Statistische Auswertung

Für die statistische Auswertung wurden die Programme Microsoft Excel (Microsoft Office 2007, Microsoft Corporation Redmond, USA) undSPSS 22.0 (SPSS Inc., Chi-cago, USA) verwendet. Die Referenzbereiche wurden basierend auf dem 2,5 %- und 97,5 %-Quantil berechnet. Darüber hinaus wurden weitere Quantile (5, 10, 25, 50, 75, 90 und 95 %) berechnet und neben den Minimum- und Maximumwerten tabellarisch dargestellt.

Datensätze in den verschiedenen Versuchsabschnittenwurden zunächst mittels Kol-mogorov-Smirnov-Test auf Normalverteilung getestet. In Abhängigkeit von der Daten-verteilung wurden Messergebnisse (z. B. die Werte einer Gruppe zu einem definierten Zeitraum) als arithmetischer Mittelwert mit Standardabweichung oder Minimum-, Me-dian- und Maximumwerte dargestellt. Für die Ergebnisdarstellung des Vorversuchs der Plättchenfunktionsanalyse im ersten Teil der Arbeit wurden nicht-parametrische, de-skriptive Verfahren (Minimum, Median und Maximum) herangezogen, obwohl der Kol-mogorov-Smirnov-Test eine Normalverteilung zeigte. Die nicht-parametrischen, de-skriptiven Verfahren stellen die Verteilung der Daten besser dar. Einzelne fehlende

Material, Tiere und Methoden

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Werte nach wiederholten Messungen mit dem Plättchenfunktionsanalysegerät im Hauptversuch wurden durch die arithmetischen Mittelwerte der benachbarten Ergeb-nisse extrapoliert.

Die vergleichende Statistik der abhängigen Datensätze in den verschiedenen Ver-suchsabschnitten erfolgte global mittels Friedman-Test und bei signifikantem Ergebnis zudem nachfolgend über post-hoc-Analysen mittels Wilcoxon-Test (zum Vergleich ein-zelner Agonistenkonzentrationen oder Zeitpunkte). Zusätzlich kam im Hautversuch des ersten Studienteils für den Vergleich der Ratio-Werte (Messwert/Ausgangswert) der Plättchenfunktionsparameter (z. B. Verschlusszeit der Plättchenfunktionsanalyse [CADP-Testkartusche], velocity der mit 2,5 µmol/l und 5 µmol/l ADP induzierten Plätt-chenaggregation) zwischen den beiden Therapiegruppen der Mann-Whitney-U-Test zum Einsatz.

Zur Überprüfung der Enge der Beziehung zwischen der Apixaban-Konzentration (ge-messen als Anti-Xa-Aktivität) und den Ergebnissen verschiedener hämostaseologi-scher Analyseverfahren wurden Rangkorrelationskoeffizienten nach Spearman be-rechnet. Als statistisch signifikant galten P-Werte (P) ≤ 0,05. Es erfolgte keine Bonfer-roni-Korrektur des Signifikanzniveaus bei multiplen Vergleichen.

Manuskript I

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Influence of a low dosage of clopidogrel on platelet function in cats as meas-ured by the platelet function analyser PFA-100 and the Multiplate analyser

M. Teuber, R. Mischke*

* Corresponding author. Address: Small Animal Clinic, University of Veterinary Medicine Foundation, Bünteweg 9, D-30559 Hannover, Germany, Tel. +49 511 953 6200; fax: +49 511 953 6204

E-mail address: Reinhard.Mischke@tiho-hannover.de (R. Mischke)

Manuskript I

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The antiplatelet drug clopidogrel is widely used for prophylaxis of arterial throm-boses in cats in a standard dosage of 18.75 mg per cat once daily. The aim of the study was to verify if a reduced daily dose of 10 mg clopidogrel per cat has a similar antiplatelet effect as the standard dosage. Platelet function was measured with the platelet function analyser PFA-100® and a novel impedance aggregometer.

Suitability of the platelet function analyser was tested in citrated blood samples of 59 healthy cats and reference ranges were established. In addition, agonist concen-trations for impedance aggregometry were optimised. In the main experiment two groups of 6 healthy cats received clopidogrel either in a dosage of 10 or 18.75 mg per cat over a period of seven days. Analyses were performed on days 1, 2, 3, 5, and 7.

In comparison to baseline both clopidogrel dosages showed an inhibitory effect on results of the platelet function analyser and velocity of ADP-induced platelet aggre-gation. Values at all times were different from baseline, with the exception of day 1 in cats receiving 10 mg clopidogrel where the closure time of the platelet function analysis and part of ADP-induced aggregation did not show a significant difference. Significant differences were not found between the two doses.

