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Changes in platelet function in Dachshunds with early stages of myxomatous mitral valve disease
Research in Veterinary Science 86 (2009) 320–324
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Changes in platelet function in Dachshunds with early stages of myxomatous mitral valve disease S.G. Moesgaard a,*, T.M. Sørensen a, A. Sterup a, I. Tarnow a, A.T. Kristensen b, A.L. Jensen b, L.H. Olsen a a b
Department of Basic Animal and Veterinary Sciences, University of Copenhagen, 7 Groennegaardsvej, Frederiksberg C DK-1870, Denmark Department of Small Animal Clinical Sciences, University of Copenhagen, Frederiksberg C DK-1870, Denmark
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Article history: Accepted 30 July 2008
Keywords: Dachshunds Platelet function Myxomatous mitral valve disease von Willebrand factor
a b s t r a c t The aim of this study was to evaluate platelet function in Dachshunds during early stages of myxomatous mitral valve disease. Clinical examination and echocardiography were performed in 34 wirehaired standard sized Dachshunds. Platelet function was evaluated using the PFA-100 (reported as closure time). In addition, whole blood platelet aggregation response and hemostatic markers were evaluated. Significant longer PFA-100 closure time (CT) was found in 12 Dachshunds with mild mitral regurgitation (MR) compared to 22 Dachshunds with minimal MR. Only five Dachshunds responded to adenosine diphosphate in the whole blood aggregation analyses. There were no differences between the two dog groups in plasma fibrinogen, plasma von Willebrand factor (vWf) or vWf multimer distribution; however, there was a significant correlation between CT and plasma vWf concentration and CT and plasma fibrinogen concentration. The higher CT found in Dachshunds with mild MR suggests a form of platelet dysfunction in Dachshunds with MR. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Myxomatous mitral valve disease (MMVD) is the most common spontaneous cardiac disease in the dog (Buchanan, 1977; Häggstrom et al., 2004). Abnormal protrusion of the mitral valve leaflets into the left atrium, i.e. mitral valve prolapse (MVP), is an important component of developing MMVD, and it is speculated that the resulting change in hemodynamic relations in MVP leads to a selfinforcing circuit of damaging events (Beardow and Buchanan, 1993; Pedersen et al., 1995, 1999b). A change in platelet function might be involved in the pathogenesis of MMVD as a consequence of the turbulent high-velocity flow and changes in fluid shear stress around the MVP. Different results have been found in previous studies concerning relations between MMVD and platelet function depending much on the assessment technique used. An increased platelet aggregation response is found in Cavalier King Charles Spaniels (CKCS; a breed predisposed to MMVD) (Nielsen et al., 2007; Olsen et al., 2001). A decreased platelet life span (Tanaka et al., 2002) indicating increased platelet activity has been shown in dogs with severe mitral regurgitation (MR). However, other studies have showed a decreased platelet aggregation response in CKCS and other dogs with severe MR (Cowan et al., 2004; Tanaka and * Corresponding author. Tel.: +45 35333884; fax: +45 35332525. E-mail address: [email protected] (S.G. Moesgaard). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.07.019
Yamane, 2000). In addition, CKCS with clinical inapparent MR have been found to have decreased ability to form a platelet plug in a hole in a membrane at high shear rates (prolonged closure times using a platelet function analyzer (PFA)) (Tarnow et al., 2003). This was associated with a selective loss of von Willebrand factor (vWf) high molecular weight multimers (HMWM) which might have been caused by high shear stress (Tarnow et al., 2005). These apparently conflicting findings may be explained by the fact that platelet aggregation at high shear rates is dependent on vWf. It is possible to detect platelet dysfunction as a result of a qualitative defect of vWf in in vitro platelet function assays based on physiologic high shear rates and adhesion to a contact surface (such as the PFA). In this case platelet aggregation tests will not be affected. Because of the high prevalence of MMVD in CKCS, this breed has been widely used in studies concerning the disease progression and the correlation with platelet function. These findings may be biased by specific breed characteristics, i.e. inherent asymptomatic thrombocytopenia (Cowan et al., 2004; Eksell et al., 1994; Pedersen et al., 2002). It is therefore important also to study the association between MMVD and changes in platelet function in other breeds with naturally occurring MMVD such as Dachshunds. MMVD is less prevalent in Dachshunds compared to CKCS (Haggstrom et al., 1992; Olsen et al., 1999; Pedersen et al., 1996). It has previously been shown that MMVD is inherited in both breeds, but the progression of the disease is slower in Dachshunds (Olsen et al., 1999; Swenson et al., 1996).
S.G. Moesgaard et al. / Research in Veterinary Science 86 (2009) 320–324
The objective of this study was to evaluate the platelet function in Dachshunds with mild MR compared to Dachshunds with no or minimal MR. More specifically, the aims were to determine (1) whether the prolonged PFA-100 closure times (CT) previously found in CKCS with MR also can be found in Dachshunds and (2) whether CT is associated with the platelet aggregation response and specific hemostatic markers. 2. Materials and methods 2.1. Animals The study population consisted of 36 client-owned wirehaired Dachshunds. The dogs were recruited through the Danish Dachshund Club. The dogs were clinically healthy, received no medication or vaccination the last month prior to the study, were not pregnant or lactating, were between 6 and 10 years of age, and were fasted for 6 h before the study. The dogs were all categorized according to their degree of MR (jet size, defined as the maximum area of a regurgitating jet in relation to the area of the left atrium) evaluated from two-dimensional echocardiography (colour flow mapping) (Pedersen et al., 1999a) and subsequently divided into two groups: A control group with no or minimal MR (jet size 6 15%) and a MR group with mild MR (jet size > 15% and 650%) (Pedersen et al., 2003). Studies were conducted between April 4th and June 19th 2006. 2.2. Examination procedures The examination comprised, in order of occurrence, collection of a urine sample1 (complete urinalysis was performed except from microscopy), an interview with the owner, collection of blood samples, physical examination, electrocardiography and echocardiography. None of the dogs were sedated for the examination and the owners were present to keep the dogs calm. Echocardiography was performed using a VividÒ32 echocardiograph as described previously (Pedersen et al., 2003). Color flow mapping of the mitral valve area was performed to assess the degree of MR (%) using the left caudal four-chamber view of the dog in left lateral recumbency (Pedersen et al., 1999a). MVP severity, left ventricular end diastolic diameter (LVEDD) and the ratio between the left atrial (LA) and aortic root (Ao) diameters were assessed from the echocardiographic recordings, as described previously (Pedersen et al., 2003). 2.3. Blood collection and handling Careful jugular venipuncture was performed by the same investigator (LHO) in all dogs using a vacutainer system connected to a 21-gauge butterfly catheter.3 Blood was collected into a tube with clot activator for clinical chemistry analysis; tubes containing 105 mM (3.2%) trisodium citrate for platelet function analysis and fibrinogen, vWf plasma and multimer analysis; and tubes containing potassium ethylenediaminetetraacetic acid (EDTA) for routine hematologic and biochemical analyses and manual platelet counts. Blood samples were centrifuged and plasma was separated within 30 min of collection. Plasma samples were frozen at 80 °C and analyzed as a batch when all dogs were examined (vWf and fibrinogen analysis). Manual platelet counts (Bürker counting chambers) were performed in duplicate by mixing 20 ll of EDTAanticoagulated blood with 380 ll Stromatol stromatolytic agent.4
