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Clopidogrel-Induced Platelet Inhibition Cannot Be Detected by the Platelet Function Analyzer-100 System in Stroke Patients
Clopidogrel-Induced Platelet Inhibition Cannot Be Detected by the Platelet Function Analyzer-100 System in Stroke Patients Nicole Kotzailias, MD,* Kirsten Elwischger, MD,* Thomas Sycha, MD,* Walter Rinner, MD,* Peter Quehenberger, MD,† Eduard Auff, MD,* and Christian Müller, MD*
The administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, is recommended for recurrent stroke patients under aspirin treatment. However, up to 25% of vascular patients have an inadequate response to clopidogrel treatment, which could be associated with increased reinfarction rates. This study investigated whether the platelet function analyzer (PFA-100) system represents an appropriate tool for monitoring clopidogrel’s antiplatelet effects in stroke patients. Sixteen stroke patients on clopidogrel therapy (75 mg/day) were included in a prospective analyst– blinded, cross-sectional study. Platelet function was assayed by collagen/epinephrine (CEPI)- and collagen/ADP (CADP)-induced closure times (CTs) using the PFA-100 system. von Willebrand factor antigen (vWF-Ag) levels were measured by enzyme immunoassay. CEPI-CT and CADP-CT values averaged 160 ⫾ 15 seconds and 102 ⫾ 10 seconds, respectively, and were in the normal range. vWF-Ag concentrations averaged 153 ⫾ 17% and correlated inversely with CTs (r ⫽ .71; P ⬍ .002 for CEPI-CT, r ⫽ .54; P ⬍ .04 for CADP-CT). Our data indicate that the current PFA-100 cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition in stroke patients. Key Words: Clopidogrel—PFA-100 —platelet function—stroke. © 2007 by National Stroke Association
Along with myocardial infarction, stroke represents one of the most important causes of death in Europe. The rate of recurrent stroke is 40% within 5 years after the acute event with up to 16% recurrence within the first year. Acetylic salicylic acid (aspirin) is the first-line anti-
From the *Department of Neurology and †Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Austria. Received October 26, 2006; accepted May 1, 2007. Supported by a research grant from the Medizinisch-Wissenschaftlicher Fonds des Buergermeisters der Bundeshauptstadt Wien (BGM project 2302). Address reprint requests to Nicole Kotzailias, MD, Department of Neurology, Medical University Vienna, Währinger Gürtel 18 –20, A-1090 Vienna, Austria. E-mail: [email protected] 1052-3057/$—see front matter © 2007 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2007.05.001
platelet therapy in secondary stroke prevention and reduces the relative risk for cerebrovascular events by approximately 23%.1,2 However, recently up to 40% of patients with recurrent ischemic stroke were identified as aspirin nonresponders.3 The stroke consensus statement (Statement for Healthcare Professionals from the American Heart Association/American Stroke Association Council on Stroke, 2006) recommends the administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, in recurrent stroke under aspirin treatment. Similar to aspirin resistence, an inadequate response to clopidogrel treatment occurs in up to 25% of cardiovascular patients,4 which could be associated with increased reinfarction rates. Thus, it may be useful to identify nonresponders in whom secondary stroke prevention can be optimized by changing to an alternative treatment regime. The platelet function analyzer (PFA-100) system is effective in detect-
Journal of Stroke and Cerebrovascular Diseases, Vol. 16, No. 5 (September-October), 2007: pp 199-202
199
N. KOTZAILIAS ET AL.
200
ing aspirin nonresponders by measuring collagen/epinephrine-induced closure time (CEPI-CT).5 In contrast, only cost- and time-inefficient methods for the detection of clopidogrel-induced platelet inhibition are available: thromboelastography, aggregometry, and flow cytometry, which can be conducted in specialized centers only.6 The PFA-100 system can provide an easy, cheap, and time-saving tool for the detection of clopidogrel’s induced platelet inhibition in a daily routine. To examine the efficacy of the PFA-100 system in detecting clopidogrel’s antiaggregatory effect, we measured collagen/ ADP-induced closure time (CADP-CT) in 16 stroke patients on clopidogrel therapy.
Methods Study Design The protocol and the consent form were approved by the Ethics Committee of the Medical University of Vienna. Written informed consent was obtained from each participant before entry into the study. The study was conducted in accordance with the Declaration of Helsinki and was designed as a prospective analyst– blinded cohort trial. The stroke patients were on clopidogrel therapy at a dose of 75 mg/day for secondary stroke prevention for a minimum of 1 week before study entry. The blood sample was drawn 2 hours after the last clopidogrel dose.
