Evaluation of binding of fibrinogen and annexin V to equine platelets in response to supramaximal treadmill exercise.

There is evidence that equine platelet reactivity is altered by strenuous exercise. Changes in platelet reactivity could impact haemostasis following exercise-induced injury and may play a role in the pathophysiology of exercise-induced pulmonary haemorrhage. Interpretation of results of previous studies is hindered by potential in vitro-induced changes in platelet activity through the choice of anticoagulant and the use of platelet inhibitors. The present study was undertaken to re-evaluate the effect of exercise on equine platelets using methodologies that minimise in vitro-induced changes in platelet activation. The percentage of platelet-neutrophil aggregates increased significantly (P = 0.01) from mean +/- s.e. 3.5 +/- 0.6% at rest to 7.2 +/- 13% during exercise. There were no significant changes in binding of anti-fibrinogen antibody or annexin V to platelets in response to exercise. An inability to detect increased binding of fibrinogen or annexin V may be a result of poor test sensitivity or low statistical power. Alternatively, activated platelets may be quickly removed from the circulation and miss detection. The significance of increased numbers of platelet-neutrophil aggregates in association with exercise is currently unknown and warrants further investigation.

Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets.

OBJECTIVE: To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. SAMPLE POPULATION: Blood samples obtained from 4 Thoroughbreds. PROCEDURE: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein-conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode. RESULTS: Number of platelets (mean +/- SEM) in samples containing LMWH (109.5+/-11.3 x 10(3) cells/microl) was significantly less than the number in samples containing sodium citrate (187.3+/-30.3 x 10(3) cells/microl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding. CONCLUSIONS AND CLINICAL RELEVANCE: A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

The effect of supramaximal exercise on equine platelet function.

When blood is collected into sodium citrate in the proportion of 9 parts blood:1 part sodium citrate, the concentration of plasma sodium citrate in the sample will depend on the packed cell volume (PCV) of the blood sample. This difference in plasma sodium citrate concentration secondary to alterations in PCV significantly affects human platelet aggregation responses. Since horses attain a high PCV in response to high-intensity exercise we investigated the effect of differences in sample plasma sodium citrate concentration on equine platelet aggregability. In addition, low molecular weight heparin (LMWH) was evaluated as an alternative anticoagulant for assessment of platelet aggregability during strenuous exercise in horses. Blood samples were collected pre-exercise and at fatigue after supramaximal treadmill exercise into either 3.8% sodium citrate (9 parts blood:1 part sodium citrate) or 20 u LMWH/ml of blood. Platelet aggregation responses to 1.25 mumol/l adenosine diphosphate (ADP) were measured via optical aggregometry. For samples collected into sodium citrate, aggregability was significantly less than pre-exercise values in samples collected at fatigue and in pre-exercise samples in which sodium citrate concentrations were adjusted to equal those in fatigue samples. However, samples collected into LMWH showed significantly increased platelet aggregability in samples collected at fatigue when compared to pre-exercise samples. In conclusion, higher plasma sodium citrate concentration had a marked inhibitory effect on equine platelet aggregation responses. Low molecular weight heparin was a good alternative anticoagulant for assessment of equine platelet function and results indicate that equine platelet aggregability was enhanced in response to supramaximal exercise.

Plasma von Willebrand factor changes during various reproductive cycle stages in mixed-breed dogs with normal von Willebrand factor and in Doberman pinschers with type-I von Willebrand's disease.

OBJECTIVE: To determine whether plasma von Willebrand factor (vWf) concentration changes during the estrous cycle and pregnancy in Doberman Pinschers with type-I von Willebrand's disease (vWd) and in mixed-breed dogs with normal vWf, and if so, whether alterations in vWf concentration are associated with changes in serum concentrations of reproductive hormones. ANIMALS: 5 sexually intact female Doberman Pinschers with type-I vWf and 8 sexually intact female mixed-breed dogs with normal vWf. PROCEDURE: Concentrations of plasma vWf and serum progesterone and estradiol-17 beta were measured during the estrous cycle of nonpregnant dogs and during pregnancy, parturition, and lactation. Serum concentrations of total triiodothyronine, total thyroxin, and free thyroxin were measured during pregnancy, parturition, and lactation. RESULTS: Von Willebrand factor concentration did not change during the estrous cycle, but during pregnancy, vWf concentration gradually increased. Peak concentrations were obtained at parturition and were 103 and 92% higher in mixed-breed dogs and dogs with type-I vWd, respectively, than were mean prepregnancy (anestrus) values. At parturition, total triiodothyronine concentration decreased from the prepregnancy value. The increase in vWf concentration during pregnancy was positively associated with changes in concentration of estradiol-17 beta and was negatively associated with changes in concentration of progesterone. CONCLUSIONS: The increase in vWf concentration in pregnant bitches may be associated with changes in concentrations of reproductive hormones. However, the increase in vWf concentration during pregnancy may involve other factors because vWf concentration did not change during the estrous cycle of nonpregnant dogs despite increases in concentrations of estradiol-17 beta and progesterone.