Hemacolor-stained5 blood smears of citrate-anticoagulated blood were examined for platelet aggregates. 2.4. Assessment of platelet function The platelet function analyzer (PFA-100Ò6) coated with collagen and adenosine diphosphate (Coll-ADP) was used to evaluate platelet function according to the manufacturer’s instructions and as described in detail elsewhere (Kundu et al., 1995; Tarnow et al., 2005). Whole-blood aggregometry was performed by standard procedures using a whole blood impedance aggregometer.7 A polystyrene plastic cuvette containing a siliconised stir bar, 500 ll of citrate-anticoagulated blood and 500 ll isotonic saline (preheated to 37 °C) was allowed to equilibrate at 37 °C for approximately 10 min. Platelet aggregation was initiated by addition of the agonist adenosine diphosphate (ADP – final concentration 20 lM). The change in impedance was recorded on a strip chart recorder for 10 min. Maximum aggregation response in ohms was measured at 10 min after addition of agonist. von Willebrand factor (vWf) multimeric profiles were assessed using sodium dodecyl sulfate–agarose gel electrophoresis (SDS– AGE), western blotting, and immunoperoxidase detection as previously described (Tarnow et al., 2005). The vWf multimer profiles were evaluated qualitatively (counting number of bands on the western blot) by a person who was unaware of the identity of the dogs and their clinical findings. Plasma concentration of vWf was measured via a turbidometric immunoassay8 as described previously (Tarnow et al., 2004). Plasma fibrinogen concentration was evaluated by use of a rabbit brain calcium tromboplastin9 to initiate clot formation. Light scattering before and after clot formation was recorded by use of an automated chemical analyzer,10 and the fibrinogen concentration was calculated by use of these values and a calibration curve. 2.5. Statistical analyses A one-way analysis of variance (ANOVA) was performed to evaluate the effect of MR status (explanatory variable) on CT, plasma vWf and fibrinogen concentration and vWf multimer bands (response variables). The response variables were all continuous. Body weight, age, platelet count, hematocrit were included as covariates. The other disease related variables (MVP, LVEDD and LA/Ao) were entered separately in multiple linear regression models. Before analysis, LVEDD was divided by (body weight)0.29 to normalize it to the size of the dog (Cornell et al., 2004), and body weight therefore was not included in that particular analysis. The initial models included the effect of all variables and their interactions. The models were reduced by manual backward, stepwise exclusion until only statistically significant effects remained. For each model, the residuals were tested for normality and homogeneity of variation and relevant transformations were used to obtain normality of the residuals. A simple linear regression analysis was used to assess possible associations between CT, vWf, vWf multimer and fibrinogen concentration. All statistical calculations were performed by statistical software.11 Results are shown as means ± SD or as median values and inter-quartile intervals when the raw data or residuals were not normally distributed. The level of significance was chosen as P < 0.05.
5 6 7
1 2 3 4
EickmeyerÒ Vet Handrefraktometer HRM 18, Tuttlingen, Germany. Vivid 3 Echocardiograph, GE Medical, Milwaukee, WI, USA. BD VacutainerÒ, No. 367844, Becton-Dickinson a/s, Brøndby, Denmark. Stromatol stromalytic agent, Mascia Brunelli S.p.a., Milano, Italy.
321
8 9 10 11
HemacolorÒ, Merck, Darmstadt, Germany. PFA-100Ò, Dade Behring Incorporated, Marburg, Germany. Whole-blood lumi-aggregometer, Chrono-Log Corporation, Haverton, PA, USA. IL Test von Willebrand factor, Instrumentation Laboratory, Warrington, UK. IL Test PT-Fibrinogen, Instrumentation Laboratory, Warrington, UK. ACL9000, Instrumentation Laboratory, Warrington, UK. SAS statistical software, version 9.1, SAS Institute, Cary, NC, USA.
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Table 1 Characteristics of the 34 Dachshunds divided into two groups according to the degree of MR
Gender (m/f) Age (years) Body weight (kg) Hematocrit Platelet count (109/l) Jet size (%)
Control, no or minimal MR (n = 22)
MR, mild MR (n = 12)
11/11 8.3 ± 1.3 9.7 ± 1.4 0.47 ± 0.04 263 ± 59 5 (5–10)
4/8 8.6 ± 1.2 9.8 ± 1.6 0.46 ± 0.03 267 ± 70 30 (25–30)*
MR, mitral regurgitation; except for gender and jet size, data are shown as means ± SD values. Jet size is shown as median and 25th to 75th percentiles. * P < 0.05 vs. control.
3. Results 3.1. Descriptive data on dogs Two dogs were excluded out of the 36 dogs enrolled in the study: one because we were not able to obtain a blood sample and another because a severe pulmonic stenosis was detected. The 34 remaining dogs were divided into two groups (control and MR) based on their jet size. Descriptive data for the two groups are shown in Table 1. None of the dogs showed any decisive abnormalities on routine biochemical analysis and urinalysis. There was a higher percentage of females in the MR group, however, this was not statistically significant. Otherwise there were no statistically significant differences between the descriptive characteristics (age, body weight, hematocrit and platelet count) of the two groups. 3.2. Platelet function Longer CT were found in the MR group compared to control (Fig. 1, Table 2, P = 0.03). The CT was not affected by age, gender, body weight, hematocrit or platelet count. Only five Dachshunds responded to ADP in the whole blood aggregometer. The five responses observed did not appear to be related to the MR status of the dogs, however, the data were too few to perform a correct statistical analysis.
Fig. 1. Closure time (s) measured by use of a platelet function analyzer (PFA-100) with collagen and adenosine diphosphate as the combined agonist. The Dachshunds with mild mitral regurgitation (MR) has significantly longer closure times compared to the Dachshunds with no or minimal MR.
Table 2 Platelet function markers in the two dog groups Dachshunds
Control, no or minimal MR (n = 22)
MR, mild MR (n = 12)
Col + ADP closure time (s) Plasma fibrinogen (mg/ml) Plasma vWf (%) vWf multimer (number of bands)
77.4 ± 16.8 1.96 (1.72–2.43) 68 ± 16 14 (12–14)
90.3 ± 14.8* 2.40 (1.98–2.90) 65 ± 21 14 (10–14)
MR, mitral regurgitation; vWf, von Willebrand factor, Col, collagen; ADP, adenosine diphosphate; data are shown as means ± SD values or as medians and 25th to 75th percentiles. * P < 0.05 vs. control.
