4.3 Materials and Methods
4.4.4 Impact of clopidogrel on platelet aggregation tested with Multiplate ® ana- ana-lyser
None of the clopidogrel dosages studied, showed a significant effect on AUC or maximum aggregation values of aggregation induced with 2.5 μmol/L ADP or 5 μmol/L ADP (Table 3). In contrast, velocity decreased significantly, except when measure-ments were performed with 5 μmol/L ADP in cats receiving 10 mg clopidogrel (P=0.071). Day 1 in cats receiving 10 mg clopidogrel was the only time point where post hoc tests did not indicate significant differences when compared to baseline (Ta-ble 3). Except day 1 in cats receiving 10 mg clopidogrel, median ratio velocity values varied between 0.28 and 0.47. Comparison of ratio velocity values between the two dosages of clopidogrel did not reveal significant differences at any of the time points, neither for analyses performed with 2.5 μmol/L ADP, nor with 5 μmol/L ADP (P<0.05;
Mann Whitney U test, Fig. 3).
In contrast, none of the parameters of the arachidonic acid-induced platelet ag-gregation showed a reduction under the influence of clopidogrel, neither with 10 mg nor 18.75 mg, regardless of which concentration of agonist was used (results not shown).
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- 56 - Table 3 Median and range (in brackets) of the parameters area under the curve (AUC), maximum platelet aggregation and velocity of the impedance aggregometry using the Multiplate® analyser with the agonist ADP (two different concentrations) under the influence of clopidogrel (10 mg or 18.75 mg once daily). Day012357Friedman- test Days which differ from baseline ADP (2.5 µmol/L) Clopidogrel 10 mg AUC [AU*min] 3763 (1866–4400)3729 (1645–4204)2739 (1432–4090)2843 (1144–3987) 2494 (1956–3038)2790 (1913–3938)0.279– Velocity [AU/min] 43.9 (11.5–53.7)19.9 (11.8–31.1)13.9 (9.5–19.9) 13.5 (8.4–23.3) 12.2 (11.0–15.5)13.2 (10.0–25.5)0.0241, 2, 3, 5, 7 Max. platelet aggregation [AU]
272.3 (134.5–303.8) 268.2 (125.6–348.8) 230.6 (108.4–354.0) 218.3 (82.1–340.5) 217.2 (175,9–301.1) 262.4 (160.5–316.5) 0.306– Clopidogrel 18.75 mg AUC [AU*min] 3188 (1969–3863)3041 (1310–3636)3408 (1397–3990)3340 (2017–4582)3823 (2222–4255)3636 (2086–4698)0.120– Velocity [AU/min] 41.6 (28.4–50.3)14.5 (9.1–15.5) 15.0 (9.1–21.1) 15.4 (10.2–24.2)16.7 (9.9–25.3) 16.6 (10.5–23.9)0.0041, 2, 3, 5, 7 Max. platelet aggregation [AU]
232.1 (153.3–275.6) 259.0 (98.3–319.5) 291.6 (114.8–314.6) 267.8 (172.5–357.0) 302.6 (202.5–351.4) 294.8 (186.4–382.5) 0.649–
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- 57 - Continuation table 3: ADP (5 µmol/L) Clopidogrel 10 mg AUC [AU*min] 3394 (1982–4811)3485 (1793–4655)2680 (1382–3668)2877 (1102–4011)2229 (1941–2989)2477 (1526–3972)0.156– Velocity [AU/min] 19.5 (12.6–34.7)12.3 (9.2–18.3) 12.3 (9.2–18.3) 13.9 (8.4–24.5) 12.1 (10.5–13.9)12.7 (8.8–21.3) 0.071(2, 3, 5, 7) Max. platelet aggregation [AU]
239.9 (152.1–336.4) 252.2 (135.8–386.6) 224.7 (102.0–318.8) 225.4 (79.1–305.3) 197.3 (163.5–331.1) 220.9 (143.3–303.8) 0.258– Clopidogrel 18.75 mg AUC [AU*min] 3383 (2595–3743)2704 (1513–3587)2807 (1737–3947)3477 (1916–4412)3471 (1457–4378)3240 (2711–4518)0.410– Velocity [AU/min] 37.6 (33.6–49.0)12.9 (10.0–17.5)13.5 (10.4–18.5)16.6 (9.4–21.6) 17.7 (9.4–26.0) 16.5 (11.1–21.9)0.0131, 2, 3, 5, 7 Max. platelet aggregation [AU]
263.1 (176.3–275.6) 231.8 (110.6–296.6) 241.2 (144.4–316.9) 282.4 (156.4–363.0) 266.5 (118.5–364.5) 273.2 (421.5–342.0) 0.185–
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Figure 3: Platelet aggregation (velocity demonstrated as ratios values, i. e., actual val-ues/baseline values before clopidogrel) induced either with 2.5 µmol/L or 5 µmol/L ADP in cats receiving clopidogrel orally once daily over 7 consecutive days: Comparison between two groups of six cats each receiving either 10 mg or 18.75 mg clopidogrel on days 1, 2, 3, 5, and 7 after onset of treatment (Statistical comparison using Mann Whitney U test between the two clopidogrel dosages did not reveal significant differences at any of the time points [P>0.05]).