Plasma von Willebrand factor in thoroughbreds in response to high-intensity treadmill exercise.

OBJECTIVE: To determine whether plasma von Willebrand factor (vWf) concentration changes in horses during and after treadmill exercise. ANIMALS: 5 mature, fit Thoroughbreds. PROCEDURE: A blood sampling catheter was placed in the right jugular vein. A warm-up period was followed by a 3-minute rest period. Horses were galloped at racing pace until fatigued (about 2 minutes). Blood samples were collected prior to warm-up, during the postwarm-up rest period, 1 minute into the run, at cessation of the run, and 5 to 120 minutes after cessation of the run. vWf activity was measured by ELISA and corrected for plasma volume changes (measured by changes in plasma albumin concentration). Platelet-poor plasma from 10 clinically normal, resting horses was pooled, assigned a value of 100 U/dl, and served as a control for all assays. RESULTS: vWf activity began increasing 1 minute after horses reached full speed. At 5 minutes after cessation of exercise, vWf values had increased by mean of 92% (P < 0.05) from baseline. vWf activity returned baseline by 15 minutes after exercise, and remained there until 90 minutes after exercise, when it began to increase. CONCLUSION AND CLINICAL RELEVANCE: The spontaneous decrease in vWf values after completion of exercise was unexpected because vWf has a long half-life in circulation. This unexpected finding is compatible with increased vWf consumption and suggests that microvascular trauma may occur in horses during strenuous exercise.

A primary production deficit in the thrombocytopenia of equine infectious anemia.

The purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (EIAV). Immunocompetent Arabian foals and Arabian foals with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were experimentally infected with EIAV. Levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. Thrombocytopenia was not dependent on the immune response: SCID foals were affected as severely as immunocompetent foals. Production of platelets, measured by metabolic incorporation of radioactive label, was significantly reduced. The decrease ranged from 35 to 89% in three SCID and two immunocompetent foals examined. Platelet survival, measured by 51Cr labeling, also declined following infection in both SCID and immunocompetent foals: 51 and 68%, respectively, relative to the preinfection life spans. The difference between immunocompetent and immunodeficient foals was not statistically significant. The number of megakaryocytes (MK) per square millimeter of bone marrow, determined by digitizing morphometry, was not significantly altered in either SCID or immunocompetent thrombocytopenic foals. Numbers of denuded MK nuclei per unit area increased, but the elevation was not statistically significant. No evidence for viral replication in MK was found. Three different parameters of intravascular coagulation (activated prothombin time, fibrin degradation products, and one-step prothombin time) remained normal until after platelet numbers had declined significantly, arguing against an important role for disseminated intravascular coagulation. The findings indicate that EIAV induces thrombocytopenia principally through an indirect, noncytocidal suppressive effect on platelet production, the mechanism of which is unknown. A shortening of platelet life span apparently contributes moderately to the platelet deficit as well. The shortening of platelet life span is multifactorial in origin, including both mechanisms that depend on an active immune response and those that do not.

Inheritance of von Willebrand factor deficiency in Doberman pinschers.