There were no significant differences in plasma fibrinogen or vWf levels between the two groups (Table 2). Furthermore, the vWf multimer numbers were the same in both groups (Table 2). There was a significant correlation between CT and plasma vWf concentration (r2 = 0.287; P = 0.0015, Fig. 2) and CT and plasma fibrinogen concentration (r2 = 0.17; P = 0.033, Fig. 2). The correlation between CT and vWf multimer numbers was borderline significant (r2 = 0.197; P = 0.066, Fig. 2). Finally, there was a significant correlation between plasma vWf concentration and vWf multimer number (r2 = 0.296; P = 0.0009). 4. Discussion In this study, the platelet function in Dachshunds with mild MR was examined and compared to Dachshunds with no or minimal MR. Previously, it has been shown that CKCS with mild to severe MR have prolonged CT (Tarnow et al., 2003, 2004). In this study, a similar prolongation of CT was observed in Dachshunds with mild MR. It therefore seems likely that CT measured with the PFA-100 is independent of breed and is associated with the degree of MR. However, it is not known whether platelet dysfunction plays a role in the progression of MMVD and further studies addressing the prognostic value of CT with regard to developing congestive heart failure are needed. In dogs with MMVD both higher and lower aggregation responses have been found (Olsen et al., 2001; Tanaka and Yamane, 2000; Tarnow et al., 2005). However, these studies differed regarding breed, control group, assessment technique and severity of disease and it is therefore difficult to draw direct comparisons. In this study, only few of the samples aggregated when ADP was added in the whole blood aggregometer, and in most samples there was a lack of aggregation response. In a previous study, we have seen different whole blood aggregation responses when comparing CKCS with Boxers, Cairn terriers and Labrador retrievers using the same equipment and ADP concentration as used in this study (Nielsen et al., 2007). The majority of the three other breeds had very low aggregation responses to ADP, and in line with these results, this study shows that there are significant breed variations in ADP-induced platelet aggregation in dogs. The mechanism of this heterogeneity in canine whole blood aggregation responses is unknown, but may be due to genetic differences which could reflect variations in ectonucleotidase NTPDase1 (CD39) activity (Glenn et al., 2005). There was a statistically significant correlation between plasma vWf concentration and vWf multimer number. Furthermore, plasma vWf concentration correlated significantly with CT as has been shown in CKCS (Tarnow et al., 2004). As can be seen in Fig. 2 there was some scattering in the correlations and the coefficients of determination were quite low. This suggests poor to moderate correlations which may also be influenced by other variables than the ones tested in this study. Previous studies have shown an association between a loss of HMWM and prolonged CT in CKCS with MR and dogs with subaortic stenosis (Tarnow et al.,2004, 2005). In this
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Fig. 2. Platelet function analyzer (PFA-100) closure times shown as a function of high molecular weight von Willebrand factor (vWf) multimers (A) (r2 = 0.197; P = 0.066), plasma vWf concentration (B) (r2 = 0.287; P = 0.0015) and plasma fibrinogen concentration (C) (r2 = 0.17; P = 0.033). Finally, plasma vWf concentration is shown as a function of high molecular weight vWf multimers (D) (r2 = 0.296; P = 0.0009).
study, the association between HMWM and CT was only borderline significant. However, the dogs only had mild MR (jet size > 15% and 650%), whereas the dogs in previous studies had moderate to severe MR (jet size P 50%) (Tarnow et al., 2004, 2005). The measurement of HMWM is not a very sensitive test and in order to see a significant effect on CT more severe stages of MR are probably needed. An increase in plasma fibrinogen and vWf concentration has been reported in human patients with MR (Goldsmith et al., 2000; Lip et al., 1996). In CKCS with MR no difference in fibrinogen concentration was found, but a decrease in vWf explained by a loss of HMWM, has been shown (Tarnow et al., 2004). In this study, there was no difference in plasma vWf and plasma fibrinogen concentration between the two groups. This may indicate that the platelet dysfunction observed in Dachshunds with MR is less dependent on changes in vWf. However, the lack of difference in plasma vWf and vWf multimers may also be due to the MR status of the Dachshunds which was less severe than in the CKCS study. A previous study has suggested that a decreased sensibility or exhaustion of the platelets may occur secondary to the chronic shear stress in MMVD (Tanaka and Yamane, 2000) and thereby explain the platelet dysfunction. This may also explain the prolonged CT associated with MR in this study. In this study, a platelet dysfunction was shown, whereas platelet aggregation did not appear to be affected in Dachshunds with MR. This may be due to the fact that the dogs only had mild MMVD
without clinical signs, and that very few dogs had a measurable aggregation response in whole blood. The consequences of the altered platelet function in dogs with MMVD are not yet clear. Studies in humans and dogs have shown a relationship between activated platelets and vascular changes such as atherosclerosis, thrombosis and intimal hyperplasia (Anderson et al., 2001; Nomura et al., 1998). Furthermore, both human and canine studies have shown an association between intramyocardial coronary arteriosclerosis and an increased risk of sudden death (Burke and Virmani, 1998; Falk and Jonsson, 2000). Dogs with heart failure due to MMVD have a significant occurrence of small vessel arteriosclerosis compared to age-matched dogs euthanized due to non-cardiac reasons (Falk et al., 2006), and a direct association between the severity of arterial changes and clinical signs of MMVD in dogs has been shown (Falk et al., 2007). Thus, it has been suggested that the arterial changes could be part of a more generalized disease process and that activated platelets might cause an accelerated disease progression by development of vascular changes and possibly microthrombi in the myocardium (Tarnow et al., 2005). However, the possible association between arterial changes and platelet dysfunction still remains to be established. This study is limited by small group size, limiting the power of the study. The fact that only one breed was used reduces the within group variation, but specific breed variations may also influence