°Outside values
* Far outside values
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According to the results of the present study, 10 mg clopidogrel per cat once daily is equally effective when compared to 18.75 mg. 10 mg per cat corresponds to approximately 2.5 mg/kg body weight. This dosage is still higher than the standard maintenance dosage in humans, consisting of one 75 mg tablet per day (Mani et al., 2006; Dyszkiewicz-Korpanty et al., 2007; Mueller et al., 2009) which corresponds to approximately 1 mg/kg body weight. It is also slightly higher than the dosage of 2 mg clopidogrel/kg bodyweight which appeared to be more effective than 1 or 0.5 mg/kg body weight in healthy dogs based on ADP-induced whole blood aggregation and ca-pillary bleeding time (Biermann and Mischke, 2014). Effective concentrations are not necessarily similar between species. Platelets in cats are larger and more reactive than those in humans and dogs (Hart and Nolte, 1991) possibly indicating a higher clopidogrel demand, although we were unable to find information regarding the density of ADP (P2Y) receptors on feline platelets in the available literature. In addition, it has to be considered that clopidogrel is a prodrug which has to be converted into the phar-macologically active metabolite by the cytochrome P450-system in the liver by oxida-tion and subsequent hydrolysis; thus well-known species differences regarding this enzyme system (van Beusekom et al., 2010) may account for species differences in dosages.
Only in the initial phase, a nonsignificant effect 8 h after the first application (when compared to baseline values) on someplatelet function tests in cats receiving 10 mg clopidogrel indicated a delayed onset of effect after applying the lower dosage.
Therefore, in cases requiring a prompt antiplatelet effect, a higher loading dosage (e.g., 18.75 or 2 × 10mg capsules) may be reasonable. Accordingly, in humans undergoing acute coronary intervention (Nylander et al., 2006; Di Sciascio et al.,2010; Mangi-acapra et al., 2010) and an experimental canine study (Steinel et al., 2013), a higher loading dosage was used prior to maintenance therapy.
Apart from impedance aggregometry, we used the platelet function analyser to monitor the effect of clopidogrel, although, in humans, this device has low sensitivity to detect a clopidogrel effect (Mani et al., 2006; Dyszkiewicz-Korpanty et al., 2007;
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Mueller et al., 2009). For example, only 3/43 (7%) or 6/43 (14%) of human patients receiving clopidogrel showed prolongation of CT using CAPD or CEPI cartridges (Mani et al., 2006), while in another study, no effect of clopidogrel therapy on CT could be detected when compared to a patient group receiving no antiplatelet therapy (Mueller et al., 2009). In this light, it was interesting, that the most remarkable effects were seen in results of the platelet function analyser. However, a significant effect of a daily ad-ministration of 18.75 mg clopidogrel on the CT of the CAPD cartridge in healthy cats was also reported in a recently published study (Ho et al., 2016). Thus, the device appears to be generally suitable to monitor clopidogrel treatment in cats, i.e. to detect possible non-responders.
Our preliminary studies on healthy cats indicate that – similar to dogs – use of this method as a screening test of primary haemostasishas limitations in feline blood.