OBJECTIVE: To determine the inheritance of von Willebrand factor (vWF) deficiency in Doberman Pinschers. DESIGN: Prospective, observational study. ANIMALS: 26 adult Doberman Pinschers or mixed-breed dogs and 101 pups produced from 18 matings between adult dogs. PROCEDURE: Measurement of plasma vWF concentrations in parents and progeny. On the basis of plasma vWF concentrations, dogs were grouped as normal (75 to 160 U of vWF/dl), midrange (> or = 30 and < 75 U of vWF/dl), or low (< 30 U of vWF/dl). RESULTS: The percentile distribution of vWF concentrations was trimodal. Distribution between dogs with low and midrange plasma vWF concentrations changed sharply, whereas the change between dogs with midrange and normal plasma vWF concentrations was gradual. Three matings between dogs with low vWF concentrations produced 13 offspring, all with low vWF concentrations. Two matings between dogs with normal plasma vWF concentrations produced 14 offspring, all with normal vWF concentrations. Eight matings between dogs with normal and low plasma vWF concentrations produced 54 offspring 40 with midrange, 13 with normal, and 1 with low vWF concentrations. There were 5 matings of dogs with midrange plasma vWF concentrations to dogs with low, midrange, or high vWF concentrations. The results of all matings were consistent with a single gene defect where each normal allele produced half the total amount of vWF when both alleles are normal and each defective allele produced < 15 U of vWF/dl. CLINICAL IMPLICATIONS: Dogs with low plasma vWF concentrations may be homozygous for the defective allele, whereas dogs with midrange plasma vWF concentrations may be heterozygous. It can be difficult to distinguish normal homozygotes from heterozygotes if evaluation is based only on plasma vWF concentration.

Temporal variation and factors affecting measurement of canine von Willebrand factor.

OBJECTIVE: To determine whether canine plasma von Willebrand factor (vWf) varies between and within individuals over time and with different blood sample collection and processing procedures. ANIMALS: 26 adult dogs and 6 pups. PROCEDURE: Blood was obtained from the jugular or cephalic vein daily for 8 to 19 days and weekly for 9 to 23 weeks in adult dogs and periodically up to 180 days of age in pups. Temporal variation in vWf concentration and the effect of vascular occlusion, venipuncture site, lipemia, hemolysis, anticoagulant, storage time, freeze-thawing, and centrifugation speed on plasma vWf concentration, measured by ELISA, were determined. RESULTS: Plasma vWf concentration varied over time. In dogs with mean vWf concentration > or = 79 U/dl, the largest intraindividual range in vWf spanned 64 U/dl with daily and 53 U/dl with weekly sample collection. In dogs with mean vWf concentration < or = 24 U/dl, the largest individual variation was 12 U/dl with daily and weekly sample collection. In dogs with mean vWf concentration > or = 53 and < or = 74 U/dl, the largest intraindividual range spanned 35 U/ dl. Mean vWf concentration of pups from 3 to 180 days of age did not change. Sample hemolysis decreased mean vWf by 37%. Mean vWf concentration was 9% higher in cephalic than jugular vein samples (P = 0.056). Other sample collection/preparation procedures did not affect vWf concentration. CONCLUSION: There was substantial temporal variation in vWf concentration within individual dogs. CLINICAL RELEVANCE: Multiple tests may be necessary to obtain a reliable estimate of vWf concentration in dogs.

Evaluation of chromogenic substrate assays for fibrinolytic analytes in dogs.

OBJECTIVE: To evaluate the ability of commercial, chromogenic kits designed to measure human fibrinolytic pathway components to measure the canine plasma fibrinolytic pathway enzymes, tissue plasminogen activator (tPA) and plasminogen (PLG), and their respective inhibitors, plasminogen activator inhibitor 1 (PAI) and alpha 2-antiplasmin (AP). ANIMALS: 20 healthy dogs of various ages and breeds. PROCEDURE: The commercial procedure was adapted to a microtitration plate. Standard curves were generated by use of a canine plasma pool. RESULTS: Modifications of the commercial kit consisted of change in incubation periods and the substitution of urokinase for the streptokinase. Plasminogen and AP procedures yielded intra- and interassay coefficients of variation (CV) ranging from 2 to 6.4%. The tPA activity gave an acceptable intra-assay CV of 4.2%, but an equivocal interassay CV of 18%. The PAI assay gave unacceptable intra-assay and interassay CV of 59 and 66%, respectively. CONCLUSIONS: Modifications of the commercial PLG and AP procedures were appropriate for use with fresh and frozen canine plasma. However, equivocal results were obtained for canine plasma tPA. Although the PAI assay was able to detect the inhibitor, it gave unacceptable quantifiable results. Human and canine plasma contained similar amounts of PLG and AP, but 25% more tPA was found in canine plasma than human plasma. CLINICAL RELEVANCE: With modifications, the commercial human PLG and AP chromogenic kits may serve to elucidate such canine fibrinolytic disorders as disseminated coagulopathy. The high cost of the chromogenic substrate limits its application.

