Marjory B. Brooks

Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia.

Hemostatic parameters were prospectively measured in 20 dogs with primary immune-mediated hemolytic anemia. Eight of 20 dogs had received prior treatment with prednisone. Activated partial thromboplastin time was increased in nine dogs; one-stage prothrombin time was increased in two dogs; fibrinogen concentration was increased in 17 dogs; and antithrombin activity was decreased in 10 dogs. Fibrin(ogen) degradation products concentration was increased in 12 dogs, and D-dimer concentration was increased in 16 dogs. Four or more laboratory criteria of disseminated intravascular coagulation (DIC) were present in nine dogs, and three criteria of DIC were found in four additional dogs. Thromboembolism was the most common finding in the dogs that died. In this study population, mortality was not significantly associated with any clinical finding or laboratory variable.

Hypercoagulability in Cats with Cardiomyopathy

BACKGROUND Arterial thromboembolism (ATE) is a common complication of feline cardiomyopathy; however, the pathogenesis of ATE is unknown. HYPOTHESIS Systemic activation of the coagulation cascade (hypercoagulability) and endothelial injury promote ATE in cardiomyopathic cats. ANIMALS Healthy cats (n = 30) and 3 groups of cardiomyopathic cats: Group (1) left atrial enlargement only (LAE [n = 11]), ie, left atrial to aortic ratio >1.4; Group (2) LAE with spontaneous echocardiographic contrast, atrial thrombi or both (SEC-T [n = 16]); and Group (3) acute ATE with LAE (n = 16). METHODS Hypercoagulability was defined by 2 or more laboratory abnormalities reflecting coagulation factor excess (high fibrinogen concentration or Factor VIII coagulant activity), inhibitor deficiency (low antithrombin activity), or thrombin generation (high thrombin-antithrombin complex [TAT] and d-dimer concentrations). High von Willebrand factor antigen concentration (vWF : Ag) was considered a marker of endothelial injury. Data were analyzed using nonparametric statistics. RESULTS The 3 groups of cats with cardiac disease had higher median fibrinogen concentrations than did the healthy cats. Criteria of hypercoagulability were found exclusively in cats with SEC-T (50%) and ATE (56%). Hypercoagulability was not associated with left atrial size or congestive heart failure (CHF). ATE cats had significantly higher median vWF : Ag concentration than did the other groups. CONCLUSION AND CLINICAL IMPORTANCE Systemic hypercoagulability is evident in many cardiomyopathic cats, often without concurrent CHF or overt ATE. Hypercoagulabilty may represent a risk factor for ATE. High vWF : Ag in ATE cats was attributed to downstream endothelial injury from the occlusive thrombus.

Scott syndrome dogs have impaired coated-platelet formation and calcein-release but normal mitochondrial depolarization