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the results. However, previous studies in another breed (CKCS) performed in the same laboratory showed similar results regarding CT suggesting that breed differences are less important (Tarnow et al., 2004). In this study, only dogs with mild MR were included. It would be interesting to include a group of Dachshunds with clinical signs of MMVD in order to study whether CT was further increased in more severe stages of the disease, and whether this might affect the platelet aggregation response. In a previous study, CKCS with moderate to severe MR (jet P 50%) and dogs with subaortic stenosis had slightly longer CT than what was shown in the MR group in this study indicating that CT may increase further as the severity of the disease increases (Tarnow et al., 2005). This study showed that mild MR in Dachshunds is associated with prolonged CT indicating platelet dysfunction. The CT was significantly correlated with plasma vWf and fibrinogen concentration. Only few of the Dachshunds responded to ADP in a whole blood aggregometer. These findings encourage further studies of the use of CT as a prognostic marker of MMVD development in dogs. Acknowledgements The work was supported by the Danish Medical Research Council. The authors thank Birgitte Holle, Department of Basic Animal and Veterinary Sciences and Natascha Errebo, Department of Small Animal Clinical Sciences, The University of Copenhagen, Denmark for their excellent technical assistance. Kirsten Christiansen and Jørgen Ingerslev, Skejby University Hospital, Århus, Denmark are thanked for assistance with the vWf multimer analysis. References Anderson, H.V., McNatt, J., Clubb, F.J., Herman, M., Maffrand, J.P., DeClerck, F., Ahn, C., Buja, L.M., Willerson, J.T., 2001. Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemicatherosclerotic canine coronary arteries. Circulation 104, 2331–2337. Beardow, A.W., Buchanan, J.W., 1993. Chronic mitral-valve disease in Cavalier King Charles spaniels – 95 cases (1987–1991). Journal of the American Veterinary Medical Association 203, 1023–1029. Buchanan, J.W., 1977. Chronic valvular disease (endocardiosis) in dogs. Advances in veterinary science and comparative medicine 21, 872–874. Burke, A.P., Virmani, R., 1998. Intramural coronary artery dysplasia of the ventricular septum and sudden death. Human Pathology 29, 1124–1127. Cornell, C.C., Kittleson, M.D., Della Torre, P., Häggstrom, J., Lombard, C.W., Pedersen, H.D., Vollmar, A., Wey, A., 2004. Allometric scaling of M-mode variables in normal adult dogs. Journal of Veterinary Internal Medicine 18, 311–321. Cowan, S.M., Bartges, J.W., Gompf, R.E., Hayes, J.R., Moyers, T.D., Snider, C.C., Gerard, D.A., Craft, R.A., Muenchen, R.A., Carroll, R.C., 2004. Giant platelet disorder in the Cavalier King Charles spaniel. Experimental Hematology 32, 344–350. Eksell, P., Haggstrom, J., Kvart, C., Karlsson, A., 1994. Thrombocytopenia in the Cavalier King Charles spaniel. Journal of Small Animal Practice 35, 153–155. Falk, T., Jonsson, L., 2000. Ischaemic heart disease in the dog: a review of 65 cases. Journal of Small Animal Practice 41, 97–103. Falk, T., Jonsson, L., Olsen, L.H., Pedersen, H.D., 2006. Arteriosclerotic changes in the myocardium, lung, and kidney in dogs with chronic congestive heart failure and myxomatous mitral valve disease. Cardiovascular Pathology 15, 185–193. Falk, T., Jonsson, L., Olsen, L.H., Tarnow, I., Ledersen, H.D., 2007. Correlation of cardiac pathology and clinical findings in dogs with naturally occurring congestive heart failure. Journal of Veterinary Internal Medicine 21, 636. Glenn, J.R., White, A.E., Johnson, A., Fox, S.C., Behan, M.W.H., Dolan, G., Heptinstall, S., 2005. Leukocyte count and leukocyte ecto-nucleotidase are major
determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood. Platelets 16, 159–170. Goldsmith, I.R.A., Blann, A.D., Patel, R.L., Lip, G.Y.H., 2000. Reduced indexes of left atrial hypercoagulability in patients with severe mitral regurgitation. American Journal of Cardiology 86, 234–236. Haggstrom, J., Hansson, K., Kvart, C., Swenson, L., 1992. Chronic valvular disease in the Cavalier King Charles spaniel in Sweden. Veterinary Record 131, 549–553. Häggstrom, J., Pedersen, H.D., Kvart, C., 2004. New insights into degenerative mitral valve disease in dogs. Veterinary Clinics of North America – Small Animal Practice 34, 1209–1226. Kundu, S.K., Heilmann, E.J., Sio, R., Garcia, C., Davidson, R.M., Ostgaard, R.A., 1995. Description of an in-vitro platelet-function analyzer – Pfa-100(Tm). Seminars in Thrombosis and Hemostasis 21, 106–112. Lip, G.Y.H., Rumley, A., Dunn, F.G., Lowe, G.D.O., 1996. Thrombogenesis in mitral regurgitation and aortic stenosis. Angiology 47, 1117–1125. Nielsen, L.A., Zois, N.E., Pedersen, H.D., Olsen, L.H., Tarnow, I., 2007. Platelet function in dogs: breed differences and effect of acetylsalisylic acid administration. Veterinary Clinical Pathology 36, 267–273. Nomura, E., Kohriyama, T., Yamaguchi, S., Kajikawa, H., Nakamura, S., 1998. Association between carotid atherosclerosis and hemostatic markers in patients with cerebral small artery disease. Blood Coagulation and Fibrinolysis 9, 55–62. Olsen, L.H., Fredholm, M., Pedersen, H.D., 1999. Epidemiology and inheritance of mitral valve prolapse in Dachshunds. Journal of Veterinary Internal Medicine 13, 448–456. Olsen, L.H., Kristensen, A.T., Haggstrom, J., Jensen, A.L., Klitgaard, B., Hansson, H., Pedersen, H.D., 2001. Increased platelet aggregation response in Cavalier King Charles spaniels with mitral valve prolapse. Journal of Veterinary Internal Medicine 15, 209–216. Pedersen, H.D., Kristensen, B.O., Lorentzen, K.A., Koch, J., Jensen, A.L., Flagstad, A., 1995. Mitral-valve prolapse in 3-year-old healthy Cavalier-King-CharlesSpaniels – an echocardiographic study. Canadian Journal of Veterinary Research – Revue Canadienne de Recherche Veterinaire 59, 294–298. Pedersen, H.D., Kristensen, B.O., Norby, B., Lorentzen, K.A., 1996. Echocardiographic study of mitral valve prolapse in dachshunds. Journal of Veterinary Medicine Series A – Zentralblatt fur Veterinarmedizin Reihe A – Physiology Pathology Clinical Medicine 43, 103–110. Pedersen, H.D., Haggstrom, J., Falk, T., Mow, T., Olsen, L.H., Iversen, L., Jensen, A.L., 1999a. Auscultation in mild mitral regurgitation in dogs: observer variation, effects of physical maneuvers, and agreement with color Doppler echocardiography and phonocardiography. Journal of Veterinary Internal Medicine 13, 56–64. Pedersen, H.D., Lorentzen, K.A., Kristensen, B.O., 1999b. Echocardiographic mitral valve prolapse in Cavalier King Charles spaniels: epidemiology and prognostic significance for regurgitation. Veterinary Record 144, 315–320. Pedersen, H.D., Haggstrom, J., Olsen, L.H., Christensen, K., Selin, A., Burmeister, M.L., Larsen, H., 2002. Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles spaniels is an autosomal recessive trait. Journal of Veterinary Internal Medicine 16, 169–173. Pedersen, H.D., Schutt, T., Sondergaard, R., Qvortrup, K., Olsen, L.H., Kristensen, A.T., 2003. Decreased plasma concentration of nitric oxide metabolites in dogs with untreated mitral regurgitation. Journal of Veterinary Internal Medicine 17, 178– 184. Swenson, L., Haggstrom, J., Kvart, C., Juneja, R.K., 1996. Relationship between parental cardiac status in Cavalier King Charles spaniels and prevalence and severity of chronic valvular disease in offspring. Journal of the American Veterinary Medical Association 208, 2009–2012. Tanaka, R., Yamane, Y., 2000. Platelet aggregation in dogs with mitral valve regurgitation. American Journal of Veterinary Research 61, 1248–1251. Tanaka, R., Murota, A., Nagashima, Y., Yamane, Y., 2002. Changes in platelet life span in dogs with mitral valve regurgitation. Journal of Veterinary Internal Medicine 16, 446–451. Tarnow, I., Kristensen, A.T., Texel, H., Olsen, L.H., Pedersen, H.D., 2003. Decreased platelet function in Cavalier King Charles spaniels with mitral valve regurgitation. Journal of Veterinary Internal Medicine 17, 680–686. Tarnow, I., Kristensen, A.T., Olsen, L.H., Pedersen, H.D., 2004. Assessment of changes in hemostatic markers in Cavalier King Charles spaniels with myxomatous mitral valve disease. American Journal of Veterinary Research 65, 1644–1652. Tarnow, I., Kristensen, A.T., Olsen, L.H., Falk, T., Haubro, L., Pedersen, L.G., Pedersen, H.D., 2005. Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution. Journal of Veterinary Internal Medicine 19, 515–522.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Changes in platelet function in Dachshunds with early stages of myxomatous mitral valve disease S.G. Moesgaard a,*, T.M. Sørensen a, A. Sterup a, I. Tarnow a, A.T. Kristensen b, A.L. Jensen b, L.H. Olsen a a b