Fibrinolytic activity in dogs after surgically induced trauma.

OBJECTIVE: To determine whether alterations in the fibrinolytic pathway analytes, plasminogen (PLG), tissue plasminogen activator, and alpha 2-antiplasmin are significant in dogs subjected to minor and major surgical trauma. ANIMALS: 18 dogs in 3 groups of 6 each. PROCEDURE: Plasma fibrinolytic pathway analytes were measured in dogs with trauma of ovariohysterectomy (minor trauma) or orthopedic surgery (major trauma) and halothane anesthesia (control group). A commercial procedure adapted to a microtitration plate was used to measure the analytes. Blood was obtained 24 hours before anesthesia, at extubation (0 hours), and again at 2, 24, and 48 hours after extubation. An analyte quality-control strategy was maintained. RESULTS: In the major trauma group, there was a significant, transient, postsurgical decrease in PLG activity at 0 and 24 hours and a return to presurgical values by 48 hours. The minor trauma group had a similar trend without significant changes, including an increase in PLG values at 48 hours that exceeded the reference range. Antiplasmin values changed significantly in the major trauma group only. Tissue plasminogen activator values remained within the reference range. CONCLUSIONS: Tissue plasminogen activator was not considered a clinical marker of interest for detection of alterations in fibrinolysis after trauma. In contrast, plasma PLG and alpha 2-antiplasmin values may be useful in the evaluation of hemostatic complications of surgery. CLINICAL RELEVANCE: Identification of altered fibrinolysis in dogs undergoing traumatic surgery may provide a baseline for preventive pre-and postsurgical hemostatic care.

Canine idiopathic thrombocytopenic purpura.

Canine idiopathic thrombocytopenic purpura (ITP) is a disease in which antibodies bound to the surface of platelets mediate premature platelet destruction by macrophages. ITP in dogs and chronic ITP in humans are analogous diseases. This article draws on information from the literature on ITP in dogs and in humans, and reviews the pathogenesis, diagnosis, and treatment of ITP in dogs.

Differential distribution of von Willebrand factor in canine vascular endothelium.

OBJECTIVE: To determine whether endothelial cell (EC) von Willebrand factor (vWf) is uniformly distributed in canine blood vessels. DESIGN: Contents of EC vWf from vascular segments was evaluated in Haütchen preparations, using immuno-histochemistry. EC from femoral arteries and veins and jugular veins were grown in culture, and the intracellular content and constitutive release of vWf from these cells were measured. The amount of vWf mRNA in the cultured EC was determined. ANIMALS: Vascular segments for Häutchen preparations and EC for culture were obtained from 5 and 10 clinically normal, mixed-breed dogs, respectively. PROCEDURES: Appropriate vascular segments were removed, fixed; processed for immunohistochemistry, using a monospecific polyclonal antibody to canine vWf, and Haütchen preparations were made. Intracellular and constitutive released vWf was measured, using an ELISA, and vWf mRNA was measured by Northern blot analysis. RESULTS: Intact endothelial linings from femoral veins, jugular veins, vena cava, and pulmonary veins stained more intensely than femoral arteries, carotid arteries, aorta, and pulmonary veins. Constitutive release and intracellular content of vWf in cultured EC from femoral veins was about 30 times higher than that from femoral arterial EC, which was barely detectable. Similar differences were seen in amounts of mRNA. CONCLUSIONS: There is marked diversity in EC vWf in canine vasculature that may result from differences in vWf mRNA. CLINICAL RELEVANCE: Low amounts of vWf in canine systemic arterial EC may contribute to thromboresistance of canine arteries.

Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura.

Canine idiopathic thrombocytopenic purpura (ITP) is clinically analogous to chronic ITP in human beings. The objective of this study was to investigate the pathogenesis of canine ITP by determining whether immunoglobulins bound to the surface of platelets from dogs with ITP (platelet-bound immunoglobulins) were directed against host platelet antigen and whether platelet glycoproteins (GP) IIb and IIIa were target antigens in dogs with ITP. Thirty-two dogs with ITP were studied. Increased platelet-bound immunoglobulin concentrations were detected in 30 cases (94%), and increased concentrations of serum platelet-bindable immunoglobulins were detected in 11 cases (34%). Immunoglobulins eluted from the surface of platelets from dogs with ITP bound to homologous normal canine platelets in 11 of 19 cases (58%). Immunoglobulins against platelet membrane GP IIb and/or IIIa were detected in serum from four of 17 affected dogs. This study provides evidence that immunoglobulins bound to surface of platelets from some dogs with ITP are directed against host platelet antigens and that platelet target antigens are, in some cases, GP IIb and IIIa. This supports the hypothesis that canine ITP is an autoimmune disease, similar to the pathogenesis of chronic ITP in human beings.