Dual agonist stimulation of platelets with thrombin plus collagen, or thrombin plus convulxin (a ligand of the collagen receptor glycoprotein VI), generates a subpopulation of highly procoagulant cells known as coated platelets [1]. Coatedplatelets represent approximately 30% of the total platelet population and display a combination of characteristic features including expression of phosphatidylserine (PS), surface retention of alpha granule proteins, permeability to calcein, and release of membrane-derived microparticles [2–6]. Furthermore, recent studies demonstrate that loss of mitochondrial membrane potential via formation of a mitochondrial permeability transition pore (MPTP), a key feature of the intrinsic apoptotic pathway, is also an integral event leading to platelet membrane PS externalization and coated platelet formation [6]. While in vitro experimental systems indicate that the proportion of coated-platelets correlates with thrombin generation [3,5], the role of coated-platelets in physiologic and pathologic thrombus formation in vivo is still under investigation. In a series of experiments, we investigated the formation of coated-platelets utilizing a canine model of Scott syndrome. This trait was identified in a colony of German shepherd dogs (GSD) with an autosomal recessive bleeding diathesis characterized by a specific deficiency of platelet procoagulant activity [7]. Affected GSD demonstrate the pathognomonic platelet phenotype of Scott syndrome (i.e. a lack of PS expression and failure of membrane microvesiculation upon activation with calcium ionophore) [8,9]. Furthermore, Scott GSD platelets do not generate prothrombinase activity in response to ionophore or the physiologic agonists thrombin and collagen [7]. We hypothesized that dual agonist stimulation of Scott GSD platelets would not only fail to elicit PS externalization, but that Scott GSD platelets would be incapable of displaying the characteristic features of coated-platelets [1]. Coated-platelets were produced by thrombin plus convulxin stimulation of platelet-rich plasma (PRP) prepared from healthy control dogs and clinically affectedGSD.All activation experiments were performed in a 100 lL (total) assay volume containing 1 lL PRP and the following reagents (final concentrations): 1 mg mL BSA, 2 mM CaCl2, 1 mM MgCl2, 1 lg mL )1 biotin-fibrinogen, 500 ng mL convulxin, 0.5 U mL bovine thrombin, 0.4 mM gly-pro-arg-pro-NH2, 150 mMNaCl, and 10 mMHEPES, pH 7.5. The reaction tubes were incubated at 37 for 10 min, and individually labeled, as previously described [4,6,7,10], with the following specific markers of coated-platelets: Annexin V-fluorescein isothicyanate (FITC) to detect PS expression; phycoerythrin-streptavidin and FITC-abciximab to label surface bound biotinylated fibrinogen and identify platelets; calcein-AM to detect calcein release; and JC-1 to monitor the loss of mitochondrial membrane potential, denoting formation of the MPTP. Scott GSD platelets failed to bind fibrinogen, externalize PS or release calcein upon dual agonist stimulation, in contrast to

Examination of hemostatic parameters to detect hypercoagulability in dogs with severe protein‐losing nephropathy

Objective – To identify hemostatic abnormalities in dogs with protein-losing nephropathies (PLN) that represent risk factors for pathologic thrombosis. Design – Cross-sectional observational study of client-owned dogs with PLN, nonprotein losing renal failure (RF), and systemic illness (SI) exclusive of primary renal disease. Setting – Urban University Referral Center. Animals – A total of 29 dogs (n=11 PLN, n=7 RF, n=11 SI) were enrolled between January 2001 and July 2002. Samples were also collected from 20 clinically normal dogs to serve as hemostasis assay controls. Interventions – None. Hemostasis Testing – Citrate anticoagulated blood was collected for point-of-care testing with a viscoelastic monitor (thromboelastograph [TEG]) and citrate plasma was prepared for coagulation screening tests and specific assay of the following hemostatic proteins: antiplasmin, antithrombin, D-dimer, Factor VIII, fibrinogen, plasminogen, protein C, and von Willebrand factor. Results – Dogs with PLN and RF demonstrated TEG abnormalities consistent with hypercoagulability (eg, short clotting time, high clot amplitude) and both groups had significantly lower antithrombin than the SI group. The PLN dogs had significantly higher protein C than either the RF or SI group. Hyperfibrinogenemia was a consistent finding among all 3 disease groups, and the coagulation index a measure of hypercoagulability derived from TEG parameters, directly correlated with fibrinogen values of all study dogs. Conclusions – Hemostatic abnormalities consistent with systemic hypercoagulability are common in dogs with RF and PLN, however, no prothrombotic factors unique to PLN were identified in our study. The thrombotic tendency of PLN may therefore involve parameters we did not directly assess such as platelet reactivity, fibrinolysis, perturbations in blood flow, and/or endothelial dysfunction. High protein C is a novel finding in PLN dogs of unknown clinical relevance.OBJECTIVE To identify hemostatic abnormalities in dogs with protein-losing nephropathies (PLN) that represent risk factors for pathologic thrombosis. DESIGN Cross-sectional observational study of client-owned dogs with PLN, nonprotein losing renal failure (RF), and systemic illness (SI) exclusive of primary renal disease. SETTING Urban University Referral Center. ANIMALS A total of 29 dogs (n=11 PLN, n=7 RF, n=11 SI) were enrolled between January 2001 and July 2002. Samples were also collected from 20 clinically normal dogs to serve as hemostasis assay controls. INTERVENTIONS None. HEMOSTASIS TESTING: Citrate anticoagulated blood was collected for point-of-care testing with a viscoelastic monitor (thromboelastograph [TEG]) and citrate plasma was prepared for coagulation screening tests and specific assay of the following hemostatic proteins: antiplasmin, antithrombin, D-dimer, Factor VIII, fibrinogen, plasminogen, protein C, and von Willebrand factor. RESULTS Dogs with PLN and RF demonstrated TEG abnormalities consistent with hypercoagulability (eg, short clotting time, high clot amplitude) and both groups had significantly lower antithrombin than the SI group. The PLN dogs had significantly higher protein C than either the RF or SI group. Hyperfibrinogenemia was a consistent finding among all 3 disease groups, and the coagulation index a measure of hypercoagulability derived from TEG parameters, directly correlated with fibrinogen values of all study dogs. CONCLUSIONS Hemostatic abnormalities consistent with systemic hypercoagulability are common in dogs with RF and PLN, however, no prothrombotic factors unique to PLN were identified in our study. The thrombotic tendency of PLN may therefore involve parameters we did not directly assess such as platelet reactivity, fibrinolysis, perturbations in blood flow, and/or endothelial dysfunction. High protein C is a novel finding in PLN dogs of unknown clinical relevance.