Department of Basic Animal and Veterinary Sciences, University of Copenhagen, 7 Groennegaardsvej, Frederiksberg C DK-1870, Denmark Department of Small Animal Clinical Sciences, University of Copenhagen, Frederiksberg C DK-1870, Denmark
a r t i c l e
i n f o
Article history: Accepted 30 July 2008
Keywords: Dachshunds Platelet function Myxomatous mitral valve disease von Willebrand factor
a b s t r a c t The aim of this study was to evaluate platelet function in Dachshunds during early stages of myxomatous mitral valve disease. Clinical examination and echocardiography were performed in 34 wirehaired standard sized Dachshunds. Platelet function was evaluated using the PFA-100 (reported as closure time). In addition, whole blood platelet aggregation response and hemostatic markers were evaluated. Significant longer PFA-100 closure time (CT) was found in 12 Dachshunds with mild mitral regurgitation (MR) compared to 22 Dachshunds with minimal MR. Only five Dachshunds responded to adenosine diphosphate in the whole blood aggregation analyses. There were no differences between the two dog groups in plasma fibrinogen, plasma von Willebrand factor (vWf) or vWf multimer distribution; however, there was a significant correlation between CT and plasma vWf concentration and CT and plasma fibrinogen concentration. The higher CT found in Dachshunds with mild MR suggests a form of platelet dysfunction in Dachshunds with MR. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Myxomatous mitral valve disease (MMVD) is the most common spontaneous cardiac disease in the dog (Buchanan, 1977; Häggstrom et al., 2004). Abnormal protrusion of the mitral valve leaflets into the left atrium, i.e. mitral valve prolapse (MVP), is an important component of developing MMVD, and it is speculated that the resulting change in hemodynamic relations in MVP leads to a selfinforcing circuit of damaging events (Beardow and Buchanan, 1993; Pedersen et al., 1995, 1999b). A change in platelet function might be involved in the pathogenesis of MMVD as a consequence of the turbulent high-velocity flow and changes in fluid shear stress around the MVP. Different results have been found in previous studies concerning relations between MMVD and platelet function depending much on the assessment technique used. An increased platelet aggregation response is found in Cavalier King Charles Spaniels (CKCS; a breed predisposed to MMVD) (Nielsen et al., 2007; Olsen et al., 2001). A decreased platelet life span (Tanaka et al., 2002) indicating increased platelet activity has been shown in dogs with severe mitral regurgitation (MR). However, other studies have showed a decreased platelet aggregation response in CKCS and other dogs with severe MR (Cowan et al., 2004; Tanaka and * Corresponding author. Tel.: +45 35333884; fax: +45 35332525. E-mail address: [email protected] (S.G. Moesgaard). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.07.019
Yamane, 2000). In addition, CKCS with clinical inapparent MR have been found to have decreased ability to form a platelet plug in a hole in a membrane at high shear rates (prolonged closure times using a platelet function analyzer (PFA)) (Tarnow et al., 2003). This was associated with a selective loss of von Willebrand factor (vWf) high molecular weight multimers (HMWM) which might have been caused by high shear stress (Tarnow et al., 2005). These apparently conflicting findings may be explained by the fact that platelet aggregation at high shear rates is dependent on vWf. It is possible to detect platelet dysfunction as a result of a qualitative defect of vWf in in vitro platelet function assays based on physiologic high shear rates and adhesion to a contact surface (such as the PFA). In this case platelet aggregation tests will not be affected. Because of the high prevalence of MMVD in CKCS, this breed has been widely used in studies concerning the disease progression and the correlation with platelet function. These findings may be biased by specific breed characteristics, i.e. inherent asymptomatic thrombocytopenia (Cowan et al., 2004; Eksell et al., 1994; Pedersen et al., 2002). It is therefore important also to study the association between MMVD and changes in platelet function in other breeds with naturally occurring MMVD such as Dachshunds. MMVD is less prevalent in Dachshunds compared to CKCS (Haggstrom et al., 1992; Olsen et al., 1999; Pedersen et al., 1996). It has previously been shown that MMVD is inherited in both breeds, but the progression of the disease is slower in Dachshunds (Olsen et al., 1999; Swenson et al., 1996).
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The objective of this study was to evaluate the platelet function in Dachshunds with mild MR compared to Dachshunds with no or minimal MR. More specifically, the aims were to determine (1) whether the prolonged PFA-100 closure times (CT) previously found in CKCS with MR also can be found in Dachshunds and (2) whether CT is associated with the platelet aggregation response and specific hemostatic markers. 2. Materials and methods 2.1. Animals The study population consisted of 36 client-owned wirehaired Dachshunds. The dogs were recruited through the Danish Dachshund Club. The dogs were clinically healthy, received no medication or vaccination the last month prior to the study, were not pregnant or lactating, were between 6 and 10 years of age, and were fasted for 6 h before the study. The dogs were all categorized according to their degree of MR (jet size, defined as the maximum area of a regurgitating jet in relation to the area of the left atrium) evaluated from two-dimensional echocardiography (colour flow mapping) (Pedersen et al., 1999a) and subsequently divided into two groups: A control group with no or minimal MR (jet size 6 15%) and a MR group with mild MR (jet size > 15% and 650%) (Pedersen et al., 2003). Studies were conducted between April 4th and June 19th 2006. 2.2. Examination procedures The examination comprised, in order of occurrence, collection of a urine sample1 (complete urinalysis was performed except from microscopy), an interview with the owner, collection of blood samples, physical examination, electrocardiography and echocardiography. None of the dogs were sedated for the examination and the owners were present to keep the dogs calm. Echocardiography was performed using a VividÒ32 echocardiograph as described previously (Pedersen et al., 2003). Color flow mapping of the mitral valve area was performed to assess the degree of MR (%) using the left caudal four-chamber view of the dog in left lateral recumbency (Pedersen et al., 1999a). MVP severity, left ventricular end diastolic diameter (LVEDD) and the ratio between the left atrial (LA) and aortic root (Ao) diameters were assessed from the echocardiographic recordings, as described previously (Pedersen et al., 2003). 2.3. Blood collection and handling Careful jugular venipuncture was performed by the same investigator (LHO) in all dogs using a vacutainer system connected to a 21-gauge butterfly catheter.3 Blood was collected into a tube with clot activator for clinical chemistry analysis; tubes containing 105 mM (3.2%) trisodium citrate for platelet function analysis and fibrinogen, vWf plasma and multimer analysis; and tubes containing potassium ethylenediaminetetraacetic acid (EDTA) for routine hematologic and biochemical analyses and manual platelet counts. Blood samples were centrifuged and plasma was separated within 30 min of collection. Plasma samples were frozen at 80 °C and analyzed as a batch when all dogs were examined (vWf and fibrinogen analysis). Manual platelet counts (Bürker counting chambers) were performed in duplicate by mixing 20 ll of EDTAanticoagulated blood with 380 ll Stromatol stromatolytic agent.4
