/L, Becton Dickinson, San José, Calif.). P-selectin expression was assessed using a phycoerithrin (PE)-conjugated anti-CD62P (0.3mg/ml, Becton Dickinson). The corresponding antibody was therefore added and incubated for 20 min in dark at room temperature. After incubation, 300 ml of 0.5% PBS-buffered paraformaldehyde was added for fixation. Samples were analyzed within 2 hours by flow cytometry and platelets were identified based on particle size (forward scatter) and complexity (side scatter). Light scatter and fluorescence data from 10,000 platelet events were collected with all detectors in logarithmic mode. Acquisition and processing data were analyzed with XL2 software (Coulter Corp.). Platelet activation was expressed as the percentage of platelets positive for antibody binding. Non specific immunostaining for negative control assessment for PAC-1 and CD62P were determined using irrelevant isotypes, IgG-FITC conjugated and IgG-PE conjugated (Becton Dickinson), respectively.¹⁰ Platelet count, fibrinogen, and von Willebrand factor assessment. Platelet count was assessed at all time points to ensure that the degree of platelet reactivity was not biased by the number of platelets in each group. Patients were not included if platelet counts were outside the range of 125 to 450 109/L. As platelet reactivity may also be affected by fibrinogen and von Willebrand factor levels, these were both assessed at baseline using the Clauss method¹¹ and a commercially available ELISA kit (Roche Diagnostics-STAGO, France), respectively. Statistical Analysis. Continuous variables are expressed as mean ± SD. Categorical variables are expressed as frequencies and percentages. Fisher’s test was used to compare qualitative variables and the Student t-test for quantitative unpaired data. A p value Platelet Aggregation. ADP-induced platelet aggregation was significantly reduced at baseline (p P-selectin expression. P-selectin expression was significantly reduced at baseline (p 1 A reduced degree of platelet function by the time of coronary intervention, as observed in the pretreatment group of our study, may contribute to the better clinical outcome early after PCI observed in clopidogrel pretreated patients in larger studies.^5,6 In particular, in the PCI-CURE study patients randomized to the pretreatment arm, received clopidogrel at a median of 10 days before undergoing PCI.⁵ At one-month followup these patients had a 30% (p = 0.03) relative risk reduction (RRR) in cardiac events and this was even greater in patients receiving a stent (RRR = 44%; p = 0.017).⁵ Of note, the clinical benefit was observed as early as 24 hours following intervention (RRR = 33%; p = 0.002) among the overall patient population of the CURE trial.¹² In the CREDO trial, the median time to clopidogrel pre-treatment was approximately 10 hours.⁶ However, at one-month follow-up although there was a 18.5% reduction in combined rate of cardiac events, this was not significant (p = 0.23), and a significant one-month clinical benefit was demonstrated only in a predefined subgroup of patients pretreated 6 to 24 hours before PCI (RRR = 38.6%; p = 0.05).⁵ Furthermore, in a recent report such benefit was not achieved unless pre-treatment was > 15 hours before PCI and this continued to increase with the number of pre-treatment hours.¹³ These data therefore strongly suggest that when there is an indication for coronary stenting, the sooner clopidogrel pre-treatment takes place, the better. The reduction in ischemic events as a consequence of a greater inhibition of platelet reactivity by the time of coronary intervention in these two major trials was highlighted in the PRONTO trial.14 In this trial, platelet function profiles were assessed in patients receiving different clopidogrel loading regimens: 300 mg clopidogrel loading dose 24 hours before, 12 hours before, 3 to 6 hours before, and 75 mg at the time of intervention, followed by a 75 mg/day regimen. This study demonstrated that a 300 mg loading dose given any time 3 to 24 hours before coronary stenting inhibited to the same extent platelet function at the time of PCI. Furthermore, a loading dose regimen reduced post-stent platelet activity more than a 75 mg dose given at intervention time and a similar degree of inhibition of platelet activity was achieved only after 5 days of treatment.¹⁴ However, at difference of the PRONTO, PCI-CURE and CREDO trials, in our study the group of non pretreated patients did not initiate clopidogrel at a 75 mg/day regimen but received a 300 mg front loading.