It was not surprising that measurement of normal feline blood using the CEPI cartridge resulted in long CTs frequently exceeding the measurement range. Different investi-gators were unable to induce a significant platelet aggregation in cats using epineph-rine (Mason and Read, 1967) or reported lack of the primary phase of aggregation and a long lag time under the influence of this agonist (MacDonald et al., 1984). For that reason, the main experiment on the influence of clopidogrel on platelet function tests was only performed with CADP cartridges. Similarly, only CADP cartridges were used in previous feline studies (Jandrey et al., 2008; Ho et al., 2015; Ho et al., 2016). In those studies, comparable lower reference range limits and median values, but slightly or remarkably shorter upper reference range limits were reported for a group of 42 healthy cats (43, 64, 176 s; minimum, median and maximum; Jandrey et al., 2008) or 46 cats (46, 69, 89 s; 2.5% quantile, median, 97.5% quantile; Ho et al., 2015) when compared to the respective values in our study (48, 58.5, 205 s). The wide reference range explains why in the present study only a limited number of CT values exceeded the reference range despite the significant prolongation. Therefore, an effective moni-toring of clopidogrel using the platelet function analyser requires comparison with baseline values.
Possibly as a consequence of the limitations of the short communication format, the first of the cited studies (Jandrey et al., 2008), does not contain any indication of
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unmeasurable results in individual feline patients which we observed. In contrast, in the second study the authors also mention that in 6/53 cats even after analyzing the samples in triplicate, no reading could be obtained and a total number of flow obstruc-tions of 37% (41/112 determinaobstruc-tions) (Ho et al., 2015). The methodological difficulties in cats may result from the high reactivity of feline platelets which tend to agglutinate and/or aggregate spontaneously in vitro (Riond et al., 2015). In addition, the physio-logically lower haematocrit values in cats may have also played a role. In dogs, haem-atocrit values of approx. 30%, corresponding to the lower reference range of cats (28–
45%), are already associated with significantly longer CT values than haematocrit val-ues between 40 and 50% (Callan and Giger, 2001; Mischke and Keidel, 2003).
Surprisingly, the effects on impedance platelet aggregation in our study were generally limited. ADP-induced aggregation is regarded as the most sensitive screen-ing test to detect clopidogrel in humans (Dyszkiewicz-Korpanty et al., 2007; Hobson et al., 2009; Mueller et al., 2009). It was even more surprising, because in a previous feline study (Hogan et al., 2004), 18.75 mg as higher dosages (75 mg, 37.5 mg) re-duced remarkably ADP-inre-duced whole blood platelet aggregation in cats. Impedance aggregometry seems to be even more sensitive to detect the clopidogrel effect than the turbidimetric/platelet rich plasma method in humans (Dyszkiewicz-Korpanty et al., 2007). However, our results are in accordance with those of the recently published PhD thesis which was unable to demonstrate an effect of 18.75 mg clopidogrel per cat on AUC values of the Multiplate analyser, irrespective of whether ADP, collagen or arachidonic acid were utilised as agonists (Ho et al., 2016). In that study, agonist con-centrations recommended by the manufacturer of the device which are optimised for use in human sample material were used.
As one explanation for this surprising result the authors discussed the use of citrate anticoagulated blood, because this is not the optimalsample material (Kalbant-ner et al., 2010). However, the samplematerial does not seem to be the main reason, because in the present study blood anticoagulated with hirudin also showed a limited effect.
Minimal agonist concentrations necessary for maximal aggregation were ad-justed for individual cats in the previous study (Hogan et al., 2004). Ourmethodological
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preliminary experiment comparing different agonist concentrations indicates a wide in-ter-individual range of responses irrespective of the agonist concentration which makes it impossible to define generally applicable “threshold values”. Therefore, an individual adjustment of agonist concentrations may increase sensitivity, but is elabo-rate and of limited practicability. Considering the high reactivity of feline platelets (Hart and Nolte, 1991), we tested agonist concentrations in the lower range of a comparable study for dogs (Kalbantner et al., 2010). Due to lacking differences, especially between the higher of the tested agonist concentrations, we used the highest and a medium concentration of the agonists for the main experiment.
Our study also demonstrates the influence of the test parameter of the device on the sensitivity to the anti-platelet effect of clopidogrel. The less commonly used velocity parameter was more sensitive than the AUC which is the standard parameter of the Multiplate analyser (Kalbantner et al., 2010; Ho et al., 2015), and also than max-imum aggregation which is in general the most commonly used parameter for presen-tation of aggregation results including part of cited studies (Hogan et al., 2004; Brainard et al., 2010).