Hemacolor-stained5 blood smears of citrate-anticoagulated blood were examined for platelet aggregates. 2.4. Assessment of platelet function The platelet function analyzer (PFA-100Ò6) coated with collagen and adenosine diphosphate (Coll-ADP) was used to evaluate platelet function according to the manufacturer’s instructions and as described in detail elsewhere (Kundu et al., 1995; Tarnow et al., 2005). Whole-blood aggregometry was performed by standard procedures using a whole blood impedance aggregometer.7 A polystyrene plastic cuvette containing a siliconised stir bar, 500 ll of citrate-anticoagulated blood and 500 ll isotonic saline (preheated to 37 °C) was allowed to equilibrate at 37 °C for approximately 10 min. Platelet aggregation was initiated by addition of the agonist adenosine diphosphate (ADP – final concentration 20 lM). The change in impedance was recorded on a strip chart recorder for 10 min. Maximum aggregation response in ohms was measured at 10 min after addition of agonist. von Willebrand factor (vWf) multimeric profiles were assessed using sodium dodecyl sulfate–agarose gel electrophoresis (SDS– AGE), western blotting, and immunoperoxidase detection as previously described (Tarnow et al., 2005). The vWf multimer profiles were evaluated qualitatively (counting number of bands on the western blot) by a person who was unaware of the identity of the dogs and their clinical findings. Plasma concentration of vWf was measured via a turbidometric immunoassay8 as described previously (Tarnow et al., 2004). Plasma fibrinogen concentration was evaluated by use of a rabbit brain calcium tromboplastin9 to initiate clot formation. Light scattering before and after clot formation was recorded by use of an automated chemical analyzer,10 and the fibrinogen concentration was calculated by use of these values and a calibration curve. 2.5. Statistical analyses A one-way analysis of variance (ANOVA) was performed to evaluate the effect of MR status (explanatory variable) on CT, plasma vWf and fibrinogen concentration and vWf multimer bands (response variables). The response variables were all continuous. Body weight, age, platelet count, hematocrit were included as covariates. The other disease related variables (MVP, LVEDD and LA/Ao) were entered separately in multiple linear regression models. Before analysis, LVEDD was divided by (body weight)0.29 to normalize it to the size of the dog (Cornell et al., 2004), and body weight therefore was not included in that particular analysis. The initial models included the effect of all variables and their interactions. The models were reduced by manual backward, stepwise exclusion until only statistically significant effects remained. For each model, the residuals were tested for normality and homogeneity of variation and relevant transformations were used to obtain normality of the residuals. A simple linear regression analysis was used to assess possible associations between CT, vWf, vWf multimer and fibrinogen concentration. All statistical calculations were performed by statistical software.11 Results are shown as means ± SD or as median values and inter-quartile intervals when the raw data or residuals were not normally distributed. The level of significance was chosen as P < 0.05.
5 6 7
1 2 3 4
EickmeyerÒ Vet Handrefraktometer HRM 18, Tuttlingen, Germany. Vivid 3 Echocardiograph, GE Medical, Milwaukee, WI, USA. BD VacutainerÒ, No. 367844, Becton-Dickinson a/s, Brøndby, Denmark. Stromatol stromalytic agent, Mascia Brunelli S.p.a., Milano, Italy.
321
8 9 10 11
HemacolorÒ, Merck, Darmstadt, Germany. PFA-100Ò, Dade Behring Incorporated, Marburg, Germany. Whole-blood lumi-aggregometer, Chrono-Log Corporation, Haverton, PA, USA. IL Test von Willebrand factor, Instrumentation Laboratory, Warrington, UK. IL Test PT-Fibrinogen, Instrumentation Laboratory, Warrington, UK. ACL9000, Instrumentation Laboratory, Warrington, UK. SAS statistical software, version 9.1, SAS Institute, Cary, NC, USA.
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Table 1 Characteristics of the 34 Dachshunds divided into two groups according to the degree of MR
Gender (m/f) Age (years) Body weight (kg) Hematocrit Platelet count (109/l) Jet size (%)
Control, no or minimal MR (n = 22)
MR, mild MR (n = 12)
11/11 8.3 ± 1.3 9.7 ± 1.4 0.47 ± 0.04 263 ± 59 5 (5–10)
4/8 8.6 ± 1.2 9.8 ± 1.6 0.46 ± 0.03 267 ± 70 30 (25–30)*
MR, mitral regurgitation; except for gender and jet size, data are shown as means ± SD values. Jet size is shown as median and 25th to 75th percentiles. * P < 0.05 vs. control.
3. Results 3.1. Descriptive data on dogs Two dogs were excluded out of the 36 dogs enrolled in the study: one because we were not able to obtain a blood sample and another because a severe pulmonic stenosis was detected. The 34 remaining dogs were divided into two groups (control and MR) based on their jet size. Descriptive data for the two groups are shown in Table 1. None of the dogs showed any decisive abnormalities on routine biochemical analysis and urinalysis. There was a higher percentage of females in the MR group, however, this was not statistically significant. Otherwise there were no statistically significant differences between the descriptive characteristics (age, body weight, hematocrit and platelet count) of the two groups. 3.2. Platelet function Longer CT were found in the MR group compared to control (Fig. 1, Table 2, P = 0.03). The CT was not affected by age, gender, body weight, hematocrit or platelet count. Only five Dachshunds responded to ADP in the whole blood aggregometer. The five responses observed did not appear to be related to the MR status of the dogs, however, the data were too few to perform a correct statistical analysis.
Fig. 1. Closure time (s) measured by use of a platelet function analyzer (PFA-100) with collagen and adenosine diphosphate as the combined agonist. The Dachshunds with mild mitral regurgitation (MR) has significantly longer closure times compared to the Dachshunds with no or minimal MR.
Table 2 Platelet function markers in the two dog groups Dachshunds
Control, no or minimal MR (n = 22)
MR, mild MR (n = 12)
Col + ADP closure time (s) Plasma fibrinogen (mg/ml) Plasma vWf (%) vWf multimer (number of bands)
77.4 ± 16.8 1.96 (1.72–2.43) 68 ± 16 14 (12–14)
90.3 ± 14.8* 2.40 (1.98–2.90) 65 ± 21 14 (10–14)
MR, mitral regurgitation; vWf, von Willebrand factor, Col, collagen; ADP, adenosine diphosphate; data are shown as means ± SD values or as medians and 25th to 75th percentiles. * P < 0.05 vs. control.