Patients Sixteen patients with a history of stroke treated with clopidogrel (Plavix) 75 mg/day with a mean age of 67 ⫾ 3 years were included in the study (Table 1). All 16 patients were free of infection and serious illness (with normal clinical chemistry and routine blood count results in all) that might have influenced the primary outcome parameter, CADP-CT. Patients with concomittant aspirin treatment were excluded.
Laboratory Analysis PFA-100 System The PFA-100 system measures in vitro bleeding time. Citrated blood flows through a membrane (at 5000 – 6000 s⫺1) coated with collagen and epinephrine (EPI) or ADP, respectively. Platelets become activated by EPI or ADP, adhere to the collagen on the membrane, and form a plug that progressively occludes the membrane. The time to occlusion is an indication of primary platelet aggregation and hemostatic activity known as the PFA closure time (CT). CT values are given in seconds. The system also measures 2 other parameters, flow rate (L/min) and total flow volume (L), from which the PFA predictive index (PI) can be calculated. The PI provides additional
Table 1. Demographic data, closure times, and von Willebrand factor levels of 16 stroke patients on clopidogrel treatment (75 mg/day) Stroke patients on clopidogrel treatment
Number of patients Male Female Age (years) Height (cm) Weight (kg) Mean duration of treatment (weeks) CEPI-CT (s) CEPI-PI CADP-CT (s) CADP-PI vWF (%) Platelet count (cells/L) Hematocrit (%)
⬍3 weeks
⬎3 weeks
5 2 2 67 ⫾ 7 174 ⫾ 6 72 ⫾ 8 2.2 ⫾ 0.5
11 9 3 67 ⫾ 4 172 ⫾ 2 80 ⫾ 2 110 ⫾ 34
16 11 5 67 ⫾ 3 173 ⫾ 2 77 ⫾ 3 76 ⫾ 26
164 ⫾ 37 0.6 ⫾ 0.1 102 ⫾ 26 0.5 ⫾ 0.05 156 ⫾ 41 227 ⫾ 12
158 ⫾ 16 0.6 ⫾ 0.02 102 ⫾ 9 0.5 ⫾ 0.03 152 ⫾ 19 219 ⫾ 14
160 ⫾ 15 0.6 ⫾ 0.02 102 ⫾ 10 0.5 ⫾ 0.03 153 ⫾ 17 222 ⫾ 10
39 ⫾ 2
41 ⫾ 2
Overall
40 ⫾ 1
Data are presented as mean ⫾ SEM.
information on real platelet function, especially when CT exceeds the measurable limit of 300 seconds. The PI is calculated as described previously.7 Measurement of Plasma von Willebrand Factor Antigen Levels Blood samples were drawn 2 hours after the last clopidogrel dose. Blood was drawn from an appropriate forearm vein with a 21-gauge butterfly needle into siliconized glass tubes (Vacutainer; Becton Dickinsor Franklin Lakes, NJ) with 0.129 mol/L of buffered sodium citrate (equivalent to 3.8% sodium citrate). The first 3 mL was discarded to avoid stasis induced changes in plasma von Willebrand factor antigen (vWF-Ag) levels. Citrated plasma was obtained by centrifugation for 15 minutes at 2000 g and 4°C. Plasma was stored at ⫺80°C until analysis. vWF concentrations were measured by enzyme immunoassay (Asserachrom vWF; Roche Diagnostics, Vienna, Austria).
Statistical Analysis Descriptive statistics were used to describe treatment effects, demographics, and laboratory values. Data are presented as mean and standard error of the mean (SEM). Spearman’s test was used for nonparametric correlations. The level of significance was set at P ⬍ .05.
STROKE THERAPY AND PFA-100
Results Demographic data for the 16 patients are given in Table 1. There were no significant differences between the shortterm– and long-term–treated patients.
