Original article 869
Changes in response to antiaggregatory treatment in patients with myeloproliferative neoplasms: a sequential study using multiple electrode aggregometry Christoph Robier, Mariana Stettin and Manfred Neubauer In the present study, we used multiple electrode aggregometry (MEA) to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with myeloproliferative neoplasms (MPNs). arachidonic acid (ASPI), ADP, and thrombin receptor activating peptide (TRAP) tests were performed at two timepoints between 32–50 months in 21 patients with MPN and 1–46 months in 29 controls. We further checked the medical records of the participants to identify a potential correlation of changes in the treatment response with clinical events. In MPN, four out of 13 patients treated with 100 mg of aspirin, no patients receiving 50 mg of aspirin, and one out of five clopidogrel-treated patients showed a therapeutic antiplatelet effect. In the subsequent examinations, five patients changed from response to nonresponse or vice versa. Initial nonresponse and changes from an initial response to nonresponse were observed in six patients with thrombotic events. In the controls, 25 out of 26 aspirin-treated patients and two out of three clopidogreltreated patients showed an initially adequate in-vitro response. Except from one patient changing from initial
Introduction Thrombotic and hemorrhagic episodes are common disease-related complications of myeloproliferative neoplasms (MPNs), such as polycythemia vera and essential thrombocythemia, and thereby represent the major causes of mortality and morbidity [1]. Thrombosis is usually independent of the platelet count and antiaggregatory treatment [2], whereas hemorrhage is frequently associated with platelet counts over 1500 109/l [3] and treatment with antiaggregatory drugs, such as acetylsalicylic acid (ASA) or clopidogrel [4]. Furthermore, various types of hypofunctional and hyperfunctional platelet abnormalities have been described in the literature. Several laboratory studies using different techniques, such as optical aggregometry [5,6], whole-blood platelet lumi-aggregometry [7], or the platelet function analyzer (PFA)-100 [6,8], have been performed to detect and characterize such functional abnormalities, in order to relate these in-vitro phenomena with the clinical events. Thereby, in-vitro platelet function tests have generally failed to identify patients at risk for thrombotic or hemorrhagic complications [9], which has been attributed to the coincidence of diverse and sometimes also oppositional defects in the same patient, changes of functional characteristics during the disease course, to the effects of antiaggregatory treatment and other nonplatelet-derived 0957-5235 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
aspirin nonresponse to response, all controls showed a stable response state. One control with two ischemic strokes showed a nonresponse to clopidogrel. In conclusion, MEA detects the response to antiaggregatory treatment, as well as its changes during the disease course in patients with MPN. An initial or subsequent nonresponse was observed in patients with thrombotic events. Blood Coagul Fibrinolysis 24:869–873 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2013, 24:869–873 Keywords: acetylsalicylic acid, clopidogrel, multiplate analyzer, myeloproliferative neoplasms, platelet function Central Laboratory, Hospital Barmherzige Brueder Graz-Eggenberg, Graz, Austria Correspondence to Christoph Robier, MD, Central Laboratory, Hospital Barmherzige Brueder, Graz-Eggenberg, Bergstrasse 27, A-8020 Graz, Austria Tel: +43 316 5989 6671; fax: +43 316 5989 1505; e-mail: [email protected] Received 12 April 2013 Revised 20 June 2013 Accepted 20 June 2013
influences [10,11]. In the clinical setting, it would be beneficial to establish valid techniques for the monitoring of antiaggregatory treatment in patients with MPN, in order to identify nonresponders and thus to prevent associated thrombotic events. Both the PFA-100 [6,8] and multiple electrode aggregometry (MEA) [12] were confirmed as rapid and reliable methods for the monitoring of ASA, whereas MEA was also regarded as appropriate for the monitoring of clopidogrel treatment [12] in patients with polycythemia vera and essential thrombocythemia. On the basis of the fact that functional platelet abnormalities in patients with MPN can change during the disease course [11], we hypothesized that changes may also occur in response to antiplatelet therapy with time. Such changes may represent temporary risk factors for thrombosis and bleeding, and may thus be of clinical relevance. In the present study, we used MEA to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with polycythemia vera and essential thrombocythemia.