Measurement of von Willebrand factor-specific mRNA and release and storage of von Willebrand factor from endothelial cells of dogs with type-I von Willebrand's disease.

OBJECTIVE: To characterize the cellular basis of the plasma von Willebrand factor (vWf) deficiency in Doberman Pinschers with type-1 von Willebrand's disease (vWd). ANIMALS: Five Doberman Pinschers with type-I vWd and 5 clinically normal dogs used as controls. PROCEDURE: Vascular endothelial cell cultures were used to measure constitutive vWf release, thrombin-stimulated vWf release, baseline intracellular vWf concentration, and vWf mRNA expression. RESULTS: Cells cultured from vWd-affected dogs were morphologically indistinguishable from cells cultured from control dogs, but had reductions in constitutive vWf release (6.5-fold) and vWf mRNA content (fivefold) that correlated to the reduction in plasma vWf concentration (sixfold) in these dogs. The 9.0-kb, canine vWf message was identified, using a polymerase chain reaction-amplified segment of the canine vWf gene and was similar in size to the human vWf message. The vWd cells also had reductions in baseline intracellular vWf concentration (15.6-fold) and thrombin-stimulated vWf release (14.5-fold). Additionally, it was observed that normal canine endothelial cells from different anatomic locations were heterogeneous with respect to vWf expression. CONCLUSIONS: These findings suggest that the plasma vWf deficit in dogs with type-I vWd results from decreased endothelial cell production of vWf resulting from either decreased transcription of the vWf gene or abnormalities in mRNA processing/stability. This is similar to findings in human beings with type-I vWd.

Detection of platelet-bound and serum platelet-bindable antibodies for diagnosis of idiopathic thrombocytopenic purpura in dogs.

The sensitivity and specificity of 2 antibody tests for diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs were investigated prospectively. An ELISA to detect antibodies bound to the surface of platelets from affected dogs (direct test) was performed in 34 dogs with a clinical diagnosis of ITP and in 21 dogs with thrombocytopenia attributable to other causes. An ELISA to detect platelet-bindable antibodies in serum from affected dogs (indirect test) was performed in 32 dogs with ITP and in 15 dogs with other causes of thrombocytopenia. The direct test was positive in 32 of 34 dogs with ITP (sensitivity, 94%) and negative in 13 of 21 dogs with other causes of thrombocytopenia (specificity, 62%). Positive direct test results were obtained in 2 dogs with systemic lupus erythematosus, and in 1 dog each with concurrent Ehrlichia canis and Babesia canis infections, dirofilariasis, myelodysplasia, disseminated intravascular coagulation (of unknown cause), and thrombocytopenia subsequent to administration of trimethoprim/sulfadiazine, as well as in 1 dog with thrombocytopenia 14 days after a whole blood transfusion. The indirect test had positive results in 11 of 32 dogs with ITP (sensitivity, 34%) and negative results in 12 of 15 dogs with other causes of thrombocytopenia (specificity, 80%). Positive indirect test results were obtained in 1 dog each with systemic lupus erythematosus, concurrent E canis and B canis infections, and thrombocytopenia subsequent to administration of trimethoprim/sulfadiazine. Detection of platelet-bound antibodies was more sensitive than detection of serum-platelet bindable antibodies in confirming a diagnosis of ITP in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimentally induced renal failure in the dog as an animal model of uremic bleeding.