PFA-100 Closure Times Normal CT values are ⬍160 seconds for CEP-CT and ⬍120 seconds for CADP-CT (according to the manufacturers’ instructions; Dade Behring, Vienna, Austria). CADP-CT exceeded the upper-normal range in 4 patients (155 ⫾ 19 seconds; range, 122–203 seconds). Overall, CADP-CT values were within the normal range in stroke patients under clopidogrel treatment (102 ⫾ 26 seconds in patients treated with clopidogrel for ⬍3 weeks and 102 ⫾ 9 seconds in those treated for ⬎3 weeks). CEPI-CT exceeded the upper-normal range in 7 patients (209 ⫾ 24 seconds; range, 163–300 seconds). Overall, CEPI-CT values were minimally prolonged or in the normal range in stroke patients under clopidogrel treatment (164 ⫾ 37 seconds in patients treated with clopidogrel for ⬍3 weeks and 158 ⫾ 16 seconds in those treated for ⬎3 weeks). CEPI-CT and CADP-CT correlated positively (Spearman’s r ⫽ .73; P ⫽ .012).8
201
troversial. In one study, PFA-100 CTs were not considered a clear indicator in this context, at least in healthy volunteers.6 In contrast, another study found that, the PFA-100 system was sensitive to clopidogrel effects in a subset of stroke patients.11 Another recent study reported a considerable time lag before relevant clopidogrelinduced antiplatelet effects could be measured in stroke patients.12 With respect to a possible delay in the detection of antiaggregatory effects, we further analyzed our data and stratified the study population into 2 groups; stroke patients treated with clopidogrel for ⬍3 weeks and ⬎3 weeks (short-term vs long-term therapy). Of our 16 patients, 4 had prolonged CADP-CT values and 7 had prolonged CEPI-CT values. This indicates respective sensitivities of 25% and 44% for detecting clopidogrel intake. In contrast, Raman et al12 showed antiplatelet effects of clopidogrel with the PFA-100 in a before after design. Evidently, this is not possible with our cross-sectional design, although it more closely reflects clinical practice. This is the first stroke population on clopidogrel therapy in which both CT and vWF-Ag levels were measured. A negative correlation between CT and vWF-Ag concentration have been reported in other populations (Fig 1).13 The present study confirms the inverse correla-
vWF-Ag vWF-Ag levels were elevated (⬎150%) in 7 patients (213 ⫾ 24%; range, 154 –308%). There was an inverse correlation between CT and vWF-Ag levels (CEPI-CT: Spearman’s r ⫽ .71, P ⫽ .002; CADP-CT: Spearman’s r ⫽ .52, P ⫽ .04).8,9
Hematocrit Hematocrit and platelet counts did not correlate with CT values in this population.
Discussion Our findings indicate that the current cartridges of the PFA-100 system are not sufficiently sensitive for monitoring clopidogrel’s antiplatelet effects in a daily routine in stroke patients. Stroke recurrence is up to 40% within the first 5 years,10 for which therapeutic failures of antiaggregtory therapy could be at least partly responsible. Plasma levels of clopidogrel cannot be routinely measured to monitor and enhance patient compliance. Methods to determine clopidogrel’s therapeutic efficacy are not available in most neurologic centers. Finally, the possible risk of stroke recurrence or bleeding cannot be quantified in individual stroke patients. Consequently, there is a need for an instrument to help optimize secondary stroke prevention. Data on the effectiveness of the PFA-100 system in detecting clopidogrel-induced platelet inhibition are con-
Figure 1. Negative correlation between vWF levels (in %) and CADP-CT (P ⫽ .04) (A) and CEPI-CT in seconds (P ⬍ .002) (B). The horizontal dashed-dotted lines in both graphs indicate the cutoff level for CADP-CT (120s) and CADP-CT (160s). The vertical dotted lines in both graphs indicate the cutoff level for vWF (150%).
