- Top of page
- Materials and Methods
Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs with spontaneous disease.
Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease.
Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay that measured TAFI activity relative to activity in standardized pooled human plasma.
Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2–70.8%) compared with 62.6% (29.1–250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual activities (>225%) were in 3 dogs with malignant neoplasms and 1 dog with thrombocytopenia. For data grouped by diagnosis, median TAFI activity was 61.7% for benign neoplasia (n=5), 64.9% for malignant neoplasia (n=8), 75.5% for Angiostrongylus vasorum infection (n=4), 68.8% for bacterial sepsis (n=7), and 58.7% for miscellaneous diseases (n=11). Compared with TAFI activity in control dogs, median TAFI activity was significantly increased only in the group of dogs with bacterial sepsis.
Conclusion: Bacterial sepsis was associated with significantly increased TAFI activity, and individual dogs with increased TAFI activities were found in all disease groups. The role of TAFI in the pathogenesis of hemostatic disorders in dogs and its value as a prognostic indicator deserve further investigation.
- Top of page
- Materials and Methods
Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U, was recently identified as a potent downregulator of fibrinolysis.1,2 Antifibrinolytic action of TAFI is primarily mediated through removal of carboxy-terminal lysine and arginine from fibrin, thereby decreasing plasminogen activation and attenuating positive feedback expressed by lys-plasminogen formation during fibrinolysis. However, at high concentrations TAFI also directly inhibits plasmin.3 Large concentrations of thrombin, generated by thrombin-mediated activation of factor XI, are required to activate TAFI.4,5 Thrombomodulin markedly enhances this activation, indicating that the thrombin-thrombomodulin complex is the likely physiologic activator of TAFI.6 TAFI antigen and activity may be measured in plasma based on binding of anti-TAFI antibody or substrate cleavage, respectively.
In people increased TAFI antigen has been associated with increased risk of venous thrombosis and recurrent pulmonary thromboembolism7,8 and has been found in various diseases with increased thrombotic potential, such as cancer,9,10 inflammatory bowel disease,11 and polycystic ovary syndrome.12 At the other end of the scale, decreased TAFI activity has been implicated as a contributor of bleeding diathesis in people with acute promyelocytic leukemia13 and in people with factor XI deficiency.4,14 Decreased TAFI antigen and TAFI activity have been identified in people with sepsis and infectious disease, respectively.15,16 Furthermore, decreased TAFI antigen has been identified in some studies of disseminated intravascular coagulation in people.15 A recent study suggests that TAFI may also serve as a downregulator of inflammation.17
TAFI represents an essential link between coagulation and fibrinolytic cascades, and determination of TAFI activity will likely provide new insight into the dynamics and pathogenesis of hemostatic disorders. TAFI activity in dogs has been described only in experimental models of thrombosis,18,19 and to the authors' knowledge there have been no studies of TAFI activity in dogs with spontaneous disease. Thus, the aim of this exploratory study was to measure and compare TAFI activity in healthy dogs and in dogs with spontaneous diseases, including some in which hemostatic aberrations were likely to be present.
- Top of page
- Materials and Methods
The observed intra- and interassay imprecision at various TAFI activities is given in Table 1. Detection limit of the assay was assessed to 7.4% (mean 2.6%, SD 1.6%). Data from dogs were retrospectively assigned to 5 different groups based on diagnoses: benign neoplasia (5 dogs), malignant neoplasia (8 dogs), angiostrongylosis (4 dogs), bacterial sepsis (7 dogs), and miscellaneous diseases (11 dogs). Sex, breed, age, final diagnosis, and TAFI activity of all dogs are listed in Table 2.
Table 1. Intra- and interassay imprecision at various TAFI activities in canine plasma samples.
| ||No. of Samples||Mean Value (%)||SD (%)||CV (%)|
Table 2. Signalment, diagnosis, and thrombin-activatable fibrinolysis inhibitor (TAFI) activity of healthy Beagles and 35 dogs with diseases.
