Study of TAMe (p-tosyl-L-arginine methyl ester) esterase activity of bovine plasma

The TAMe (p-tosyl-L-arginine methyl ester) esterase activity of mature and immature bovine plasma was studied and compared with the activity of this enzyme in human plasma. Kaolin activation of 2 minutes was required to produce maximal activation in cattle, as compared with 1 minute activation in man. The kaolin-activated TAMe esterase values in bovine plasma were approximately one-half the values found in human plasma. The activity of this enzyme was statistically greater in immature than in mature cattle (P less than 0.05) at kaolin activation times of 1, 2, 15, and 20 minutes.     

Glanzmann thrombasthenia in an Oldenbourg filly

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.     

Endotoxin-induced changes in the hemostatic system in neonatal calves: the effect of antiserum to a mutant Escherichia coli (J-5)

Changes in the hemostatic system were studied in 22 neonatal calves given a small dosage of Escherichia coli endotoxin (0.5 microgram/kg) by slow (5-hour) IV infusion. The effect of pretreatment with an antiserum to mutant of E coli O111:B4 (J-5) was evaluated. The platelet count, plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time changed significantly from base line during and after endotoxin infusions in all calves. The mean platelet count was significantly decreased from 1 through 24 hours after endotoxin infusion was started. Mean plasma fibrinogen was decreased 2 through 12 hours after endotoxin infusion was started. The mean prothrombin time and activated partial thromboplastin time were significantly greater than base line at 3 to 6 hours and 3 to 12 hours, respectively, after endotoxin infusion was started. Serum concentration of fibrinolytic degradation products remained less than 10 micrograms/ml. Bovine J-5 antiserum did not prevent the endotoxin-induced changes in the hemostatic system of these neonatal calves.     

Preoperative and postoperative hemostatic profiles of dogs undergoing ovariohysterectomy.

Hemostatic profiles were evaluated in 15 healthy dogs immediately before and 24 hours after celiotomy for routine ovariohysterectomy. Prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin degradation products, antithrombin III activity, platelet count, and hemogram were measured. There were no significant changes in prothrombin time, activated partial thromboplastin time, fibrin degradation products, antithrombin III activity, or platelet count. Fibrinogen concentration was significantly higher following surgery. Postoperative leukocyte differential counts were typical of stress leukograms, and were characterized by leukocytosis, neutrophilia, lymphopenia and eosinopenia. Mild decreases in packed cell volume, red blood cell count and hemoglobin concentration were consistent with minor blood loss during surgery or fluid retention and hemodilution postoperatively. It was concluded that celiotomy and routine ovariohysterectomy in healthy dogs did not alter hemostatic profiles 24 hours after surgery. Abnormal postoperative hemostatic profiles should not be attributed to surgery alone; other causes of abnormal hemostatic profiles should be investigated.     

Influence of an omega-3 fatty acid-enriched ration on in vivo responses of horses to endotoxin.

Because certain inflammatory processes are dependent on the fatty acid composition of the cellular membrane, dietary manipulations that replace omega-6 fatty acids with omega-3 fatty acids may modify inflammatory responses. We investigated the effect of supplemental dietary linseed oil, containing the omega-3 fatty acid, alpha-linolenic acid, on in vivo responses of horses to endotoxin. One group of horses (n = 6) was fed a control pelleted ration (0% linseed oil), and another group of horses (n = 6) was fed an 8% linseed oil pelleted ration. After 8 weeks of consuming these rations, all horses were given 0.03 micrograms of Escherichia coli 055:B5 endotoxin/kg of body weight, infused over 30 minutes. Horses were monitored over 24 hours. Compared with baseline values within each ration group, endotoxin infusion caused significant (P less than 0.05) increase in rectal temperature, heart rate, and plasma concentration of thromboxane B2, 6-keto-prostaglandin F1 alpha, and fibrinogen and significant (P less than 0.05) decrease in total WBC count. Compared with baseline values within each ration group, endotoxin infusion failed to cause significant changes in prothrombin, activated partial thromboplastin, thrombin, or whole blood recalcification times, serum concentration of fibrin degradation products, PCV, or plasma total protein concentration. Before and after endotoxin infusion, horses given the linseed oil ration had longer mean whole blood recalcification time and activated partial thromboplastin time than did horses fed the control ration.     