Materials and methods Study population
We studied samples from 21 patients with MPN (polycythemia vera, n ¼ 15; essential thrombocythemia, n ¼ 6) DOI:10.1097/MBC.0b013e328364713a
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
870 Blood Coagulation and Fibrinolysis 2013, Vol 24 No 8
from the hematologic outpatient clinic and the Department of Internal Medicine of the Hospital Barmherzige Brueder in Graz-Eggenberg, Austria. There were 14 women and seven men, ranging from 44 to 90 years. The majority of the study participants were diagnosed according to the criteria of the World Health Organization from 2001 [13]. Four patients with polycythemia vera were tested for the JAK2 V617F mutation and were all positive, whereas one of three tested patients with essential thrombocythemia showed this mutation. All included patients received long-time treatment with ASA and clopidogrel because of medical indication and confirmed their compliance with therapy prior to blood collection. The detailed basic and clinical characteristics, including further data on the disease course, cardiovascular and thrombophilic risk factors, the occurrence of bleeding and thrombotic events, and the antiaggregatory dosage regimens of the study population, are presented in Table 1. In order to determine control individuals without MPN, we retrospectively viewed our laboratory database for patients who had routine sequential examinations by MEA within the study period. We identified 29 controls (ASA 100 mg, n ¼ 26; clopidogrel 75 mg, n ¼ 3), who were characterized as follows: median age 75 years (52–95), female sex n ¼ 22 (76%), and median interval between the two examinations 16 months (1–46). Patients with thrombocytopenia (platelet count 2 mg/dl), renal insufficiency (serum creatinine level >2 mg/dl), monoclonal gammopathy, as well as patients who received other antithrombotic drugs than ASA and clopidogrel, nonsteroidal anti-inflammatory drugs, and corticosteroids within 2 weeks prior to the Clinical characteristics and demographics of the patients with myeloproliferative neoplasms
Table 1
Demographics Study participants, n Age in yearsa Female sex, n (%) Disease-related characteristics Myeloproliferative neoplasm Essential thrombocythemia, n (%) Polycythemia vera, n (%) Disease course in yearsa Observation period in monthsa Clinical events, n (%) Bleeding episodes Thrombotic events Platelet count (109/l)a Hematocrit (%)a Antiaggregatory treatment, n (%) ASA 100 mg ASA 50 mg Clopidogrel 75 mg ASA 100 mg þ clopidogrel 75 mg Cardiovascular risk factors, n (%) Arterial hypertension Dyslipidemia Diabetes mellitus Hereditary thrombophilia (protein S deficiency) ASA, acetylsalicylic acid.
a
Median (range).
21 71 (44–90) 14 (66.66)
6 15 10 40 6 7 424 48
(28.57) (71.43) (3–32) (32–50) (28.57) (33.33) (195–919) (36–57)
11 5 3 2
(52.38) (23.81) (14.29) (9.52)
11 11 4 1
(53.38) (53.38) (19) (4.76)
blood drawings, were excluded from the study to eliminate potential bias because of secondary thrombocytopathy. Blood sampling
Venous blood samples were collected with a 21-gauge needle, the first at least 3 ml were discarded. MEA was performed within 1.5 h after blood collection. In MPN patients, blood specimens were obtained at two timepoints as follows: at study entry and after a median time period of 40 months (range 32–50 months). Prior to each venipuncture, written informed consent was obtained from all study participants in accordance with the Declaration of Helsinki. This study was approved by the institutional ethics committee. Materials and platelet aggregation studies
For aggregation testing, hirudin-anticoagulated blood (25 mg/ml hirudin; Sarstedt, Nuembrecht, Germany) was used; platelet aggregation was measured from whole blood using MEA on the Multiplate analyzer (Roche Diagnostics GmbH, Vienna, Austria) according to the manufacturer’s instructions. Details of this method have been described previously [14]. We performed three test procedures with different agonists on each sample: ASPI test (arachidonic acid, 0.5 mmol/l), ADP test (adenosine diphosphate, 6.5 mmol/l), and TRAP test (thrombin receptor activating peptide, TRAP-6, 32 mmol/l). In MEA, after adding the agonist, impedance changes, representing platelet adhesion on the paired electrodes, are detected and recorded for 6 min. Platelet aggregation is quantified by the area under the aggregation curve, expressed as arbitrary units and converted to units (1 U ¼ 10 arbitrary units min) [14]. The reference range of MEA using hirudinized whole blood is 75–136 U in the ASPI test, 53–122 U in the ADP test, and 95–156 U in the TRAP test [15]. A cutoff value of less than 30 U in the ASPI test was determined for a therapeutic antiplatelet effect of ASA, whereas values less than 45 U in the ADP test were defined as a sufficient response to clopidogrel [16].