There were no significant differences in plasma fibrinogen or vWf levels between the two groups (Table 2). Furthermore, the vWf multimer numbers were the same in both groups (Table 2). There was a significant correlation between CT and plasma vWf concentration (r2 = 0.287; P = 0.0015, Fig. 2) and CT and plasma fibrinogen concentration (r2 = 0.17; P = 0.033, Fig. 2). The correlation between CT and vWf multimer numbers was borderline significant (r2 = 0.197; P = 0.066, Fig. 2). Finally, there was a significant correlation between plasma vWf concentration and vWf multimer number (r2 = 0.296; P = 0.0009). 4. Discussion In this study, the platelet function in Dachshunds with mild MR was examined and compared to Dachshunds with no or minimal MR. Previously, it has been shown that CKCS with mild to severe MR have prolonged CT (Tarnow et al., 2003, 2004). In this study, a similar prolongation of CT was observed in Dachshunds with mild MR. It therefore seems likely that CT measured with the PFA-100 is independent of breed and is associated with the degree of MR. However, it is not known whether platelet dysfunction plays a role in the progression of MMVD and further studies addressing the prognostic value of CT with regard to developing congestive heart failure are needed. In dogs with MMVD both higher and lower aggregation responses have been found (Olsen et al., 2001; Tanaka and Yamane, 2000; Tarnow et al., 2005). However, these studies differed regarding breed, control group, assessment technique and severity of disease and it is therefore difficult to draw direct comparisons. In this study, only few of the samples aggregated when ADP was added in the whole blood aggregometer, and in most samples there was a lack of aggregation response. In a previous study, we have seen different whole blood aggregation responses when comparing CKCS with Boxers, Cairn terriers and Labrador retrievers using the same equipment and ADP concentration as used in this study (Nielsen et al., 2007). The majority of the three other breeds had very low aggregation responses to ADP, and in line with these results, this study shows that there are significant breed variations in ADP-induced platelet aggregation in dogs. The mechanism of this heterogeneity in canine whole blood aggregation responses is unknown, but may be due to genetic differences which could reflect variations in ectonucleotidase NTPDase1 (CD39) activity (Glenn et al., 2005). There was a statistically significant correlation between plasma vWf concentration and vWf multimer number. Furthermore, plasma vWf concentration correlated significantly with CT as has been shown in CKCS (Tarnow et al., 2004). As can be seen in Fig. 2 there was some scattering in the correlations and the coefficients of determination were quite low. This suggests poor to moderate correlations which may also be influenced by other variables than the ones tested in this study. Previous studies have shown an association between a loss of HMWM and prolonged CT in CKCS with MR and dogs with subaortic stenosis (Tarnow et al.,2004, 2005). In this
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Fig. 2. Platelet function analyzer (PFA-100) closure times shown as a function of high molecular weight von Willebrand factor (vWf) multimers (A) (r2 = 0.197; P = 0.066), plasma vWf concentration (B) (r2 = 0.287; P = 0.0015) and plasma fibrinogen concentration (C) (r2 = 0.17; P = 0.033). Finally, plasma vWf concentration is shown as a function of high molecular weight vWf multimers (D) (r2 = 0.296; P = 0.0009).
study, the association between HMWM and CT was only borderline significant. However, the dogs only had mild MR (jet size > 15% and 650%), whereas the dogs in previous studies had moderate to severe MR (jet size P 50%) (Tarnow et al., 2004, 2005). The measurement of HMWM is not a very sensitive test and in order to see a significant effect on CT more severe stages of MR are probably needed. An increase in plasma fibrinogen and vWf concentration has been reported in human patients with MR (Goldsmith et al., 2000; Lip et al., 1996). In CKCS with MR no difference in fibrinogen concentration was found, but a decrease in vWf explained by a loss of HMWM, has been shown (Tarnow et al., 2004). In this study, there was no difference in plasma vWf and plasma fibrinogen concentration between the two groups. This may indicate that the platelet dysfunction observed in Dachshunds with MR is less dependent on changes in vWf. However, the lack of difference in plasma vWf and vWf multimers may also be due to the MR status of the Dachshunds which was less severe than in the CKCS study. A previous study has suggested that a decreased sensibility or exhaustion of the platelets may occur secondary to the chronic shear stress in MMVD (Tanaka and Yamane, 2000) and thereby explain the platelet dysfunction. This may also explain the prolonged CT associated with MR in this study. In this study, a platelet dysfunction was shown, whereas platelet aggregation did not appear to be affected in Dachshunds with MR. This may be due to the fact that the dogs only had mild MMVD
without clinical signs, and that very few dogs had a measurable aggregation response in whole blood. The consequences of the altered platelet function in dogs with MMVD are not yet clear. Studies in humans and dogs have shown a relationship between activated platelets and vascular changes such as atherosclerosis, thrombosis and intimal hyperplasia (Anderson et al., 2001; Nomura et al., 1998). Furthermore, both human and canine studies have shown an association between intramyocardial coronary arteriosclerosis and an increased risk of sudden death (Burke and Virmani, 1998; Falk and Jonsson, 2000). Dogs with heart failure due to MMVD have a significant occurrence of small vessel arteriosclerosis compared to age-matched dogs euthanized due to non-cardiac reasons (Falk et al., 2006), and a direct association between the severity of arterial changes and clinical signs of MMVD in dogs has been shown (Falk et al., 2007). Thus, it has been suggested that the arterial changes could be part of a more generalized disease process and that activated platelets might cause an accelerated disease progression by development of vascular changes and possibly microthrombi in the myocardium (Tarnow et al., 2005). However, the possible association between arterial changes and platelet dysfunction still remains to be established. This study is limited by small group size, limiting the power of the study. The fact that only one breed was used reduces the within group variation, but specific breed variations may also influence