Values of three coagulation screening tests of precolostral calves.

Prothrombin times, partial thromboplastin times and platelet counts were performed to determine normal values and to screen for coagulation defects of precolostral calves. The precolostral calves were in two groups: one group of a few calves was tested two years before the second larger group. The results for both groups were similar. The tests were performed on postcolostral calves and on mature cows to compare their values with those of precolostral calves. The mean values of prothrombin times and partial thromboplastin times of precolostral calves in the first group were 18.8 seconds and 54.8 seconds respectively. The mean values of prothrombin times and partial thromboplastin times of precolostral calves in the second group were 18.8 seconds and 50.8 seconds respectively. The mean platelet count was 422,400/cmm for the first group and 482,800/cmm for the second group.     

Evaluation of point-of-care tests for diagnosis of disseminated intravascular coagulation in dogs admitted to an intensive care unit.

OBJECTIVE: To evaluate the accuracy of point-of-care tests for the diagnosis of disseminated intravascular coagulation (DIC) in dogs and assess the correlation and agreement of results between point-of-care and laboratory tests in the evaluation of hemostatic function. DESIGN: Prospective case series. ANIMALS: 59 critically ill dogs (affected dogs) with clinical signs of diseases known to predispose to DIC and 52 clinically normal dogs. PROCEDURES: Accuracy of the point-of-care tests (activated clotting time [ACT], estimated platelet count and number of schizocytes from a blood smear, plasma total solids [TS] concentration, and the protamine sulfate test) was evaluated, using receiver operating characteristic curves and likelihood ratios. A strategy, using likelihood ratios to calculate a posttest probability of DIC, was tested with 65% used as a threshold for initiation of treatment. Results of laboratory tests (coagulogram and plasma antithrombin III activity) were used as the standard for comparison in each dog. RESULTS: ACT and estimated platelet count provided the best accuracy for detection of DIC. The plasma TS concentration, schizocyte number, and protamine sulfate test had poor accuracy. The strategy using post-test probability of DIC identified 12 of 16 affected dogs that had DIC. Estimated platelet count was correlated and had acceptable clinical agreement with automated platelet count (r = 0.70). The plasma TS (r = 0.28) concentration and serum albumin (r = 0.63) concentration were not accurate predictors of plasma antithrombin III activity. The ACT did not correlate with activated partial thromboplastin time (r = 0.28). CONCLUSIONS AND CLINICAL RELEVANCE: Strategic use of likelihood ratios from point-of-care tests can assist clinicians in making treatment decisions for dogs suspected to have DIC when immediate laboratory support is unavailable.     

The Evaluation of Coagulation Profiles in Calves with Suspected Septic Shock

The purpose of the study reported here was to evaluate the haemostatic function in calves with suspected septic shock and to reflect the occurrence of disseminated intravascular coagulation (DIC). Twenty-six calves suspected of having septic shock (experimental group) and 10 clinically healthy calves (control group) were used. On admission, the experimental group of calves had been ill for an average of 2 days. Therapy was applied to the experimental group of calves. The packed cell volume (PCV), haemoglobin (Hb), white blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts were determined. Blood smears for toxic neutrophil and schistocyte intensity were evaluated. For the coagulation profile, plasma activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and fibrin/fibrinogen degradation products (FDPs) values were determined. Toxic neutrophils in blood smears were observed in 12 calves of the experimental group. APTT was prolonged in the experimental group compared with the control group (p < 0.05). Fibrinogen concentration was found to be higher in the experimental group than in the control group (p < 0.001). Total leukocyte counts were higher in the experimental group compared with the control group (p < 0.01). Platelet counts in the experimental group were lower than the control group (p < 0.001). However, when the individual values of coagulation profiles of each calf were evaluated, 8 calves had at least three abnormal coagulation profiles (APTT >72 s, PT >34.5 s, TT >33.7 s, FDPs >5 microg/ml, PLT < or = 150 x 10(3)/mm(3)) and abnormal erythrocyte morphology (schistocytes > or = 1). The most common abnormal tests in the coagulation profile were APTT and PT (7 cases), FDPs (6 cases), thrombocytopenia (4 cases), and schistocytes in blood smears (8 cases) in these 8 calves. The results of this study indicate that DIC might be a significant risk factor for mortality in calves with suspected septic shock.     