Results The complete results of the platelet aggregation studies are presented in Table 2 for the study participants with MPN and in Supplemental Table 1 for the controls. Response to antiaggregatory treatment
In the MPN group, four out of 13 patients (31%) treated with 100 mg of ASA, but no patients receiving a reduced dose of 50 mg of ASA because of medical indication, initially had ASPI values less than 30 U, being consistent with a response. In the subsequent examinations in the 100 mg group, three patients turned to a nonresponder state, one patient did not change, and two initially nonresponders showed a response. One patient treated with 50 mg, who was identified as a nonresponder at the first
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Aggregation changes in myeloproliferative neoplasms Robier et al. 871
Table 2
Aggregation data and clinical events of the study participants with myeloproliferative neoplasms First analysis, U
Drug, dose, and patients ASA 100 mg 1 2 3 4 5 6 7 8 9 10 11 ASA 50 mg 1 2 3 4 5 Clop 75 mg 1 2 3 ASA 100 mg þ clop 75 mg 1 2
Sequential analysis, U Clinical event
ASPI
ADP
TRAP
ASPI
ADP
TRAP
51 34 24 32 50 29 58 64 97 122 47
131 136 112 114 64 89 124 111 93 156 126
127 122 129 109 72 122 131 116 102 131 129
26 8 61 66 79 25 77 89 99 71 74
118 174 100 124 133 79 114 114 51 106 168
138 130 126 122 126 132 117 123 95 131 186
42 111 134 100 100
70 134 166 103 86
96 132 158 105 104
48 116 126 114 13
118 123 171 96 72
113 110 147 101 110
Ischemic stroke MCI þ hemorrhage Bleeding diathesis
101 102 106
84 67 92
100 139 115
97 100 97
73 47 75
82 118 111
Upper GI bleeding Upper GI bleeding Ischemic stroke
12 18
87 27
97 121
50 32
81 26
100 50
MCI
TIA
Splenic infarction
Epistaxis
ASA, acetylsalicylic acid; ASPI, arachidonic acid; clop, clopidogrel; GI, gastrointestinal; MCI, myocardial infarction; TIA, transient ischemic attack; TRAP, thrombin receptor activating peptide; U, units.
timepoint, had a result below the cutoff at the second timepoint and thus fulfilled the defined criterion of response. One out of five clopidogrel-treated patients had an initial response and was confirmed as a responder at the second evaluation. In the control group, 25 out of 26 ASA-treated patients (96%) and two out of three clopidogrel-treated patients (66%) showed an initial response. In the sequential examinations, all of the 26 ASA-treated patients yielded a sufficient response and the clopidogrel-treated patients did not change in their response patterns. Thrombotic and hemorrhagic events
Overall, in a review of the medical records of the 21 study participants with MPN, 11 clinical events were observed, with six thrombotic and five bleeding episodes. One patient developed both thrombotic and hemorrhagic events during the disease course. Thereby, except from one patient with a splenic infarction and one with a general enhanced bleeding diathesis who endured their events between the two measurements, all the other participants’ clinical events occurred prior to the study period. In case of the splenic infarction, a protein S deficiency has been found as an additional thrombophilic risk factor. An initial nonresponse and changes from an initial response to a nonresponse to ASA or clopidogrel were found in six of 10 patients with arterial or venous thrombotic events. One patient with a bleeding event showed a change from an initial ASA nonresponse to a response at
the second measure point. A trend toward an association with the occurrence of hemorrhagic complications was not found. All clinical episodes of the MPN patients and its association with MEA results are shown in Table 2. All patients who endured myocardial infarctions, a transient ischemic attack, or ischemic strokes had one or more additional cardiovascular risk factors, such as diabetes mellitus, arterial hypertension, and dyslipidemia. In the medical records of the controls, eight arterial thromboses (myocardial infarction, n ¼ 3; ischemic stroke, n ¼ 5), but no bleeding or venous thrombotic events, were found. Except from one patient who was defined as a clopidogrel nonresponder, and endured two ischemic strokes, all the other patients with vascular events showed a sufficient response to ASA or clopidogrel. In those patients, other than solely platelet-associated risk factors may have contributed to the clinical events.