A sensitive immunoassay for von Willebrand factor

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.

Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005–2006)

OBJECTIVE To characterize clinical signs, clinicopathologic features, treatments, and survival in dogs with naturally acquired foodborne aflatoxicosis. DESIGN Retrospective case series. ANIMALS 72 dogs that consumed aflatoxin-contaminated commercial dog food. PROCEDURES Medical records of affected dogs were reviewed. Between December 2005 and March 2006, dogs were identified as having foodborne aflatoxin hepatotoxicosis on the basis of the history of consumption of contaminated food or characteristic histopathologic lesions (subject dog or a recently deceased dog in the same household or kennel). Recorded information included signalment, clinical features, clinicopathologic test results, treatments, and survival. Data were analyzed by survival status. RESULTS Most dogs were of large breeds from breeding kennels. No significant differences were found in age or weight between 26 (36%) survivor dogs and 46 (64%) nonsurvivor dogs. Severity of clinical signs varied widely; 7 dogs died abruptly. In order of onset, clinical features included anorexia, lethargy, vomiting, jaundice, diarrhea (melena, hematochezia), abdominal effusion, peripheral edema, and terminal encephalopathy and hemorrhagic diathesis. Common clinicopathologic features included coagulopathic and electrolyte disturbances, hypoproteinemia, increased serum liver enzyme activities, hyperbilirubinemia, and hypocholesterolemia. Cytologic hepatocellular lipid vacuolation was confirmed in 11 dogs examined. In comparisons of clinicopathologic test results between survivor and nonsurvivor dogs, only granular cylindruria (7/21 dogs) consistently predicted death. Best early markers of aflatoxicosis were low plasma activities of anticoagulant proteins (protein C, antithrombin) and hypocholesterolemia. Despite aggressive treatment, many but not all severely affected dogs died. CONCLUSIONS AND CLINICAL RELEVANCE Serum liver enzyme activities and bilirubin concentration were unreliable early markers of aflatoxin hepatotoxicosis in dogs. Hypocholesterolemia and decreased plasma protein C and antithrombin activities may function as exposure biomarkers.

Effect of a Single Plasma Transfusion on Thromboembolism in 13 Dogs With Primary Immune-Mediated Hemolytic Anemia

Thirteen dogs with primary immune-mediated hemolytic anemia received fresh-frozen plasma within 12 hours of admission, in addition to unfractionated heparin and other therapies, such as prednisone, azathioprine, and packed red blood cell transfusion. Antithrombin activity was quantified prior to transfusion and at 30 minutes and 48 hours after transfusion. Plasma antithrombin activity did not change significantly after a single plasma transfusion. There were no deaths in the first 48 hours of treatment. Thromboembolism was identified at necropsy in six of 10 dogs that died within 12 months of admission. There was no significant difference in the incidence of thromboembolism between the current treatment group and a historical control group.