Study of haemostatic disorders in experimentally induced leishmaniasis in Beagle dogs

Haemostatic alterations in dogs experimentally infected with Leishmania infantumwere studied before and after therapy with meglumine antimonate. Haemostatic function tests including platelet count, collagen-induced platelet aggregation, prothrombin time, activated partial thromboplastin time, thrombin time, plasma fibrinogen determination, and serum fibrinogen/fibrin degradation products concentration were performed. In the course of infection and before treatment, moderate thrombocytopenia (P<0·00001) decreased collagen induced platelet aggregation (P=0·0003), prolonged thrombin time (P=0·0117) and increased fibrinogen/fibrin degradation products were observed. Statistically significant differences of plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time were not encountered. Haemostatic parameters returned to normal values after therapy. The results indicate that Leishmania infection may impair haemostasis suggesting induction of disseminated intravascular coagulation (DIC), and that treating dogs in an early stage of infection may potentially avoid the possibility of developing an uncompensated DIC.     

Correlation of haemostatic abnormalities with tumour stage and characteristics in dogs with mammary carcinoma

Sixty female dogs with untreated mammary carcinoma, comprising equal numbers of dogs in tumour stages I to IV, were evaluated for haemostatic abnormalities using the following tests: platelet count, prothrombin time, activated partial thromboplastin time, thrombin time, plasma activity of factor V, VIII and X, plasma concentration of fibrinogen, fibrin monomers and fibrinogen degradation products, and plasma antithrombin III activity. Two-thirds of all dogs had one or more haemostatic test abnormality of which the likelihood and frequency was increased in those with stage III and IV neoplasia. Haemostatic abnormalities were more frequently observed in dogs which had mammary tumours with distant metastases, extended tumour necrosis, inflammatory carcinomas, tumours fixed to underlying structures, or tumours in which there was penetration of the tumour capsule by tumour cells. As in humans with mammary carcinoma, these haemostatic abnormalities might be used as prognostic indicators, but their clinical importance remains unknown.     

Reduction of the red blood cell mass of horses: toxic effect of heparin anticoagulant therapy

This study was designed to test the efficacy of heparin anticoagulant therapy in the horse and its effect on the formed elements of blood. Nine clinically normal, nontraumatized adult horses were subjected to 4 different heparin maintenance regimens (dosages of 320, 240, 160, and 40 U/kg of body weight). Porcine intestinal mucosa heparin (20,000 U/ml) was injected subcutaneously every 12 hours for 96 hours (total 9 times). A loading dose of one-third the maintenance dose was given IV just before the first heparin injection. Three control horses were given an equivalent volume of 0.9% saline solution. The 2 large doses of heparin (320, 240 U/kg) resulted in an extension of the therapeutic range for heparin anticoagulant therapy (1.5 to 2.5 X data base-line prolongation of the activated partial thromboplastin time [APTT]). The 160-U/kg dose maintained the APTT in the therapeutic range, and the 40-U/kg dose had no effect on the APTT. Heparin was shown to exert a profound influence on the RBC mass of the horse. Three of the heparin regimens (320, 240, and 160 U/kg) resulted in a significant decrease in RBC numbers, PCV, and total hemoglobin content. Platelet count also was reduced in the horses when given the 320 and 240 U/kg doses. The observed increase in the mean corpuscular volume was associated with decreasing RBC numbers. Plasma proteins, serum bilirubin, free hemoglobin (plasma), haptoglobin (plasma), and urine and fecal hemoglobin values remained unchanged in all groups. Heparin anticoagulation therapy with the smallest dose (40 U/kg) had no detectable effects on the measured values, nor did the saline solution.     