Discussion In the present study, we confirmed that MEA is able to detect responses as well as nonresponses to antiaggregatory treatment with ASA and clopidogrel in patients with polycythemia vera and essential thrombocythemia. To our knowledge, this is the first study in which sequential evaluations of platelet aggregation in MPN were performed by MEA, and thereby showed changes in the drug responses during the disease course. Our findings correspond well with a prior study, highlighting that changes in the platelet function over time
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872 Blood Coagulation and Fibrinolysis 2013, Vol 24 No 8
are frequently occurring in patients with MPN [11]. In light of that trial and our current data, MEA may represent a useful tool in the management of patients with MPN and it may thus be beneficial to re-evaluate the platelet function of patients with MPN during the further course of the disease. One main goal of platelet function testing is the early identification of nonresponders to antiaggregatory drugs, in order to prevent thrombotic events and thus to avoid associated morbidity and mortality. In several study populations with cardiovascular diseases, low responses to antiplatelet agents assessed with MEA were significantly associated with an increased risk of stent thrombosis [17–19]. In MPN, previous studies using different devices frequently showed various platelet function abnormalities in vitro, but failed to identify a correlation with the hemostatic complications [1–3,9,10]. In the current study, only a few patients treated with ASA and clopidogrel showed an adequate initial or sustained response to treatment. We observed that all of the six patients who endured an arterial or venous thrombotic episode such as myocardial infarction, ischemic stroke, or splenic infarction during their disease course were at least at one timepoint of the study period classified as nonresponders according to the defined cutoffs. In consideration of the fact that only one of 29 control individuals developed a change in the response state, the observed treatment response changes in the study participants with polycythemia vera and essential thrombocythemia support the hypothesis of a disease-related phenomenon. However, because of the small number of clinical events, the coincidence with further cardiovascular and thrombophilic risk factors, and because of the fact that only two events occurred between the two MEA measurements, further prospective studies using MEA in a larger MPN population are needed to prove a correlation between changes in platelet aggregation and thrombotic and hemorrhagic events. This study had limitations that merit mention. First, the interval between the two examinations in the MPN group was quite long (median 40 months, range 32–50 months). Hence, further changes in platelet aggregation emerging within that time period may have been missed. Second, the MPN group and the controls were not matched in sex and age. In our opinion, that does not add a significant bias to our trial, because several previous studies clearly showed that age and sex do not exert a significant influence on MEA tests in patients with MPN [12] and healthy individuals [20]. Third, we did not focus on platelet function abnormalities in our population, because of our study design, which was restricted to long-time treated patients with MPN, a valid exploration of primary defects in the various platelet activation
pathways was not reasonable. Further examinations using MEA in an untreated MPN study population would be necessary to elucidate that question. In conclusion, MEA is a valid technique to examine the response to antiaggregatory treatment with ASA and clopidogrel, as well as its changes over a long observation period in patients with polycythemia vera and essential thrombocythemia. Furthermore, in the current study, an initial or subsequent nonresponse to treatment was observed in patients with a history of arterial and venous thrombotic events. It thus appears useful to re-evaluate the platelet function of patients with MPN during the disease course.
Acknowledgements Funding: The MEA test cells were obtained from Dynabyte Medical (Munich, Germany) and Ekomed (Vienna, Austria), and the sample tubes were provided by Sarstedt (Nuembrecht, Germany). The companies were not involved in the study design, sample collection, data analysis, writing process, and release of this article. Conflicts of interest
The authors declare no competing direct and indirect conflicts of interest concerning this study.