Apart fromthe agonist ADP, we investigated the effect of clopidogrel on arachi-donic acid-induced platelet aggregation, and thereby on the “acetylic salicylic acid (ASA) specific” pathway as well. Significant effects on arachidonic acid-induced plate-let activation have been demonstrated in dogs by use of impedance aggregometry (Biermann and Mischke, 2014) and in humans by use of thrombelastogram Plate-letMapping (Hobson et al., 2009). In humans and dogs, in addition to ASA-synergistic or -potentiating antiplatelet effects, clopidogrel has ASA-independent inhibitory activity on arachidonic acid-induced platelet activation. The lacking effect of clopidogrel on the
“ASA specific” pathway in cats is plausible, when considering the fact that ASA itself has a limited inhibitory effect on feline platelet activity (Hart et al., 1995; Cathcart et al., 2012). It is suggested, that feline platelets may require lower thromboxane A2 concen-trations due to higher sensitivity, or that they experience less pronounced suppression of cyclooxygenase-1-derived thromboxane or require higher dose of ASA to achieve sufficient cyclooxygenase inhibition (Cathcart et al., 2012).
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One limitation of the study is the lack of power analysis reflecting its character as a pilot study. As a further limitation of this study, it has to be considered, that alt-hough the in vitro testswe used are considered standard tests of platelet function, the effectiveness of the tested dosage has to be confirmed in vivo in clinical studies as already performed for the standard dosage of 18.75 mg (Hogan et al., 2015). Clinical studies will also consider the aspect that potentially activated platelets in cats with dis-eases associated with hypercoagulability such as cardiomyopathy (Stokol et al., 2008) or manifest thromboembolic diseases may react differently and may have different ef-fective blood levels of antiplatelet drugs such as clopidogrel. This is, for example, indi-cated by the fact that in vitro activation of platelets from cardiomyopathic cats required lower dosages of collagen (Welles et al., 1994), whereas for ADP both increased (Helenski and Ross, 1987) and decreased responsiveness (aggregation and [14C] ser-otonin release) (Welles et al., 1994) were reported.
Clinical trials can also clarify, whether the reduced 10 mg dosage is significantly better tolerated when compared to the standard dosage, which according to Hamel-Jolette et al. (2009) and our unpublished observations, causes gastrointestinal signs in someanimals. For example, 3 of 10 experimental cats receiving 18.75mg clopidogrel for three consecutive days developed mild self-limiting diarrhea (Hamel-Jolette et al., 2009). The reduced dosage has the disadvantage that 10 mg tablets have to be spe-cifically produced by pharmacists and are, therefore,more expensive than usage of a quarter of the commercially available tablet.
4.6 Conclusion
The in vitro results of the present study indicate that,with the exception of the first day, 10 mg clopidogrel has an equal antiplatelet effectivity as the present standard dosage of 18.75 mg per cat. Therefore, possibly after a higher loading dosage, a re-duced dosage may be appropriate although this has to be confirmed in clinical trials.
Platelet function analysis is significantly influenced by clopidogrel, and thereby seems to be a suitable method to monitor clopidogrel effect in individual cats, although general applicability of this device in cats has limitations. The limited effect of clopidogrel on
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impedance aggregometry was surprising, whereby it turned out that velocity is the most sensitive parameter.
4.7 References
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Di Sciascio, G., Patti, G., Pasceri, V., Gatto, L., Colonna, G., Montinaro, A., ARMYDA-5 PRELOAD Investigators, 2010. Effectiveness of in-laboratory high-dose clopidogrel loading versus routine pre-load in patients undergoing percutaneous coronary intervention: results of the ARMYDA-5 PRELOAD (Antiplatelet therapy for Reduction of Myocardial Damage during Angioplasty) randomized trial. J.
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Dyszkiewicz-Korpanty, A., Olteanu, H., Frenkel, E.P., Sarode, R., 2007. Clopidogrel anti-platelet effect: An evaluation by optical aggregometry, impedance ag-gregometry, and the Platelet Function Analyzer (PFA-100®). Platelets 18, 491–
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Falconer, L., Atwell, R., 2003. Feline aortic Thromboembolism. Aust. Vet. Pract. 33, 20–32.
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Harris, P., 2005. Platelet function analysis. Blood Reviews 19, 111-123.
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Ho, K.K., Abrams-Ogg. A.C., Wood, R.D., O'Sullivan, M.L., Kirby, G.M., Blois, S.L.., 2016. Assessment of platelet function in healthy cats in response to commonly prescribed antiplatelet drugs using three point-of-care platelet function tests. J.