Clinicopathologic evidence of disseminated intravascular coagulation in horses with acute colitis

OBJECTIVE: To detect subclinical disseminated intravascular coagulation (DIC) in horses with colitis and to determine any association between the diagnosis of subclinical DIC and outcome or occurrence of complications in horses with colitis. DESIGN: Prospective study. ANIMALS: 37 horses admitted to a veterinary teaching hospital for treatment of acute colitis. PROCEDURE: Coagulation profiles were obtained on each horse 0, 24, and 48 hours after admission. Six tests were performed: platelet count, plasma fibrinogen concentration, prothrombin time, activated partial thromboplastin time, antithrombin activity, and serum fibrin degradation products concentration. RESULTS: A clinicopathologic diagnosis of subclinical DIC was made if 3 of the 6 tests had abnormal results at any 1 sample period. No horse had clinical signs of DIC at the time of sampling. Twelve of 37 (32%) horses met the criteria for diagnosis of subclinical DIC within a 1-year period. Outcome was defined as survival or nonsurvival. Five of 12 horses with subclinical DIC and 2 of 25 horses without subclinical DIC did not survive. Crude odds ratio analysis revealed a horse with acute colitis was 8 times as likely to die or be euthanatized if a diagnosis of subclinical DIC was made. CONCLUSIONS AND CLINICAL RELEVANCE: Clinicopathologic evidence of DIC is common and is significantly associated with a poor outcome in horses with acute colitis. Treatment of subclinical DIC may influence outcome in horses with acute colitis.     

Effect of fusarium T-2 toxin on hematological and biochemical parameters in the rabbit.

The single intravenous administration of purified T-2 toxin to rabbits to 0.5 mg per kg body weight produced a decrease in hematocrit, while blood cell count, and serum alkaline phosphatase activity. The plasma clotting time, as measured by the activated partial thromboplastin time assay, was prolonged after intravenous T-2 toxin administration. In contrast, the administration of T-2 toxin to rabbits at 2.0 mg per kg body weight by gastric intubation produced oral lesions, diarrhea and anorexia in the animals but did not cause significant alteration in hematological and biochemical parameters. The results suggest that the rabbit may be a suitable model for further examination of the biochemical mechanisms involved in the cytotoxic action of T-2 toxin.     

Effect of transport loading or noise on blood biochemical variables in calves

The influence of 2 transport stressors (loading and noise) on several serum and plasma biochemical variables was studied in 4 calves. When calves were loaded and kept for 30 minutes on a motionless transport simulator or were exposed to the noise of the working simulator, increases in plasma epinephrine and serum cortisol and nonesterified fatty acids concentrations were similar to changes observed in calves subjected to simulated transport for 30 minutes. Results indicate that loading and noise have an important role in transport stress.     

Effects of Escherichia coli endotoxin on leukocyte and platelet counts, fibrinogen concentrations, and blood clotting in colostrum-fed and colostrum-deficient neonatal calves

Effects of a single IV injection of Escherichia coli endotoxin on hemogram and clotting function were compared in colostrum-fed and colostrum-deficient neonatal calves. Before endotoxin administration, the 2 groups of calves only differed in their prothrombin times. After endotoxin administration, there were significant differences (P less than 0.005) between colostrum-fed and colostrum-deficient calves in total leukocyte, segmented neutrophil, nonsegmented neutrophil, and lymphocyte (P less than 0.05) counts and partial thromboplastin time. Significant time dependent changes were observed in the aforementioned parameters and in platelet count and fibrinogen concentration. Seemingly, colostrum feeding improved the calf's ability to respond to endotoxin challenge exposure probably because of improved granulopoietic activity.     

Evaluation of the effect of storage at -70 degrees C for six months on hemostatic function testing in dogs.

Freezing is a routine method of storage for plasma that is to be used in evaluating certain aspects of hemostatic function in many species. The purpose of this study was to evaluate the effect of storage at -70 degrees C for 6 mo on canine plasma samples. On fresh and frozen plasma from 12 clinically healthy dogs, prothrombin time, activated partial thromboplastin time, thrombin clotting time, fibrinogen determination, antithrombin III activity, fragment D and E assay, and protamine sulfate test were performed. Clinical agreement analysis was utilized to determine the effect of such storage on all assays. Individual differences detected between fresh and frozen samples were all within 2 standard deviations of the mean difference. With the exception of the activated partial thromboplastin time, storing canine plasma at -70 degrees C for 6 mo has no significant effect on hemostatic function, as assessed by these tests.     