Postoperative Bleeding in Retired Racing Greyhounds

BACKGROUND Some retired racing Greyhounds (RRG) that undergo surgery bleed excessively. HYPOTHESIS Greyhounds that bleed excessively will have one or more preoperative hemostatic abnormalities that can be used to predict the risk and severity of postoperative bleeding. ANIMALS Eighty-eight RRG undergoing ovariohysterectomy or castration. METHODS All dogs were evaluated preoperatively with a physical exam, CBC, platelet count, OSPT, APTT, platelet function with PFA-100(a); fibrinogen, d-dimer, plasminogen (Plmg), antiplasmin (AP), antithrombin (AT), and vWF concentration (vWF:Ag); vWF collagen binding assay (vWF:CBA), and Factor XIII assay. Assays were repeated in the dogs that bled, and in an age- and sex-matched control group of RRG. RESULTS Twenty-six percent of the dogs had bleeding 36-48 hours after surgery. AP (P <.0001) and AT concentration (P= .007) were significantly lower, and vWF:CBA (P= .0284) was higher preoperatively in the dogs with excessive hemorrhage. A lower platelet count (P= .001) and hematocrit (P= .002), shorter OSPT (P= .0002) and higher plasma fibrinogen (P <.0001), and AP (P= .001) concentration were detected at the time of bleeding compared with preoperative values in the dogs that bleed excessively. The same findings were observed postoperatively for the control group, except for the decrease in hematocrit. CONCLUSIONS AND CLINICAL IMPORTANCE The results indicate that this excessive postoperative bleeding is not attributable to a primary or secondary hemostatic defect, but could result from altered fibrinolysis.

Incidence of Transfusion Reactions and Retention of Procoagulant and Anticoagulant Factor Activities in Equine Plasma

BACKGROUND The extent of preservation of clotting factors and incidence of transfusion reactions to noncommercial equine plasma is not documented. HYPOTHESIS Equine frozen plasma would retain its coagulation factor activity within the reference range and the incidence of transfusion reactions would be low. ANIMALS Ten plasma donor horses. Fifty clinically ill hospitalized horses receiving plasma were reviewed to determine the incidence of reactions. METHODS In vitro study and retrospective case review. Plasma was prepared by gravity sedimentation from whole blood refrigerated for 48 hours. The activities of factors VII through XII, antithrombin (AT), and Protein C were measured. Factor activities were compared for plasma samples obtained before blood collection (S0), after 48 hours of gravity sedimentation at 5 degrees C and after plasma separation (S1), and after 90 days of storage at -20 degrees C (S90). The medical records of 50 consecutive clinically ill horses receiving frozen plasma were reviewed to determine the incidence of transfusion reactions. RESULTS The combined effect of plasma harvest, gravity sedimentation, decantation, and freezing caused significant reductions in factors IX, (43%P= .0013), X, (33%P= .0001), XI, (48%P= .0008), AT, (10%P= .02), and Protein C (26%P= .0001). Activities for all factors analyzed, except factor X, remained within the reference ranges. Transfusion reactions were recorded for 5/50 horses. CONCLUSIONS AND CLINICAL RELEVANCE Clotting factors, AT, and Protein C were well preserved. The incidence of reactions to frozen plasma was 10%.

A Line 1 insertion in the Factor IX gene segregates with mild hemophilia B in dogs.

We undertook the biochemical and molecular characterization of hemophilia in a large pedigree of German wirehaired pointers. Males affected with hemophilia B had approximately 5% normal Factor IX coagulant activity and a proportional reduction of Factor IX protein concentration, indicative of a mild hemophilia B phenotype. Using Southern blot analyses and PCR amplification of genomic DNA, we discovered a large, 1.5-kb insertion in intron 5 of the Factor IX gene of an affected male. The insert consists of a 5′ truncated canine Line-1 followed by an approximately 200-bp 3′ poly (A) tract, flanked by a 15-bp direct repeat. The insert can be traced through at least five generations and segregates with the hemophilia B phenotype in this breed. This is the first description of a Factor IX mutation associated with mild hemophilia B in a non-human species and provides evidence for a recent Line-1 insertion event in the canine genome.