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Changes in response to antiaggregatory treatment in patients with myeloproliferative neoplasms: a sequential study using multiple electrode aggregometry Christoph Robier, Mariana Stettin and Manfred Neubauer In the present study, we used multiple electrode aggregometry (MEA) to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with myeloproliferative neoplasms (MPNs). arachidonic acid (ASPI), ADP, and thrombin receptor activating peptide (TRAP) tests were performed at two timepoints between 32–50 months in 21 patients with MPN and 1–46 months in 29 controls. We further checked the medical records of the participants to identify a potential correlation of changes in the treatment response with clinical events. In MPN, four out of 13 patients treated with 100 mg of aspirin, no patients receiving 50 mg of aspirin, and one out of five clopidogrel-treated patients showed a therapeutic antiplatelet effect. In the subsequent examinations, five patients changed from response to nonresponse or vice versa. Initial nonresponse and changes from an initial response to nonresponse were observed in six patients with thrombotic events. In the controls, 25 out of 26 aspirin-treated patients and two out of three clopidogreltreated patients showed an initially adequate in-vitro response. Except from one patient changing from initial
Introduction Thrombotic and hemorrhagic episodes are common disease-related complications of myeloproliferative neoplasms (MPNs), such as polycythemia vera and essential thrombocythemia, and thereby represent the major causes of mortality and morbidity [1]. Thrombosis is usually independent of the platelet count and antiaggregatory treatment [2], whereas hemorrhage is frequently associated with platelet counts over 1500 109/l [3] and treatment with antiaggregatory drugs, such as acetylsalicylic acid (ASA) or clopidogrel [4]. Furthermore, various types of hypofunctional and hyperfunctional platelet abnormalities have been described in the literature. Several laboratory studies using different techniques, such as optical aggregometry [5,6], whole-blood platelet lumi-aggregometry [7], or the platelet function analyzer (PFA)-100 [6,8], have been performed to detect and characterize such functional abnormalities, in order to relate these in-vitro phenomena with the clinical events. Thereby, in-vitro platelet function tests have generally failed to identify patients at risk for thrombotic or hemorrhagic complications [9], which has been attributed to the coincidence of diverse and sometimes also oppositional defects in the same patient, changes of functional characteristics during the disease course, to the effects of antiaggregatory treatment and other nonplatelet-derived 0957-5235 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
aspirin nonresponse to response, all controls showed a stable response state. One control with two ischemic strokes showed a nonresponse to clopidogrel. In conclusion, MEA detects the response to antiaggregatory treatment, as well as its changes during the disease course in patients with MPN. An initial or subsequent nonresponse was observed in patients with thrombotic events. Blood Coagul Fibrinolysis 24:869–873 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2013, 24:869–873 Keywords: acetylsalicylic acid, clopidogrel, multiplate analyzer, myeloproliferative neoplasms, platelet function Central Laboratory, Hospital Barmherzige Brueder Graz-Eggenberg, Graz, Austria Correspondence to Christoph Robier, MD, Central Laboratory, Hospital Barmherzige Brueder, Graz-Eggenberg, Bergstrasse 27, A-8020 Graz, Austria Tel: +43 316 5989 6671; fax: +43 316 5989 1505; e-mail: [email protected] Received 12 April 2013 Revised 20 June 2013 Accepted 20 June 2013
influences [10,11]. In the clinical setting, it would be beneficial to establish valid techniques for the monitoring of antiaggregatory treatment in patients with MPN, in order to identify nonresponders and thus to prevent associated thrombotic events. Both the PFA-100 [6,8] and multiple electrode aggregometry (MEA) [12] were confirmed as rapid and reliable methods for the monitoring of ASA, whereas MEA was also regarded as appropriate for the monitoring of clopidogrel treatment [12] in patients with polycythemia vera and essential thrombocythemia. On the basis of the fact that functional platelet abnormalities in patients with MPN can change during the disease course [11], we hypothesized that changes may also occur in response to antiplatelet therapy with time. Such changes may represent temporary risk factors for thrombosis and bleeding, and may thus be of clinical relevance. In the present study, we used MEA to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with polycythemia vera and essential thrombocythemia.