Feline Med. Surg. (in press). pii: 1098612X16648182 [Epub ahead of print].
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"aspirin specific" pathways of platelet inhibition. Platelets 20, 386–390.
Hogan, D.F., Andrews, D.A., Green, H.W., Talbot, K.K., Ward, M.P., Calloway, B.M., 2004. Antiplatelet effect and pharmacodynamics of Clopidogrel in cats. J. Am.
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Hogan, D.F., Fox, P.R., Jacob, K., Keene, B., Laste, N.J., Rosenthal, S., Sederquist K., Weng, H.Y., 2015. Secondary prevention of cardiogenic arterial thromboem-bolism in the cat: The double-blind, randomized, positive-controlled feline arte-rial thromboembolism; clopidogrel vs. aspirin tarte-rial (FAT CAT). J. Vet. Cardiol. 17 Suppl 1, S306–S317.
Jandrey, K.E., Norris, J.W., MacDonald, K.A., Kittleson, M.D., Tablin, F., 2008. Platelet function in clinically healthy cats and cats with hypertrophic cardiomyopathy:
analysis using the Platelet Function Analyzer-100. Vet. Clin. Pathol. 37, 385–
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Luis Fuentes, V., 2012. Arterial Thromboembolism: Risks, realities and a rational first-line approach. J. Fefirst-line Med. Surg. 14, 459–470.
MacDonald, M.L., Rogers, Q.R., Morris, J.G., 1984. Effect of dietary arachidonate de-ficiency on the aggregation of cat platelets. Comp. Biochem. Physiol. C Toxicol.
Pharmacol. 78, 123–126.
Mangiacapra, F., Muller, O., Ntalianis, A., Trana, C., Heyndrickx, G.R., Bartunek, J., Vanderheyden, M., Wijns, W., De Bruyne, B., Barbato, E., 2010. Comparison of 600 versus 300-mg Clopidogrel loading dose in patients with ST-segment ele-vation myocardial infarction undergoing primary coronary angioplasty. Am. J.
Cardiol. 106, 1208–1211.
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Pereillo, J.-M., Maftouh, M., Andrieu, A., Uzabiaga, M.-F., Fedeli, O., Savi, P., Pascal, M., Herbert, J.-M., Maffrand, J.-P., Picard, C., 2002. Structure and stereo-chemistry of the active metabolite of clopidogrel. Drug Metab. Dispos. 30, 1288–
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Measurements of endogenous thrombin potential using the CAT method in cats:
reference values and influence of the direct factor Xa inhibitor apixaban
Corresponding address: Small Animal Clinic, University of Veterinary Medicine Foun-dation, 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)
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The aim of this study was to establish the thrombin generation test (endogenous thrombin potential) measured by the Calibrated Automated Thrombogram (CAT) tech-nique in cats by establishing reference values, examination of its reproducibility and sensitivity to anticoagulant treatment with the factor Xa inhibitor apixaban. The CAT method was performed on citrated plasma with different commercial tissue factor rea-gents (PPP Reagent 1 pM [LOW], PPP Reagent 5 pM, PPP Reagent 20 pM [HIGH]) according to the manufacturers` test instruction.
Measurements in triplicate were performed in platelet free plasma of 58 healthy cats and in 6 cats at different times following the oral administration of 2.5 mg apixaban.
The median CVs calculated for threefold measurements of healthy cats usually were
< 10 % with the exception of thrombin peak height measured using PPP Reagent 1 pM (14.6 %). Reference values of all parameters depended largely on the tissue factor concentration of the used activating reagent. Thrombin generation was significantly influenced by apixaban and reacted more sensitively than other tests of haemostasis including the prothrombin time, aPTT, and rotationelastometry.
In conclusion, thrombin generation measured by the CAT method using com-mercially available reagents seems a suitable for examination of feline platelet free plasma and may be a valuable method to establish effective anticoagulant therapies for the feline patient and monitoring of such therapies in cats.
Keywords: Haemostasis, feline, anticoagulation, monitoring,Calibrated Automated Thrombography
5.2 Introduction
The generation of thrombin is a fundamental part of the clotting cascade and, thereby, the estimation of endogenous thrombin potential (ETP), i. e. the concentration of thrombin in clotting plasma, in an individual may help to predict possible risks of