| ||Breed||Age||Sex||Clinical Status||TAFI Activity (%)|
|Controls (n=20)||Beagles||2–5 years||12 F 8 MC||Clinically healthy||32.2–70.9 (range)|
| 1||Dachshund||1.5 years||F||Lymphoma (stage IVa)||250.0|
| 2||Bullmastiff||9 years||MC||Lymphoma (stage IIIb)||60.5|
| 3||Dachshund||9 years||F||Mammary adenocarcinoma (stage II)||35.1|
| 4||West Highland White Terrier||13 years||F||Bronchoalveolar carcinoma||227.0|
| 5||American Bulldog||4.5 years||MC||Malignant mesenchymal tumor (oral)||58.0|
| 6||Labrador Retriever||9 years||F||Nasal carcinoma||38.0|
| 7||Mixed breed||8 years||M||Scrotal mastocytoma grade III (stage I)||250.0|
| 8||Cairn Terrier||4 years||MC||Cutaneous melanoma||69.2|
| 9||Maltese||8.5 years||FS||Mammary gland adenoma||55.2|
|10||Golden Retriever||9.5 years||F||Mammary gland adenoma||88.3|
|11||Rhodesian Ridgeback||8.5 years||F||Mammary gland adenoma and lipoma||81.7|
|12||Border Collie||2.5 years||FS||Squamous papilloma (vulva)||61.7|
|13||Mixed breed||10 years||FS||Skin hamartoma, not infected or inflamed||37.7|
|14||Basset Fauve de Bretagne||1.3 years||M||Angiostrongylus vasorum||54.9|
|15||Labrador Retriever||1 year||M||Angiostrongylus vasorum||39.6|
|16||Dachshund||14 years||FS||Angiostrongylus vasorum||117.0|
|17||Mixed breed||6 months||F||Angiostrongylus vasorum||96.1|
|18||Portuguese Water Dog||12 years||FS||Bacterial sepsis||82.9|
|19||Dachshund||14.2 years||FS||Bacterial sepsis||110.0|
|20||Basset Hound||9 years||M||Bacterial sepsis||29.1|
|21||Dalmatian||9.8 years||FS||Bacterial sepsis||57.7|
|22||Doberman||10 years||FS||Bacterial sepsis||62.6|
|23||Mixed breed||12 years||FS||Bacterial sepsis||68.8|
|24||Australian Shepherd||8 years||MC||Bacterial sepsis||96.1|
|25||Standard Poodle||6.6 years||FS||GDV||40.5|
|26||German Shepherd||1.7 years||MC||Hemorrhagic gastroenteritis||29.1|
|27||Mixed breed||6 years||M||Heatstroke||140.0|
|28||Dachshund||12.8 years||M||Bite wound||70.8|
|29||Labrador Retriever||12 years||M||Pancreatitis||119.0|
|30||West Highland White Terrier||5 years||FS||Snake bite||30.6|
|31||Border Collie||9.3 years||MC||Thrombocytopenia||58.7|
|32||Labrador Retriever||9 years||MC||Thrombocytopenia||250.0|
|33||Labrador Retriever||9.8 years||FS||Hit by car||32.2|
|34||Labrador Retriever||6.9 years||MC||GDV with gastric perforation||98.6|
|35||Boxer||1.2 years||FS||Hit by car||40.5|
Median TAFI activity in control dogs was 46.1% (range 32.2–70.8%). Median (range) for all dogs diagnosed with clinical abnormalities was 62.6% (29.1–250%). TAFI activity in dogs with clinical abnormalities was significantly increased compared with control dogs (P=0.023). There were 14 of 35 (40%) affected dogs with values above the range of controls and 3 dogs (8.6%) with values slightly below. The highest individual TAFI activities (above 225%) were observed in 3 dogs with malignant neoplasms and 1 dog with thrombocytopenia. Analyzed by groups, median (range) of TAFI activities were: 61.7% (37.7–88.3%) for benign neoplasia; 64.9% (35.1–250%); for malignant neoplasia; 75.5% (39.6–117%) for A. vasorum infection; 68.8% (29.1–110%) for bacterial sepsis; and 58.7% (29.1–250%) for miscellaneous diseases (Figure 1). Compared with TAFI activity in control dogs, median TAFI activity was significantly increased only in the group of dogs with bacterial sepsis (P=.025; Figure 1), but only 3 of those 7 dogs had values greater than the upper limit of controls and 1 dog had a low value.
Figure 1. Box and whisker plot of thrombin-activatable fibrinolysis inhibitor (TAFI) activity in healthy dogs and in dogs with various diseases. The top and bottom of the box indicate the upper and lower quartile, respectively, and the horizontal line is the median value. Whiskers are minimum and maximum values. Compared with healthy dogs, P-values are .023 for all diseases, .126 for benign neoplasia, .088 for malignant neoplasia, .188 for angiostrongylosis, .025 for bacterial sepsis, and .563 for miscellaneous diseases.
Download figure to PowerPoint
- Top of page
- Materials and Methods
More than one-third of the dogs in this study had TAFI activities above the upper limit of control dogs. Interestingly, 3 of the 4 TAFI activities of greatest magnitude were found in dogs with malignant neoplasms. In people significantly increased TAFI activities and plasma antigen concentrations have been found to contribute to the hypercoagulable state in various types of lung cancers.9,10 A recent study of 30 dogs with neoplasia demonstrated that the majority of dogs with malignant neoplasms are hypercoagulable.20 Whether the same relationship between malignancies, thrombosis, and TAFI activity exists in dogs has yet to be investigated.