Materials and methods Study population
We studied samples from 21 patients with MPN (polycythemia vera, n ¼ 15; essential thrombocythemia, n ¼ 6) DOI:10.1097/MBC.0b013e328364713a
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
870 Blood Coagulation and Fibrinolysis 2013, Vol 24 No 8
from the hematologic outpatient clinic and the Department of Internal Medicine of the Hospital Barmherzige Brueder in Graz-Eggenberg, Austria. There were 14 women and seven men, ranging from 44 to 90 years. The majority of the study participants were diagnosed according to the criteria of the World Health Organization from 2001 [13]. Four patients with polycythemia vera were tested for the JAK2 V617F mutation and were all positive, whereas one of three tested patients with essential thrombocythemia showed this mutation. All included patients received long-time treatment with ASA and clopidogrel because of medical indication and confirmed their compliance with therapy prior to blood collection. The detailed basic and clinical characteristics, including further data on the disease course, cardiovascular and thrombophilic risk factors, the occurrence of bleeding and thrombotic events, and the antiaggregatory dosage regimens of the study population, are presented in Table 1. In order to determine control individuals without MPN, we retrospectively viewed our laboratory database for patients who had routine sequential examinations by MEA within the study period. We identified 29 controls (ASA 100 mg, n ¼ 26; clopidogrel 75 mg, n ¼ 3), who were characterized as follows: median age 75 years (52–95), female sex n ¼ 22 (76%), and median interval between the two examinations 16 months (1–46). Patients with thrombocytopenia (platelet count 2 mg/dl), renal insufficiency (serum creatinine level >2 mg/dl), monoclonal gammopathy, as well as patients who received other antithrombotic drugs than ASA and clopidogrel, nonsteroidal anti-inflammatory drugs, and corticosteroids within 2 weeks prior to the Clinical characteristics and demographics of the patients with myeloproliferative neoplasms
Table 1
Demographics Study participants, n Age in yearsa Female sex, n (%) Disease-related characteristics Myeloproliferative neoplasm Essential thrombocythemia, n (%) Polycythemia vera, n (%) Disease course in yearsa Observation period in monthsa Clinical events, n (%) Bleeding episodes Thrombotic events Platelet count (109/l)a Hematocrit (%)a Antiaggregatory treatment, n (%) ASA 100 mg ASA 50 mg Clopidogrel 75 mg ASA 100 mg þ clopidogrel 75 mg Cardiovascular risk factors, n (%) Arterial hypertension Dyslipidemia Diabetes mellitus Hereditary thrombophilia (protein S deficiency) ASA, acetylsalicylic acid.
a
Median (range).
21 71 (44–90) 14 (66.66)
6 15 10 40 6 7 424 48
(28.57) (71.43) (3–32) (32–50) (28.57) (33.33) (195–919) (36–57)
11 5 3 2
(52.38) (23.81) (14.29) (9.52)
11 11 4 1
(53.38) (53.38) (19) (4.76)
blood drawings, were excluded from the study to eliminate potential bias because of secondary thrombocytopathy. Blood sampling
Venous blood samples were collected with a 21-gauge needle, the first at least 3 ml were discarded. MEA was performed within 1.5 h after blood collection. In MPN patients, blood specimens were obtained at two timepoints as follows: at study entry and after a median time period of 40 months (range 32–50 months). Prior to each venipuncture, written informed consent was obtained from all study participants in accordance with the Declaration of Helsinki. This study was approved by the institutional ethics committee. Materials and platelet aggregation studies
For aggregation testing, hirudin-anticoagulated blood (25 mg/ml hirudin; Sarstedt, Nuembrecht, Germany) was used; platelet aggregation was measured from whole blood using MEA on the Multiplate analyzer (Roche Diagnostics GmbH, Vienna, Austria) according to the manufacturer’s instructions. Details of this method have been described previously [14]. We performed three test procedures with different agonists on each sample: ASPI test (arachidonic acid, 0.5 mmol/l), ADP test (adenosine diphosphate, 6.5 mmol/l), and TRAP test (thrombin receptor activating peptide, TRAP-6, 32 mmol/l). In MEA, after adding the agonist, impedance changes, representing platelet adhesion on the paired electrodes, are detected and recorded for 6 min. Platelet aggregation is quantified by the area under the aggregation curve, expressed as arbitrary units and converted to units (1 U ¼ 10 arbitrary units min) [14]. The reference range of MEA using hirudinized whole blood is 75–136 U in the ASPI test, 53–122 U in the ADP test, and 95–156 U in the TRAP test [15]. A cutoff value of less than 30 U in the ASPI test was determined for a therapeutic antiplatelet effect of ASA, whereas values less than 45 U in the ADP test were defined as a sufficient response to clopidogrel [16].