N. KOTZAILIAS ET AL.
202 8
tion not only with CEPI-CT, but also, to a lesser degree, with CADP-CT in stroke patients (see Fig 1). The PFA-100 CTs strongly depend on vWF-Ag levels. Platelets bind to vWF-Ag through the gplb receptor under high shear stress. vWF-Ag has high affinity to collagen and thereby provides a bridge for the primary thrombus. Therefore, decreased vWF-Ag levels (in, eg, von Willebrand disease) or increased concentrations (as found in, eg, severe atherosclerosis) shorten or prolong CTs. vWF-Ag levels were above the the upper normal range in 7 of our 16 patients. Therefore, the failure of the PFA-100 system to detect clopidogrel’s platelet inhibition effect could be due to increased vWF-Ag levels in approximately 44% of our stroke patients. A recent report suggests that the current PFA-100 cartridges could be modified to enable the detection of P2Y12 inhibition.14 Such modified cartridges are currently in clinical evaluation. Interestingly, even within the normal ranges of the endothelial marker vWF-Ag, we found an inverse correlation with platelet counts. Previous reports described this relationship in patients with thrombocytopenia or myeloproliferative disorders and suggested an association with abnormal vWF multimers.15,16 However, this observation requires further study in stroke patients. Our study has some obvious limitations. We cannot exclude the possibility that we detected therapeutic failures only, although this seems unlikely. The medication was taken under direct observation on the study day; however, drug intake previous to that study day was not controlled in all patients. Unfortunately, it is not possible to routinely measure clopidogrel plasma levels to confirm compliance with drug therapy. In conclusion, our data indicate that the current PFA100 system cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition. Modified cartridges are currently in clinical development. Acknowledgment: We thank Prof. Dr. Bernd Jilma, expert in the field of platelet research (Department of Clinical Pharmacology, Medical University of Vienna), for proofreading the manuscript.
References 1. Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet ther-
2.
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4.
5.
6.
7.
8.
9.
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11.
12.
13.
14.
15.
16.
apy for prevention of death, myocardial infarction and stroke in high-risk patients. BMJ 2002;324:71-86. Antiplatelet Trialists’ Collaboration. Collaborative overview of randomised trials of antiplatelet therapy, I: prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. BMJ 1994;308:81-106. Grundmann K, Jaschonek K, Kleine B, et al. Aspirin non-responder status in patients with recurrent cerebral ischemic attacks. J Neurol 2003;250:63-66. Muller I, Besta F, Scholz C, et al. Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placement. Thromb Haemost 2003;89:783-787. Crowe B, Abbas S, Meany B, et al. Detection of aspirin resistance by PFA-100: prevalence and aspirin compliance in patients with chronic stable angina. Semin Thromb Haemost 2005;31:420-425. Geiger J, Teichmann L, Grossmann R, et al. Monitoring of clopidogrel action: comparison of methods. Clin Chem 2005;51:957-965. Borzini P, Lazzaro A, Mazzucco L. Evaluation of the hemostatic function of stored platelet concentrates using the platelet function analyzer (PFA-100). Haematologica 1999;84:1104-1109. Homoncik M, Jilma B, Hergovich N, et al. Monitoring of aspirin (ASA) pharmacodynamics with the platelet function analyzer PFA-100. Thromb Haemost 2000;83:316321. Nitu-Whalley IC, Lee CA, Brown SA, et al. The role of the platelet function analyser (PFA-100™) in the characterization of patients with von Willebrand’s disease and its relationship with von Willebrand factor and the ABO blood group. Haemophilia 2003;9:298-302. Burn J, Dennis M, Bamford J, et al. Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community Stroke Project. Stroke 1994;25:333-337. Grau AJ, Reiners S, Lichy C, et al. Platelet function under aspirin, clopidogrel and both after ischemic stroke: a cross-over study. Stroke 2003;34:849-854. Raman S, Jilma B. Time lag in platelet function inhibition by clopidogrel in stroke patients as measured by PFA100. J Thromb and Haemost 2004;2:2278-2279. Bemardo A, Bergeron AL, Sun CW, et al. Von Willebrand factor present in fibrillar collagen anhances platelet adhesion to collagen and collagen-induced platelet aggregation. J Thromb Haemost 2004;2:660-669. Pidcock M, Harrison P. Can the PFA-100 be modified to detect P2Y12 inhibition? J Thromb Haemost 2006;4:14241426. Budde U, Scharf RE, Franke P, et al. Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma. Blood 1993;82:17491757. Casonato A, Fabris F, Boscaro M, et al. Increased factor VIII/vWf levels in patients with reduced platelet number. Blut 1987;54:281-288.
The administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, is recommended for recurrent stroke patients under aspirin treatment. However, up to 25% of vascular patients have an inadequate response to clopidogrel treatment, which could be associated with increased reinfarction rates. This study investigated whether the platelet function analyzer (PFA-100) system represents an appropriate tool for monitoring clopidogrel’s antiplatelet effects in stroke patients. Sixteen stroke patients on clopidogrel therapy (75 mg/day) were included in a prospective analyst– blinded, cross-sectional study. Platelet function was assayed by collagen/epinephrine (CEPI)- and collagen/ADP (CADP)-induced closure times (CTs) using the PFA-100 system. von Willebrand factor antigen (vWF-Ag) levels were measured by enzyme immunoassay. CEPI-CT and CADP-CT values averaged 160 ⫾ 15 seconds and 102 ⫾ 10 seconds, respectively, and were in the normal range. vWF-Ag concentrations averaged 153 ⫾ 17% and correlated inversely with CTs (r ⫽ .71; P ⬍ .002 for CEPI-CT, r ⫽ .54; P ⬍ .04 for CADP-CT). Our data indicate that the current PFA-100 cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition in stroke patients. Key Words: Clopidogrel—PFA-100 —platelet function—stroke. © 2007 by National Stroke Association
Along with myocardial infarction, stroke represents one of the most important causes of death in Europe. The rate of recurrent stroke is 40% within 5 years after the acute event with up to 16% recurrence within the first year. Acetylic salicylic acid (aspirin) is the first-line anti-
From the *Department of Neurology and †Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Austria. Received October 26, 2006; accepted May 1, 2007. Supported by a research grant from the Medizinisch-Wissenschaftlicher Fonds des Buergermeisters der Bundeshauptstadt Wien (BGM project 2302). Address reprint requests to Nicole Kotzailias, MD, Department of Neurology, Medical University Vienna, Währinger Gürtel 18 –20, A-1090 Vienna, Austria. E-mail: [email protected] 1052-3057/$—see front matter © 2007 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2007.05.001
platelet therapy in secondary stroke prevention and reduces the relative risk for cerebrovascular events by approximately 23%.1,2 However, recently up to 40% of patients with recurrent ischemic stroke were identified as aspirin nonresponders.3 The stroke consensus statement (Statement for Healthcare Professionals from the American Heart Association/American Stroke Association Council on Stroke, 2006) recommends the administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, in recurrent stroke under aspirin treatment. Similar to aspirin resistence, an inadequate response to clopidogrel treatment occurs in up to 25% of cardiovascular patients,4 which could be associated with increased reinfarction rates. Thus, it may be useful to identify nonresponders in whom secondary stroke prevention can be optimized by changing to an alternative treatment regime. The platelet function analyzer (PFA-100) system is effective in detect-
Journal of Stroke and Cerebrovascular Diseases, Vol. 16, No. 5 (September-October), 2007: pp 199-202
199
N. KOTZAILIAS ET AL.
200
ing aspirin nonresponders by measuring collagen/epinephrine-induced closure time (CEPI-CT).5 In contrast, only cost- and time-inefficient methods for the detection of clopidogrel-induced platelet inhibition are available: thromboelastography, aggregometry, and flow cytometry, which can be conducted in specialized centers only.6 The PFA-100 system can provide an easy, cheap, and time-saving tool for the detection of clopidogrel’s induced platelet inhibition in a daily routine. To examine the efficacy of the PFA-100 system in detecting clopidogrel’s antiaggregatory effect, we measured collagen/ ADP-induced closure time (CADP-CT) in 16 stroke patients on clopidogrel therapy.
Methods Study Design The protocol and the consent form were approved by the Ethics Committee of the Medical University of Vienna. Written informed consent was obtained from each participant before entry into the study. The study was conducted in accordance with the Declaration of Helsinki and was designed as a prospective analyst– blinded cohort trial. The stroke patients were on clopidogrel therapy at a dose of 75 mg/day for secondary stroke prevention for a minimum of 1 week before study entry. The blood sample was drawn 2 hours after the last clopidogrel dose.
Patients Sixteen patients with a history of stroke treated with clopidogrel (Plavix) 75 mg/day with a mean age of 67 ⫾ 3 years were included in the study (Table 1). All 16 patients were free of infection and serious illness (with normal clinical chemistry and routine blood count results in all) that might have influenced the primary outcome parameter, CADP-CT. Patients with concomittant aspirin treatment were excluded.