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the results. However, previous studies in another breed (CKCS) performed in the same laboratory showed similar results regarding CT suggesting that breed differences are less important (Tarnow et al., 2004). In this study, only dogs with mild MR were included. It would be interesting to include a group of Dachshunds with clinical signs of MMVD in order to study whether CT was further increased in more severe stages of the disease, and whether this might affect the platelet aggregation response. In a previous study, CKCS with moderate to severe MR (jet P 50%) and dogs with subaortic stenosis had slightly longer CT than what was shown in the MR group in this study indicating that CT may increase further as the severity of the disease increases (Tarnow et al., 2005). This study showed that mild MR in Dachshunds is associated with prolonged CT indicating platelet dysfunction. The CT was significantly correlated with plasma vWf and fibrinogen concentration. Only few of the Dachshunds responded to ADP in a whole blood aggregometer. These findings encourage further studies of the use of CT as a prognostic marker of MMVD development in dogs. Acknowledgements The work was supported by the Danish Medical Research Council. The authors thank Birgitte Holle, Department of Basic Animal and Veterinary Sciences and Natascha Errebo, Department of Small Animal Clinical Sciences, The University of Copenhagen, Denmark for their excellent technical assistance. Kirsten Christiansen and Jørgen Ingerslev, Skejby University Hospital, Århus, Denmark are thanked for assistance with the vWf multimer analysis. References Anderson, H.V., McNatt, J., Clubb, F.J., Herman, M., Maffrand, J.P., DeClerck, F., Ahn, C., Buja, L.M., Willerson, J.T., 2001. Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemicatherosclerotic canine coronary arteries. Circulation 104, 2331–2337. Beardow, A.W., Buchanan, J.W., 1993. Chronic mitral-valve disease in Cavalier King Charles spaniels – 95 cases (1987–1991). Journal of the American Veterinary Medical Association 203, 1023–1029. Buchanan, J.W., 1977. Chronic valvular disease (endocardiosis) in dogs. Advances in veterinary science and comparative medicine 21, 872–874. Burke, A.P., Virmani, R., 1998. Intramural coronary artery dysplasia of the ventricular septum and sudden death. Human Pathology 29, 1124–1127. Cornell, C.C., Kittleson, M.D., Della Torre, P., Häggstrom, J., Lombard, C.W., Pedersen, H.D., Vollmar, A., Wey, A., 2004. Allometric scaling of M-mode variables in normal adult dogs. Journal of Veterinary Internal Medicine 18, 311–321. Cowan, S.M., Bartges, J.W., Gompf, R.E., Hayes, J.R., Moyers, T.D., Snider, C.C., Gerard, D.A., Craft, R.A., Muenchen, R.A., Carroll, R.C., 2004. Giant platelet disorder in the Cavalier King Charles spaniel. Experimental Hematology 32, 344–350. Eksell, P., Haggstrom, J., Kvart, C., Karlsson, A., 1994. Thrombocytopenia in the Cavalier King Charles spaniel. Journal of Small Animal Practice 35, 153–155. Falk, T., Jonsson, L., 2000. Ischaemic heart disease in the dog: a review of 65 cases. Journal of Small Animal Practice 41, 97–103. Falk, T., Jonsson, L., Olsen, L.H., Pedersen, H.D., 2006. Arteriosclerotic changes in the myocardium, lung, and kidney in dogs with chronic congestive heart failure and myxomatous mitral valve disease. Cardiovascular Pathology 15, 185–193. Falk, T., Jonsson, L., Olsen, L.H., Tarnow, I., Ledersen, H.D., 2007. Correlation of cardiac pathology and clinical findings in dogs with naturally occurring congestive heart failure. Journal of Veterinary Internal Medicine 21, 636. Glenn, J.R., White, A.E., Johnson, A., Fox, S.C., Behan, M.W.H., Dolan, G., Heptinstall, S., 2005. Leukocyte count and leukocyte ecto-nucleotidase are major
determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood. Platelets 16, 159–170. Goldsmith, I.R.A., Blann, A.D., Patel, R.L., Lip, G.Y.H., 2000. Reduced indexes of left atrial hypercoagulability in patients with severe mitral regurgitation. American Journal of Cardiology 86, 234–236. Haggstrom, J., Hansson, K., Kvart, C., Swenson, L., 1992. Chronic valvular disease in the Cavalier King Charles spaniel in Sweden. Veterinary Record 131, 549–553. Häggstrom, J., Pedersen, H.D., Kvart, C., 2004. New insights into degenerative mitral valve disease in dogs. Veterinary Clinics of North America – Small Animal Practice 34, 1209–1226. Kundu, S.K., Heilmann, E.J., Sio, R., Garcia, C., Davidson, R.M., Ostgaard, R.A., 1995. Description of an in-vitro platelet-function analyzer – Pfa-100(Tm). Seminars in Thrombosis and Hemostasis 21, 106–112. Lip, G.Y.H., Rumley, A., Dunn, F.G., Lowe, G.D.O., 1996. Thrombogenesis in mitral regurgitation and aortic stenosis. Angiology 47, 1117–1125. Nielsen, L.A., Zois, N.E., Pedersen, H.D., Olsen, L.H., Tarnow, I., 2007. Platelet function in dogs: breed differences and effect of acetylsalisylic acid administration. Veterinary Clinical Pathology 36, 267–273. Nomura, E., Kohriyama, T., Yamaguchi, S., Kajikawa, H., Nakamura, S., 1998. Association between carotid atherosclerosis and hemostatic markers in patients with cerebral small artery disease. Blood Coagulation and Fibrinolysis 9, 55–62. Olsen, L.H., Fredholm, M., Pedersen, H.D., 1999. Epidemiology and inheritance of mitral valve prolapse in Dachshunds. Journal of Veterinary Internal Medicine 13, 448–456. Olsen, L.H., Kristensen, A.T., Haggstrom, J., Jensen, A.L., Klitgaard, B., Hansson, H., Pedersen, H.D., 2001. Increased platelet aggregation response in Cavalier King Charles spaniels with mitral valve prolapse. Journal of Veterinary Internal Medicine 15, 209–216. Pedersen, H.D., Kristensen, B.O., Lorentzen, K.A., Koch, J., Jensen, A.L., Flagstad, A., 1995. Mitral-valve prolapse in 3-year-old healthy Cavalier-King-CharlesSpaniels – an echocardiographic study. Canadian Journal of Veterinary Research – Revue Canadienne de Recherche Veterinaire 59, 294–298. Pedersen, H.D., Kristensen, B.O., Norby, B., Lorentzen, K.A., 1996. Echocardiographic study of mitral valve prolapse in dachshunds. Journal of Veterinary Medicine Series A – Zentralblatt fur Veterinarmedizin Reihe A – Physiology Pathology Clinical Medicine 43, 103–110. Pedersen, H.D., Haggstrom, J., Falk, T., Mow, T., Olsen, L.H., Iversen, L., Jensen, A.L., 1999a. Auscultation in mild mitral regurgitation in dogs: observer variation, effects of physical maneuvers, and agreement with color Doppler echocardiography and phonocardiography. Journal of Veterinary Internal Medicine 13, 56–64. Pedersen, H.D., Lorentzen, K.A., Kristensen, B.O., 1999b. Echocardiographic mitral valve prolapse in Cavalier King Charles spaniels: epidemiology and prognostic significance for regurgitation. Veterinary Record 144, 315–320. Pedersen, H.D., Haggstrom, J., Olsen, L.H., Christensen, K., Selin, A., Burmeister, M.L., Larsen, H., 2002. Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles spaniels is an autosomal recessive trait. Journal of Veterinary Internal Medicine 16, 169–173. Pedersen, H.D., Schutt, T., Sondergaard, R., Qvortrup, K., Olsen, L.H., Kristensen, A.T., 2003. Decreased plasma concentration of nitric oxide metabolites in dogs with untreated mitral regurgitation. Journal of Veterinary Internal Medicine 17, 178– 184. Swenson, L., Haggstrom, J., Kvart, C., Juneja, R.K., 1996. Relationship between parental cardiac status in Cavalier King Charles spaniels and prevalence and severity of chronic valvular disease in offspring. Journal of the American Veterinary Medical Association 208, 2009–2012. Tanaka, R., Yamane, Y., 2000. Platelet aggregation in dogs with mitral valve regurgitation. American Journal of Veterinary Research 61, 1248–1251. Tanaka, R., Murota, A., Nagashima, Y., Yamane, Y., 2002. Changes in platelet life span in dogs with mitral valve regurgitation. Journal of Veterinary Internal Medicine 16, 446–451. Tarnow, I., Kristensen, A.T., Texel, H., Olsen, L.H., Pedersen, H.D., 2003. Decreased platelet function in Cavalier King Charles spaniels with mitral valve regurgitation. Journal of Veterinary Internal Medicine 17, 680–686. Tarnow, I., Kristensen, A.T., Olsen, L.H., Pedersen, H.D., 2004. Assessment of changes in hemostatic markers in Cavalier King Charles spaniels with myxomatous mitral valve disease. American Journal of Veterinary Research 65, 1644–1652. Tarnow, I., Kristensen, A.T., Olsen, L.H., Falk, T., Haubro, L., Pedersen, L.G., Pedersen, H.D., 2005. Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution. Journal of Veterinary Internal Medicine 19, 515–522.