Results The complete results of the platelet aggregation studies are presented in Table 2 for the study participants with MPN and in Supplemental Table 1 for the controls. Response to antiaggregatory treatment
In the MPN group, four out of 13 patients (31%) treated with 100 mg of ASA, but no patients receiving a reduced dose of 50 mg of ASA because of medical indication, initially had ASPI values less than 30 U, being consistent with a response. In the subsequent examinations in the 100 mg group, three patients turned to a nonresponder state, one patient did not change, and two initially nonresponders showed a response. One patient treated with 50 mg, who was identified as a nonresponder at the first
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Aggregation changes in myeloproliferative neoplasms Robier et al. 871
Table 2
Aggregation data and clinical events of the study participants with myeloproliferative neoplasms First analysis, U
Drug, dose, and patients ASA 100 mg 1 2 3 4 5 6 7 8 9 10 11 ASA 50 mg 1 2 3 4 5 Clop 75 mg 1 2 3 ASA 100 mg þ clop 75 mg 1 2
Sequential analysis, U Clinical event
ASPI
ADP
TRAP
ASPI
ADP
TRAP
51 34 24 32 50 29 58 64 97 122 47
131 136 112 114 64 89 124 111 93 156 126
127 122 129 109 72 122 131 116 102 131 129
26 8 61 66 79 25 77 89 99 71 74
118 174 100 124 133 79 114 114 51 106 168
138 130 126 122 126 132 117 123 95 131 186
42 111 134 100 100
70 134 166 103 86
96 132 158 105 104
48 116 126 114 13
118 123 171 96 72
113 110 147 101 110
Ischemic stroke MCI þ hemorrhage Bleeding diathesis
101 102 106
84 67 92
100 139 115
97 100 97
73 47 75
82 118 111
Upper GI bleeding Upper GI bleeding Ischemic stroke
12 18
87 27
97 121
50 32
81 26
100 50
MCI
TIA
Splenic infarction
Epistaxis
ASA, acetylsalicylic acid; ASPI, arachidonic acid; clop, clopidogrel; GI, gastrointestinal; MCI, myocardial infarction; TIA, transient ischemic attack; TRAP, thrombin receptor activating peptide; U, units.
timepoint, had a result below the cutoff at the second timepoint and thus fulfilled the defined criterion of response. One out of five clopidogrel-treated patients had an initial response and was confirmed as a responder at the second evaluation. In the control group, 25 out of 26 ASA-treated patients (96%) and two out of three clopidogrel-treated patients (66%) showed an initial response. In the sequential examinations, all of the 26 ASA-treated patients yielded a sufficient response and the clopidogrel-treated patients did not change in their response patterns. Thrombotic and hemorrhagic events
Overall, in a review of the medical records of the 21 study participants with MPN, 11 clinical events were observed, with six thrombotic and five bleeding episodes. One patient developed both thrombotic and hemorrhagic events during the disease course. Thereby, except from one patient with a splenic infarction and one with a general enhanced bleeding diathesis who endured their events between the two measurements, all the other participants’ clinical events occurred prior to the study period. In case of the splenic infarction, a protein S deficiency has been found as an additional thrombophilic risk factor. An initial nonresponse and changes from an initial response to a nonresponse to ASA or clopidogrel were found in six of 10 patients with arterial or venous thrombotic events. One patient with a bleeding event showed a change from an initial ASA nonresponse to a response at
the second measure point. A trend toward an association with the occurrence of hemorrhagic complications was not found. All clinical episodes of the MPN patients and its association with MEA results are shown in Table 2. All patients who endured myocardial infarctions, a transient ischemic attack, or ischemic strokes had one or more additional cardiovascular risk factors, such as diabetes mellitus, arterial hypertension, and dyslipidemia. In the medical records of the controls, eight arterial thromboses (myocardial infarction, n ¼ 3; ischemic stroke, n ¼ 5), but no bleeding or venous thrombotic events, were found. Except from one patient who was defined as a clopidogrel nonresponder, and endured two ischemic strokes, all the other patients with vascular events showed a sufficient response to ASA or clopidogrel. In those patients, other than solely platelet-associated risk factors may have contributed to the clinical events.