Experimental canine renal failure was studied as a potential animal model for human uremic bleeding. Renal failure accompanied by hemostatic alterations was induced in eight dogs by means of two surgical techniques of renal mass reduction. The hemostatic deficits consisted of immediate and marked reduction of the platelet glass bead retention (PR) to less than 10% of normal and gradual prolongation of the buccal mucosal bleeding time (BMBT) to approximately four times the normal value. Platelet count, volume, aggregation responses, and coagulation were normal. A packed cell volume (PCV) of less than 30% was observed in three dogs. Elevation of the PCV normalized the BMBT in two dogs, but because the PR was unchanged and the BMBT effect was temporary, anemia was not considered the primary cause of the prolonged bleeding time. There was a significant, positive correlation between BMBT and BUN, suggesting that the altered hemostasis may be related to the accumulation of urea or other uremic toxins of protein origin. The finding of a defect in PR and BMBT--tests that require normal platelet adhesion and aggregation--in azotemic dogs were platelet numbers and aggregation are normal indirectly implicates platelet adhesion as the primary hemostatic defect.

von Willebrand factor is not altered in azotemic dogs with prolonged bleeding time.

The effects of azotemia on von Willebrand factor (vWf) plasma concentration, structure, and function were studied by utilizing canine models for both uremic bleeding and type I vWf deficiency (vWd). Seventy-five percent to 80% renal mass reduction in eight mixed-breed dogs induced marked azotemia (blood urea nitrogen [BUN] 103 +/- 7 mg/dl [mean +/- SEM]; creatinine 5.8 +/- 1 mg/dl) and prolonged mean buccal mucosal bleeding time (BMBT) from 1.8 +/- 0.2 minutes to 7.0 +/- 0.4 minutes. The mean vWf plasma concentration increased from 0.88 +/- 0.11 U/ml to 1.26 +/- 0.14 U/ml. The pre- and postsurgical sodium dodecyl sulfate-agarose gel electrophoresis multimeric patterns were similar in all dogs. Administration of cryoprecipitate from pooled azotemic mixed-breed dog plasma to five Doberman pinschers with type I vWd increased the mean plasma vWf from 0.14 +/- 0.01 U/ml to 0.48 +/- 0.04 U/ml and decreased the BMBT from 7.1 +/- 0.6 minutes to 3.14 +/- 0.09 minutes. After renal mass reduction, five type I vWd Dobermans developed marked azotemia (BUN 79 +/- 8.6 mg/dl; creatinine 3.7 +/- 0.6 mg/dl) and prolonged BMBT (16.1 +/- 3.6 minutes). Findings in the eight azotemic mixed-breed dogs indicated that (1) vWf plasma levels were normal to increased in azotemic dogs; (2) vWf structure and multimeric distribution were not altered in canine azotemia; and (3) vWf was functional when placed in a non-azotemic environment. The prolongation of the BMBT in azotemic vWd dogs indicated that factors other than alteration of vWf function were responsible for the prolonged BMBT in canine azotemia.

Evaluation of an additive solution for preservation of canine red blood cells.

The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4 degrees C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (51Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage (P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively (P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.

Effect of anticoagulant and blood storage time on platelet-bound antibody concentrations in clinically normal dogs.

A solid-phase ELISA to detect antibodies bound to the surface of canine platelets (platelet-bound antibodies) is described. Using this assay, the effect of anticoagulant and storage time of anticoagulant blood on the concentration of antibodies bound to the surface of platelets from clinically normal dogs was investigated. Blood from 3 clinically normal dogs was anticoagulated with acid citrate dextrose, Na3 citrate, and aqueous K3 EDTA and stored on ice for up to 48 hours. Platelet-bound antibody concentration was measured on platelets isolated from anticoagulated blood immediately after venipuncture and subsequent to storage of blood for 24 and 48 hours. Differences in platelet-bound antibody concentrations were investigated among dogs, anticoagulants, and storage times by ANOVA and Bonferroni pair-wise comparison of means. There was no effect of dog on platelet-bound antibody concentration. The effect of time was significant (P < 0.0001), with higher concentration of platelet-bound antibodies detected with increasing storage time. Effect of anticoagulant on platelet-bound antibody concentration was not statistically significant; however, there was a trend to increasing concentration of antibodies bound to platelets isolated from Na3 citrate- and K3 EDTA-anticoagulated blood. Moreover, there was significant (P = 0.02) interaction between anticoagulant and time. Platelet-bound antibody concentration increased with storage of anticoagulated blood prior to platelet isolation and with use of Na3 citrate and K3 EDTA anticoagulants. The preferred anticoagulant for platelet-bound antibody measurement is acid citrate dextrose. Platelet-bound antibody concentration should be determined not longer than 24 hours after blood collection.