The anti-aggregating effect of clopidogrel (75 mg/day) on platelet function was monitored in 33 solid tumor patients. Clopidogrel irreversibly blocks the platelet P2Y12 receptor and inhibits platelet aggregation induced by adenosine diphosphate (ADP). Whole blood aggregation was measured using a Siemens PFA-100 aggregometer including the Innovance PFA P2Y cartridge. The mean age of patients was 62 ± 13 years. Among them, there were 19 (58%) males and 14 (42%) females. The values of platelet aggregation >106 seconds and 106 sekunda pokazala je klopidogrelski antiagregacijski učinak u 22 (67%) bolesnika; a vrijednost <106 sekunda uočena je u 11 (33%) bolesnika koji nisu reagirali. Prosječni klopidogrelski antiagregacijski učinak bio je 219 ± 110 sekunda. Agregacijskih razlika između muškaraca i žena nije bilo (p=0;784). Interindividualna agregacijska varijacija bila je 50%. Nije bilo statistički značajne razlike između dvaju mjerenja 7 uzoraka istih osoba u razmaku od mjesec ili više dana; što pokazuje intraindividualnu stabilnost klopidogrelskog djelovanja na trombocitnu agregaciju (p=1;000)

Banović, Miro

Banović, Miroslav

Krajačić-Karas, Gorana

Lesar, Nikola

Veliki-Dalić, Irena

English

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1ARTICLE Libri Oncol., Vol. 39 (2011), No 1-3, 1 – 6PLATELET RESISTANCE TO ANTI-AGGREGATING EFFECT OF CLOPIDOGREL IN SOLID TUMOR PATIENTSMIROSLAV BANOVI]1, GORANA KRAJA^I] KARAS1, IRENA VELIKI DALI]1, MIRO BANOVI]2 and NIKOLA LESAR2“Sestre milosrdnice” University Hospital Center, University Hospital for Tumors, Zagreb, Croatia2Cand. Med. School of Medicine University of Rijeka, CroatiaSummaryThe anti-aggregating effect of clopidogrel (75 mg/day) on platelet function was monitored in 33 solid tumor patients. Clopidogrel irreversibly blocks the platelet P2Y12 receptor and inhibits platelet aggregation induced by adenosine diphos-phate (ADP). Whole blood aggregation was measured using a Siemens PFA-100 aggregometer including the Innovance PFA P2Y cartridge. The mean age of patients was 62 ± 13 years. Among them, there were 19 (58%) males and 14 (42%) females. The values of platelet aggregation >106 seconds and <106 seconds showed the anti-aggregating effect of clopidogrel in 22 (67%) and no effect (no response) in 11 (33%) patients, respectively. The average anti-aggregating effect of clopidogrel was 219 ± 110 seconds. No differences in platelet aggregation between males and females were observed (p=0.784). The interin-dividual variation in platelet aggregation was 50%. There was no statistically significant difference between two measure-ments on 7 samples of the same individuals performed at an interval of a month or more, showing the intraindividual stabil-ity of clopidogrel activity on platelet aggregation (p=1.000).KEYWORDS: tumors, clopidogrel resistance, PFA-100 aggregometer, Innovance PFA P2YTROMBOCITNA REZISTENCIJA NA KLOPIDOGRELSKI ANTIAGREGACIJSKI U^INAK U BOLESNIKA SA SOLIDNIM TUMORIMASa`etakPra}en je klopidogrelski (75 mg/dan) antiagregacijski u~inak na trombocite u 33 bolesnika sa solidnim tumorima. Klopidogrel ireverzibilno blokira trombocitni receptor P2Y12 i sprje~ava trombocitnu agregaciju pokrenutu adenozin difos-fatom (ADP). Agregacija je mjerena u punoj krvi agregometrom Siemens PFA-100 s ulo{kom Innovance PFA P2Y. Prosje~na dob bolesnika bila je 62 ± 13 godine. Mu{karaca je bilo 19 (58%), a `ena 14 (42%). Vrijednost trombocitne agregacije >106 sekunda pokazala je klopidogrelski antiagregacijski u~inak u 22 (67%) bolesnika, a vrijednost <106 sekunda uo~ena je u 11 (33%) bolesnika koji nisu reagirali. Prosje~ni klopidogrelski antiagregacijski u~inak bio je 219 ± 110 sekunda. Agregacijskih razlika izme|u mu{karaca i `ena nije bilo (p=0,784). Interindividualna agregacijska varijacija bila je 50%. Nije bilo statisti~ki zna~ajne razlike izme|u dvaju mjerenja 7 uzoraka istih osoba u razmaku od mjesec ili vi{e dana, {to pokazuje intraindivi-dualnu stabilnost klopidogrelskog djelovanja na trombocitnu agregaciju (p=1,000).KLJU^NE RIJE^I: tumori, rezistencija na klopidogrel, PFA-100 agregometar, Innovance P2YINTRODUCTIONAdenosine diphosphate (ADP) is important for platelet function. Three ADP receptors have been identified: P2Yl, P2Y12 and P2X1. Stimulation of the P2Y12 receptor can lead to irreversible plate-let aggregation and the development of blood clots (1-3). Approximately 15% of clopidogrel is Libri Oncol., Vol. 39 (2011), No 1–3, 1 – 62metabolized by cytochrome P450 3A4 (CYP3A4) liver enzyme, and about 85% is excreted in the stool (4-6, 8). Its metabolites bind to the P2Y12 re-ceptor and inhibit ADP-induced aggregation (7). Resistance to clopidogrel can be due to P450 3A4 polymorphisms, and polymorphisms of the pla-telet P2Y12 receptor. The reduced effect of clopi-dogrel can be due to irregular drug intake, inap-propriate dosage or drug interactions, poor ab-sorption, and variable conversion to its active metabolite or increased clearance of the active me-tabolite. The anti-aggregating effect of clopidogrel has a normal, bell-shaped Gaussian distribution, which means that some people develop a weaker and others a stronger response to the same dosage (9). Resistance to clopidogrel may be a marker of an increased risk for a thrombotic event.MATERIAL AND METHODSBlood samples were collected from patients with cancer who, for their blood circulation prob-lems, were taking clopidogrel anti-aggregation therapy at a dose of 75 mg/daily. During 18 months, 33 such patients were reg-istered, of whom 19 (58%) were male and 14 (42%) were female (Fig. 1). The mean age of patients was 62 ± 13 years (Fig. 2). The intraindividual stability of aggregation parameters was tested in the same 7 patients whose blood was taken on two occa-sions, at an interval of a month or more. Blood was collected in 4.5 mL glass Vacutainer blood collec-tion tubes with 0.105 molar buffered sodium ci-trate (3.2%). Platelet aggregation was measured in whole blood using the Siemens PFA-100 System (Platelet Function Analyzer) including the In-novance PFA P2Y cartridge. Clopidogrel prolongs the aggregation time. The analyzer and reagent manufacturer has set the reference value for clopi-dogrel aggregation at 106 seconds, which was taken as the cut-off for the purpose of this study. Results below the value were assumed to be nor-mal or showing no effect of clopidogrel, and were labeled as clopidogrel resistant. The obtained re-sults were statistically processed using the Stu-dent’s t-test and Mann-Whitney Rank Sum Test to compare mean values (x), standard deviations (sd), coefficient of variation (CV), minimum (min) and maximum (max).Figure 1. Representation of patients by gender. Out of 33 pa-tients, 19 (58%) were male, and 14 (42%) were female (N = number of patients)Figure 2. Patient distribution by age. The mean age of 33 pa-tients was 62 ± 13 years; age variability = 21%. The youngest patient was 22, and the oldest one was 84 years of age. (N = number of patients, x = mean value, sd = standard deviation, CV = coefficient of variation, min = minimum, max = maximum).3Libri Oncol., Vol. 39 (2011), No 1–3, 1 – 6RESULTSFigure 3. Age-adjusted distribution of male patients. The mean age of 19 male patients was 61 ± 13 years; age variability = 21%. The youngest male patient was 32, and the oldest one is 84 years of age. (N = number of patients, x = mean value, sd = standard deviation, CV = coefficient of variation, min = minimum, max = maximum).Figure 4. Age-adjusted distribution