Observed TAFI activities in dogs with bacterial sepsis in our study were significantly different from TAFI activities in healthy dogs. Knowledge of the role of TAFI in host responses to severe infections is limited. In people reduced levels of TAFI antigen and TAFI activity have been identified in septic patients and in patients with infectious disease, respectively.15,16 In the sepsis study a low median TAFI antigen level was identified in a group of septic patients compared with a group of nonseptic patients though most of the individual TAFI antigen levels in septic patients were within the reference interval.16 In that study low TAFI antigen levels were speculated to be secondary to consumption due to a positive correlation between TAFI antigen and various coagulation factors (FII and FXI) though there was no correlation between overt DIC and TAFI antigen levels.16 Other possible explanations for low TAFI antigen levels in human sepsis include extravasation of proteins due to increased vascular permeability and genetic polymorphisms. In contrast upregulation of TAFI mRNA and circulating TAFI plasma concentrations have been found in response to endotoxin exposure in rodents and in a murine model of Escherichia coli peritonitis.21 In the latter model TAFI plasma antigen levels decreased by 20% in the early phase of infection but increased 2.5-fold after 20 hours.21 One out of 7 septic dogs in our study had a TAFI activity below the minimum value for healthy dogs and 3/7 septic dogs had TAFI activities above the maximum level of healthy dogs. Based on these results it seems reasonable to assume that bacterial sepsis in dogs likely leads to upregulation of TAFI at some stage and that plasma TAFI activity is determined by the balance between production and consumption or loss at a given point in time.
In our study a wide range of TAFI activity was established in a homogenous group of healthy research beagle dogs. Polymorphism in the promoter gene of TAFI has been identified in people; however, variation in TAFI antigen levels in healthy people22 may correlate with immunoreactivity of various isoforms when measuring antigen concentrations rather than being true differences in activity.23 Other risks of assay-related bias have been identified, but the application of an activity-based kinetic assay with substrates selectively detecting TAFI activity in the present study minimized those risks.23 Whether breed-related differences in TAFI antigen levels or TAFI activity occur in dogs is currently unknown. However, use of an unmatched control group with regard to phenotype, genotype, and environment is a weakness of this study. For future clinical studies, TAFI reference intervals should be established in a larger group of dogs that represent the population of dogs where the assay is applied.
The assay used in this study measured TAFI with intra-assay variation that is comparable to that reported by the manufacturer for human plasma samples and is considered acceptable for diagnostic purposes. On the other hand imprecision, as indicated by the interassay variation, may pose a problem if the assay is used for diagnostic purposes. The assay will only be able to consistently detect differences exceeding the interassay CV, and there is substantial overlap of TAFI activities between groups of possible clinical interest. However, running samples in batches permits detection of much smaller differences between groups. Thus, the assay seems applicable for group comparison of TAFI activity in future studies of the pathophysiologic significance of abnormal TAFI activity. The detection limit appears acceptable as it was well below observed activities. A study of linearity was not performed in the present study to confirm the quantitative nature of the assay. However, it is assumed that observed levels in the study are ranked correctly, and thus assumptions of nonparametric statistics are fulfilled.
In this study we used plasma that had been stored for up to 21 months for the majority of the diseased dogs and 32 months for a single dog with A. vasorum infection. The effect of long-term plasma storage on TAFI activity is unknown and could potentially result in protein degradation. Thus, prolonged storage and differences in plasma storage time between control and disease groups are weaknesses of this study. However, except for the dogs with A. vasorum infection, the disease groups were comparable with regard to plasma storage time both within and between groups, and the effect of prolonged storage should theoretically affect all groups in a similar way. If the effect of storage time on TAFI activity was substantial enough to cause a statistically significant difference between control and disease groups, we would have expected to see this difference in all disease groups, not the sepsis group only. Therefore, the significant difference between the sepsis and controls was attributed to factors associated with sepsis.
In conclusion, bacterial sepsis was associated with significantly increased TAFI activity, and at the same time individual dogs with abnormal TAFI activities were found in all disease groups. Further investigation of TAFI activity may help elucidate the complex pathophysiologic mechanisms leading to hemostatic dysfunction in disease. The role of TAFI in the pathogenesis of thrombosis and hemorrhagic diathesis in dogs with primary hemostatic disorders and hemostatic disorders secondary to systemic, inflammatory, or neoplastic diseases deserves further investigation. The clinical consequences of increased or decreased TAFI activity is currently unknown for dogs, and changes in activity potentially have prognostic implications for individual animals.