^5,6,14 Therefore this allowed these patients to achieve a similar degree of inhibition of platelet function to that of pretreated patients after 24 hours, and not after several days. Also, our findings are in accordance with that of the PRONTO trial, as non pretreated patients present an increased degree of platelet aggregation and activation in the earliest hours after intervention, despite receiving a 300 mg loading dose, and therefore may present an increased thrombotic risk. Although clopidogrel pre-treatment is associated with a reduced risk of early ischemic events, as it ensures a greater degree of inhibition of platelet function by the time of intervention, such strategy within the required time frame is not always feasible. Although this study represents the experience of our center, many centers are dealing with the same issue and clopidogrel treatment does not begin until intervention time, and not before as ideally should. Furthermore, coronary stenting is associated with an increase in platelet activity and there is a higher number of suboptimal responders to a 300 clopidogrel loading dose in the earliest hours after intervention.^14–16 Therefore, a 300 mg may not be able to provide an adequate inhibition of platelet function in the initial hours after intervention, suggesting the use of higher periprocedural loading doses.¹⁷ Higher clopidogrel loading doses may be especially warranted in those patients who were not able to benefit from a clopidogrel pretreatment approach. Previous reports assessed the functional effects on platelet reactivity using higher than a standard 300 mg clopidogrel loading dose in patients undergoing coronary stenting.^18–20 In particular, we have previously reported that a 600 mg clopidogrel loading dose was associated with a more accelerated inhibition of platelet reactivity early after front loading and this was maintained significantly lower throughout the first 48 hours following treatment compared to a 300 mg loading dose regimen.¹⁸ Recent clinical data on the optimal timing for PCI when using intense antiplatelet treatment have also been reported. In the ISAR-COOL study,²² deferral of intervention for prolonged antithrombotic pre-treatment (including a high, 600 mg, clopidogrel loading dose and intravenous tirofiban) does not improve clinical outcome compared with immediate (23 Interestingly, there was no further improvement in outcome with the number of hours of pre-treatment.²⁴ Overall, these studies question the need of a prolonged antiplatelet pre-treatment strategy as this is not associated with a better outcome when a more intense antiplatelet regimen is used. Last, but not least, a higher clopidogrel loading dose regimen is safe as this has not been associated with an increased risk of hemorrhagic complications compared to a standard 300 mg loading dose.^18,20,21Study Limitations. The main limitation of the present study is that it is the result of a retrospective analysis of platelet function assays performed in patients with different clopidogrel treatment strategies which were defined in a non-randomized fashion, but according to the referring physicians’ criteria. In addition, the small sample size of the study does not allow us to assert significant clinical differences (i.e stent thrombosis). However, since thrombotic events are associated with an increased platelet reactivity,²⁴ our data remain clinically relevant. Larger studies with assessment of platelet function and use of different loading-dose regimens, preferably in a randomized fashion, are warranted to define the optimal clopidogrel loading dose to be administered at intervention time. Ultimately, platelet agonists used during platelet function studies may vary. This may lead to different results according to the type and concentration of the agonist used, representing therefore a limitation for all platelet function studies. However, the methodology carried out in the present study has been previously reported and ADP was chosen as platelet agonist since it acts upon the same receptor as clopidogrel.^9,17,18 Conclusions Clopidogrel pre-treatment is associated with an enhanced degree of inhibition of platelet function profiles in the early hours after PCI compared to patients receiving a 300 mg clopidogrel treatment at intervention time. However, a clopidogrel pre-treatment strategy is not always feasible, suggesting the use of higher peri-procedural clopidogrel loading doses to minimize early thrombotic risk. Larger and randomized platelet function studies are warranted to assess the clinical impact of higher clopidogrel loading doses compared to a standard 300 mg approach in order to define the optimal loading dose regimen.