of female patients. The mean age of 14 female patients was 62 ± 13 years; age variability = 21%. The youngest female patient was 22, and the oldest one was 77 years of age. (N = number of patients, x = mean value, sd = standard deviation, CV = coefficient of variation, min = mini-mum, max = maximum).Figure 5. Comparison between age and gender groups. There is no statistically significant age-related difference between males and females (t-test: p=0.784). (N = number of patients, x = mean value, sd = standard deviation, p = probability).Figure 6. Distribution of platelet aggregation times. Out of 33 patients, 11 (33%) had platelet aggregation time <106 sec, show-ing no response to clopidogrel therapy. The mean platelet aggre-gation time in 33 patients was 219 ± 110 sec; aggregation vari-ability = 50%. The lowest and the highest aggregation values were 52 sec and 300 sec, respectively.Libri Oncol., Vol. 39 (2011), No 1–3, 1 – 64Figure 10. Comparison between two aggregation measurements from the same individuals. The measurements were performed at an interval of a month or more. There was no statistically sig-nificant measurement between the first and the second measure-ment (Mann-Whitney rank sum test: p=1.000). The platelet ag-gregation time in patients on clopidogrel therapy showed the intraindividual stability in the time pattern.Figure 7. Distribution of platelet aggregation times in male pa-tients. The mean platelet aggregation time in 19 male patients was 202 ± 118 sec; aggregation variability = 58%. The lowest and the highest aggregation values were 52 sec and 300 sec, re-spectively.Figure 8. Distribution of platelet aggregation times in female patients on clopidogrel therapy The mean platelet aggregation time in 14 female patients was 240 ± 99 sec; aggregation vari-ability = 41%. The lowest and the highest aggregation values were 55 sec and 300 sec, respectively.Figure 9. Comparison between platelet aggregation time and gender groups. There is no statistically significant aggregation time-related difference between male and female patients on clop-idogrel therapy (Mann-Whitney rank sum test: p=0.390).5Libri Oncol., Vol. 39 (2011), No 1–3, 1 – 6DISCUSSIONIn our previous study, we found platelet ag-gregation resistance to acetylsalicylic acid (ASA) in about 60% of study subjects (11). In this study, 33% resistance to clopidogrel anti-aggregation therapy was shown. Measurements performed by other authors using a different method, i.e. plate-let aggregation flow cytometry, showed the cut-off value for clopidogrel platelet aggregation of 15%. Measurements of platelet aggregation using a simple test such as the VerifyNow-P2Y12 test demonstrated a poor response in 20% of study subjects (12). Also, some additional studies show-ed 20% resistance to clopidogrel (13-15). The Vaso-dilator-Stimulated Phosphoprotein (VASP) phos-phorylation assay and the VerifyNow-P2Y12 31 test revealed resistance to clopidogrel of 29% and 6%, respectively (16). Clopidogrel resistance vari-ability, as shown by other authors, ranges from 5-63% (16-24). Our result of 33% clopidogrel resis-tance conforms to average results of worldwide measurements.CONCLUSIONThe metabolites of clopidogrel irreversibly block the platelet P2Y12 receptor and inhibit the binding of ADP to this receptor, resulting in the absence of platelet aggregation. It is important to know that there is a certain proportion of people who do not respond to a clopidogrel dose of 75 mg/day and are therefore at an increased risk of developing thrombosis. 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Trombocitna rezistencija na klopidogrelski antiagregacijski učinaku bolesnika sa solidnim tumorima

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