Laboratory Analysis PFA-100 System The PFA-100 system measures in vitro bleeding time. Citrated blood flows through a membrane (at 5000 – 6000 s⫺1) coated with collagen and epinephrine (EPI) or ADP, respectively. Platelets become activated by EPI or ADP, adhere to the collagen on the membrane, and form a plug that progressively occludes the membrane. The time to occlusion is an indication of primary platelet aggregation and hemostatic activity known as the PFA closure time (CT). CT values are given in seconds. The system also measures 2 other parameters, flow rate (L/min) and total flow volume (L), from which the PFA predictive index (PI) can be calculated. The PI provides additional
Table 1. Demographic data, closure times, and von Willebrand factor levels of 16 stroke patients on clopidogrel treatment (75 mg/day) Stroke patients on clopidogrel treatment
Number of patients Male Female Age (years) Height (cm) Weight (kg) Mean duration of treatment (weeks) CEPI-CT (s) CEPI-PI CADP-CT (s) CADP-PI vWF (%) Platelet count (cells/L) Hematocrit (%)
⬍3 weeks
⬎3 weeks
5 2 2 67 ⫾ 7 174 ⫾ 6 72 ⫾ 8 2.2 ⫾ 0.5
11 9 3 67 ⫾ 4 172 ⫾ 2 80 ⫾ 2 110 ⫾ 34
16 11 5 67 ⫾ 3 173 ⫾ 2 77 ⫾ 3 76 ⫾ 26
164 ⫾ 37 0.6 ⫾ 0.1 102 ⫾ 26 0.5 ⫾ 0.05 156 ⫾ 41 227 ⫾ 12
158 ⫾ 16 0.6 ⫾ 0.02 102 ⫾ 9 0.5 ⫾ 0.03 152 ⫾ 19 219 ⫾ 14
160 ⫾ 15 0.6 ⫾ 0.02 102 ⫾ 10 0.5 ⫾ 0.03 153 ⫾ 17 222 ⫾ 10
39 ⫾ 2
41 ⫾ 2
Overall
40 ⫾ 1
Data are presented as mean ⫾ SEM.
information on real platelet function, especially when CT exceeds the measurable limit of 300 seconds. The PI is calculated as described previously.7 Measurement of Plasma von Willebrand Factor Antigen Levels Blood samples were drawn 2 hours after the last clopidogrel dose. Blood was drawn from an appropriate forearm vein with a 21-gauge butterfly needle into siliconized glass tubes (Vacutainer; Becton Dickinsor Franklin Lakes, NJ) with 0.129 mol/L of buffered sodium citrate (equivalent to 3.8% sodium citrate). The first 3 mL was discarded to avoid stasis induced changes in plasma von Willebrand factor antigen (vWF-Ag) levels. Citrated plasma was obtained by centrifugation for 15 minutes at 2000 g and 4°C. Plasma was stored at ⫺80°C until analysis. vWF concentrations were measured by enzyme immunoassay (Asserachrom vWF; Roche Diagnostics, Vienna, Austria).
Statistical Analysis Descriptive statistics were used to describe treatment effects, demographics, and laboratory values. Data are presented as mean and standard error of the mean (SEM). Spearman’s test was used for nonparametric correlations. The level of significance was set at P ⬍ .05.
STROKE THERAPY AND PFA-100
Results Demographic data for the 16 patients are given in Table 1. There were no significant differences between the shortterm– and long-term–treated patients.
PFA-100 Closure Times Normal CT values are ⬍160 seconds for CEP-CT and ⬍120 seconds for CADP-CT (according to the manufacturers’ instructions; Dade Behring, Vienna, Austria). CADP-CT exceeded the upper-normal range in 4 patients (155 ⫾ 19 seconds; range, 122–203 seconds). Overall, CADP-CT values were within the normal range in stroke patients under clopidogrel treatment (102 ⫾ 26 seconds in patients treated with clopidogrel for ⬍3 weeks and 102 ⫾ 9 seconds in those treated for ⬎3 weeks). CEPI-CT exceeded the upper-normal range in 7 patients (209 ⫾ 24 seconds; range, 163–300 seconds). Overall, CEPI-CT values were minimally prolonged or in the normal range in stroke patients under clopidogrel treatment (164 ⫾ 37 seconds in patients treated with clopidogrel for ⬍3 weeks and 158 ⫾ 16 seconds in those treated for ⬎3 weeks). CEPI-CT and CADP-CT correlated positively (Spearman’s r ⫽ .73; P ⫽ .012).8
201
troversial. In one study, PFA-100 CTs were not considered a clear indicator in this context, at least in healthy volunteers.6 In contrast, another study found that, the PFA-100 system was sensitive to clopidogrel effects in a subset of stroke patients.11 Another recent study reported a considerable time lag before relevant clopidogrelinduced antiplatelet effects could be measured in stroke patients.12 With respect to a possible delay in the detection of antiaggregatory effects, we further analyzed our data and stratified the study population into 2 groups; stroke patients treated with clopidogrel for ⬍3 weeks and ⬎3 weeks (short-term vs long-term therapy). Of our 16 patients, 4 had prolonged CADP-CT values and 7 had prolonged CEPI-CT values. This indicates respective sensitivities of 25% and 44% for detecting clopidogrel intake. In contrast, Raman et al12 showed antiplatelet effects of clopidogrel with the PFA-100 in a before after design. Evidently, this is not possible with our cross-sectional design, although it more closely reflects clinical practice. This is the first stroke population on clopidogrel therapy in which both CT and vWF-Ag levels were measured. A negative correlation between CT and vWF-Ag concentration have been reported in other populations (Fig 1).13 The present study confirms the inverse correla-