Abnormal Hemostatic Profiles and Gastric Necrosis in Canine Gastric Dilatation-Volvulus

Hemostatic profiles (prothrombin time, activated partial thromboplastin time, fibrinogen concentration, fibrin degradation product concentration, platelet count, and antithrombin III activity) were acquired prospectively in 20 dogs with a diagnosis of gastric dilatation-volvulus. Eighteen dogs had abnormal results of one or more hemostatic test, including eight dogs that had hemostatic profiles consistent with a diagnosis of disseminated intravascular coagulation. During surgery, or at necropsy, the dogs' stomachs were evaluated for gross abnormalities, and lesions were graded subjectively as mild, moderate, or severe. Eight dogs had mild gastric lesions, five had moderate lesions, and seven had severe changes indicating gastric necrosis. Seventy percent (7/10) of the dogs with two to six abnormal hemostatic test results had gastric necrosis, whereas none of the 10 dogs with no or one abnormality had gastric necrosis (p < .001). A multiple linear regression equation, based on fibrin degradation product concentration, activated partial thromboplastin time, and antithrombin III activity was derived to predict gastric necrosis. This equation correctly identified gastric necrosis with 86% sensitivity, 100% specificity, 100% positive predictive value, and 93% negative predictive value.     

Use of hexadimethrine bromide as a heparin-neutralizing agent in canine plasma

Hexadimethrine bromide was evaluated as a heparin-neutralizing agent in a simple modification of the activated partial thromboplastin time test in canine plasma. Addition of various amounts of heparin in vitro to canine plasma indicated that heparin could be neutralized by adding 0.5 micrograms of hexadimethrine bromide 15 s before CaCl2 was added to the reaction mixture of the activated partial thromboplastin time test. In 8 dogs given (subcutaneous injection) 500 USP units of sodium heparin/kg, marked individual variations in clotting time prolongations were observed over the 12-hour period of study. The hexadimethrine bromide modification effectively neutralized the heparin-related clotting time prolongations to values that were not significantly different from base-line (preheparin) activated partial thromboplastin time values. The modification seems to be useful in confirming the presence of heparin and in monitoring heparin therapy in dogs.     

Clotting profile in cattle showing chronic enzootic haematuria (CEH) and bladder neoplasms

Primary haemostasis (bleeding and blood clotting time), activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin III (ATIII), protein C, protein S, fibrinogen and D-dimer were determined in 13 cattle affected by chronic enzootic haematuria (CEH) and bladder neoplasms and 10 healthy cattle (control group). Increases in antithrombin III and protein S activities (P<0.01) and protein C and fibrinogen plasma levels (P<0.05) were observed in sick animals, while activated partial thromboplastin time, prothrombin time, and D-dimer did not show significant differences when compared to healthy animals. The clotting profile observed does not seem responsible for the chronic bleeding typical of CEH. The observed modification of some coagulation markers may derive from multiple interactions among cancer, inflammation and viral infection status typical of this syndrome.     

Studies on Some Blood Parameters of Crossbred Calves with Experimental Theileria annulata Infections

Subcutaneous inoculation of 1 ml of ground Theileria annulata tick tissue stabilate (0.75 tick equivalent) into crossbred calves (n = 6, average age 53 days) resulted in the development of acute theileriosis. The percentage parasitaemia was 71.7%±3.3% on day 20 after inoculation. Macroschizonts were observed in lymphocytes and monocytes. Phagocytosed schizonts were observed in neutrophils, along with cytoplasmic vacuolation in monocytes and neutrophils. There was progressive decrease (p<0.05) in the haemoglobin and packed cell volume, along with a marked reticulocytosis. Serum analysis revealed a decrease (p<0.05) in the concentrations of calcium, cholesterol and triglycerides, while there was an increase (p<0.05) in the concentrations of blood urea nitrogen as compared to day 0 values. The total serum proteins, albumin and serum immunoglobulin concentrations and the albumin-to-immunoglobulin ratio showed marked decreases (p<0.05). Coagulopathies included thrombocytopenia and an increased prothrombin time, along with a non-significant increase in the bleeding time and activated partial thromboplastin time during the terminal stages of the disease. There was an increase in the osmotic fragility of erythrocytes during the disease. Morphological alterations in the erythrocytes were observed with the developing parasitaemia.