Discussion In the present study, we confirmed that MEA is able to detect responses as well as nonresponses to antiaggregatory treatment with ASA and clopidogrel in patients with polycythemia vera and essential thrombocythemia. To our knowledge, this is the first study in which sequential evaluations of platelet aggregation in MPN were performed by MEA, and thereby showed changes in the drug responses during the disease course. Our findings correspond well with a prior study, highlighting that changes in the platelet function over time
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872 Blood Coagulation and Fibrinolysis 2013, Vol 24 No 8
are frequently occurring in patients with MPN [11]. In light of that trial and our current data, MEA may represent a useful tool in the management of patients with MPN and it may thus be beneficial to re-evaluate the platelet function of patients with MPN during the further course of the disease. One main goal of platelet function testing is the early identification of nonresponders to antiaggregatory drugs, in order to prevent thrombotic events and thus to avoid associated morbidity and mortality. In several study populations with cardiovascular diseases, low responses to antiplatelet agents assessed with MEA were significantly associated with an increased risk of stent thrombosis [17–19]. In MPN, previous studies using different devices frequently showed various platelet function abnormalities in vitro, but failed to identify a correlation with the hemostatic complications [1–3,9,10]. In the current study, only a few patients treated with ASA and clopidogrel showed an adequate initial or sustained response to treatment. We observed that all of the six patients who endured an arterial or venous thrombotic episode such as myocardial infarction, ischemic stroke, or splenic infarction during their disease course were at least at one timepoint of the study period classified as nonresponders according to the defined cutoffs. In consideration of the fact that only one of 29 control individuals developed a change in the response state, the observed treatment response changes in the study participants with polycythemia vera and essential thrombocythemia support the hypothesis of a disease-related phenomenon. However, because of the small number of clinical events, the coincidence with further cardiovascular and thrombophilic risk factors, and because of the fact that only two events occurred between the two MEA measurements, further prospective studies using MEA in a larger MPN population are needed to prove a correlation between changes in platelet aggregation and thrombotic and hemorrhagic events. This study had limitations that merit mention. First, the interval between the two examinations in the MPN group was quite long (median 40 months, range 32–50 months). Hence, further changes in platelet aggregation emerging within that time period may have been missed. Second, the MPN group and the controls were not matched in sex and age. In our opinion, that does not add a significant bias to our trial, because several previous studies clearly showed that age and sex do not exert a significant influence on MEA tests in patients with MPN [12] and healthy individuals [20]. Third, we did not focus on platelet function abnormalities in our population, because of our study design, which was restricted to long-time treated patients with MPN, a valid exploration of primary defects in the various platelet activation
pathways was not reasonable. Further examinations using MEA in an untreated MPN study population would be necessary to elucidate that question. In conclusion, MEA is a valid technique to examine the response to antiaggregatory treatment with ASA and clopidogrel, as well as its changes over a long observation period in patients with polycythemia vera and essential thrombocythemia. Furthermore, in the current study, an initial or subsequent nonresponse to treatment was observed in patients with a history of arterial and venous thrombotic events. It thus appears useful to re-evaluate the platelet function of patients with MPN during the disease course.
Acknowledgements Funding: The MEA test cells were obtained from Dynabyte Medical (Munich, Germany) and Ekomed (Vienna, Austria), and the sample tubes were provided by Sarstedt (Nuembrecht, Germany). The companies were not involved in the study design, sample collection, data analysis, writing process, and release of this article. Conflicts of interest
The authors declare no competing direct and indirect conflicts of interest concerning this study.
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