In conclusion, our study indicates that 10 mg clopidogrel per day may be as effective as 18.75 mg although the latter may be advantageous as an initial loading dosage to achieve effective levels more rapidly.

Keywords: impedance aggregometer; reference interval; platelet agonist; antiplatelet treatment; feline

4.2 Introduction

Antithrombotic drugs play an essential role in prophylaxis and therapy of throm-botic disorders due to cardiovascular diseases in cats (Luis Fuentes, 2012). Cats suf-fering from hypertrophic cardiomyopathy frequently develop arterial thromboembolism.

Manuskript I

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For prevention and therapy of thromboembolism anticoagulant drugs such as vitamin k-antagonists and heparin are used as well as antiplatelet drugs (Falconer and Atwell, 2003). Clopidogrel is a modern antiplatelet drug which is used widely for the treatment of acute coronary syndromes in humans (Wallentin et al., 2009). It is a thienopyridine which expresses its effect by irreversible inhibition of ADP receptors on the platelet membrane (Pereillo et al., 2002). After oral administration, clopidogrel undergoes he-patic transformation to active metabolites (Pereillo et al., 2002; Brainard et al., 2010).

In a pilot study using an experimental model of feline aortic thrombosis, clopidogrel in a daily dosage of 75 mg per cat improved clinical signs indicating a beneficial effect on pelvic limb circulation and, thereby, an in vivo vasomodulating effect (Hogan et al., 2006).

The available veterinary literature on the antiplatelet effect of clopidogrel in cats comprises several experimental studies (Hogan et al., 2004; Hogan et al., 2006; Ha-mel-Jolette et al., 2009; Ho et al., 2016) as well as a positive-controlled clinical study (Hogan et al., 2015). Short term (Hogan et al., 2004; Hamel-Jolette et al., 2009) as well as long term administration (Hogan et al., 2015) of the active ingredient is usually well tolerated by cats, but gastrointestinal signs and occasionally icterus may occur.

Clopidogrel in different dosages (18.75–75 mg/cat once daily) significantly reduced platelet function as measured using buccal mucosal bleeding time and ADP- and col-lagen-induced whole blood aggregometry (Hogan et al., 2004). Analogously, Hamel-Jolette et al. (2009) found significantly decreased platelet aggregation in healthy cats receiving 18.75 mg clopidogrel per day using a screening test for antiplatelet medica-tion (plateletworks). A recent study describes the effect of the same dosage of clopidogrel on different platelet function tests (platelet function analyser, plateletworks, and impedance aggregometry) in healthy cats (Ho et al., 2016).

Based on these results, clopidogrel is usually applied in a standard dosage of 18.75 mg (¼ tablet clopidogrel 75 mg) per cat once daily for therapy of arterial throm-boembolism due to hypertrophic cardiomyopathy (Smith, 2012; Hogan et al., 2015). In a double-blind, randomised, positive-controlled feline study it was demonstrated that this dosagesignificantly reduced the likelihood of recurrent ardiogenic arterial throm-boembolism when compared with aspirin (Hogan et al., 2015). However, it remains

Manuskript I

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unclear, whether a lower dosage may be equally effective. Definition of the lowest ef-fective dosage may be particular important for cats showing side effects during treat-ment with the standard dosage.

Therefore, the present study aimed to compare the inhibitory effect of a dosage of 10 mg once daily per cat with the above stated standard dosage. Our hypothesiswas that this reduced dosage is equally effective as the currently used standard dosage.

Monitoring of platelet function was performed using the platelet function analyser PFA-100® and the modern impedance aggregometer Multiplate® analyser. The PFA-PFA-100®

evaluates platelet function in whole blood as determined by closure time (CT). That is the time required to form a platelet plug and to occlude the aperture within a membrane coated with collagen and ADP (CADP cartridge) or collagen and epinephrine (CEPI cartridge) and, thereby, stop the blood flow through a capillary (Harris, 2005). The CT seemed to have potential to monitor clopidogrel effect in cats as confirmed in a recently published feline study (Ho et al., 2016). Reference values for the platelet function an-alyser have rarely been published for cats and with slightly discrepant results (Jandrey et al., 2008; Ho et al., 2015). In preliminary experiments of the present study, laboratory specific reference values for the platelet function analyser in cats were established using both cartridge types. In addition, in the methodological part of this study, imped-ance aggregometry with the new aggregometer was performed with a wide range of agonist concentrations in order to define the optimal concentration to investigate cat blood.