vWF-Ag vWF-Ag levels were elevated (⬎150%) in 7 patients (213 ⫾ 24%; range, 154 –308%). There was an inverse correlation between CT and vWF-Ag levels (CEPI-CT: Spearman’s r ⫽ .71, P ⫽ .002; CADP-CT: Spearman’s r ⫽ .52, P ⫽ .04).8,9
Hematocrit Hematocrit and platelet counts did not correlate with CT values in this population.
Discussion Our findings indicate that the current cartridges of the PFA-100 system are not sufficiently sensitive for monitoring clopidogrel’s antiplatelet effects in a daily routine in stroke patients. Stroke recurrence is up to 40% within the first 5 years,10 for which therapeutic failures of antiaggregtory therapy could be at least partly responsible. Plasma levels of clopidogrel cannot be routinely measured to monitor and enhance patient compliance. Methods to determine clopidogrel’s therapeutic efficacy are not available in most neurologic centers. Finally, the possible risk of stroke recurrence or bleeding cannot be quantified in individual stroke patients. Consequently, there is a need for an instrument to help optimize secondary stroke prevention. Data on the effectiveness of the PFA-100 system in detecting clopidogrel-induced platelet inhibition are con-
Figure 1. Negative correlation between vWF levels (in %) and CADP-CT (P ⫽ .04) (A) and CEPI-CT in seconds (P ⬍ .002) (B). The horizontal dashed-dotted lines in both graphs indicate the cutoff level for CADP-CT (120s) and CADP-CT (160s). The vertical dotted lines in both graphs indicate the cutoff level for vWF (150%).
N. KOTZAILIAS ET AL.
202 8
tion not only with CEPI-CT, but also, to a lesser degree, with CADP-CT in stroke patients (see Fig 1). The PFA-100 CTs strongly depend on vWF-Ag levels. Platelets bind to vWF-Ag through the gplb receptor under high shear stress. vWF-Ag has high affinity to collagen and thereby provides a bridge for the primary thrombus. Therefore, decreased vWF-Ag levels (in, eg, von Willebrand disease) or increased concentrations (as found in, eg, severe atherosclerosis) shorten or prolong CTs. vWF-Ag levels were above the the upper normal range in 7 of our 16 patients. Therefore, the failure of the PFA-100 system to detect clopidogrel’s platelet inhibition effect could be due to increased vWF-Ag levels in approximately 44% of our stroke patients. A recent report suggests that the current PFA-100 cartridges could be modified to enable the detection of P2Y12 inhibition.14 Such modified cartridges are currently in clinical evaluation. Interestingly, even within the normal ranges of the endothelial marker vWF-Ag, we found an inverse correlation with platelet counts. Previous reports described this relationship in patients with thrombocytopenia or myeloproliferative disorders and suggested an association with abnormal vWF multimers.15,16 However, this observation requires further study in stroke patients. Our study has some obvious limitations. We cannot exclude the possibility that we detected therapeutic failures only, although this seems unlikely. The medication was taken under direct observation on the study day; however, drug intake previous to that study day was not controlled in all patients. Unfortunately, it is not possible to routinely measure clopidogrel plasma levels to confirm compliance with drug therapy. In conclusion, our data indicate that the current PFA100 system cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition. Modified cartridges are currently in clinical development. Acknowledgment: We thank Prof. Dr. Bernd Jilma, expert in the field of platelet research (Department of Clinical Pharmacology, Medical University of Vienna), for proofreading the manuscript.
References 1. Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet ther-
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
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