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Abstract

Objective

To characterize the cellular basis of the plasma von Willebrand factor (vWf) deficiency in Doberman Pinschers with type-I 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.

Free access
in American Journal of Veterinary Research

Summary

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.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether endothelial cell (EC) von Willebrand factor (vWf) is uniformly distributed in canine blood vessels.

Design

Content of EC vWf from vascular segments was evaluated in Haütchen preparations, using immunohistochemistry. 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 Hautchen 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. (Am J Vet Res 1996; 57:750–755)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether alterations in the fibrinolytic pathway analytes, plasminogen (PLG), tissue plasminogen activator, and α2-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 α2-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. (Am J Vet Res 1996;57:1137-1140)

Free access
in American Journal of Veterinary Research

Abstract

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 ≥ 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 ≤ 24 U/dl, the largest individual variation was 12 U/dl with daily and weekly sample collection. In dogs with mean vWf concentration ≥ 53 and ≤ 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. (Am J Vet Res 1996;57:1288-1293)

Free access
in American Journal of Veterinary Research

Abstract

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 α2-anti-plasmim (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. (Am J Vet Res 1996;57: 1124-1130)

Free access
in American Journal of Veterinary Research

SUMMARY

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 vWd and 8 sexually intact female mixed-breed dogs with normal vWf.

Procedure

Concentrations of plasma vWf and serum progesterone and estradiol-17β 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-17β 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-17β and progesterone. (Am J Vet Res 1998;59:111–118)

Free access
in American Journal of Veterinary Research

Abstract

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 to 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. (Am J Vet Res 1997;58:71–76)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.

Sample Population—Blood samples from 6 Thoroughbreds.

Procedure—The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITCannexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187- activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry.

Results—In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours.

Conclusion and Clinical Relevance—Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results. (Am J Vet Res 2002;63:840–844)

Full access
in American Journal of Veterinary Research

Abstract

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.

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 × 103 cells/µl) was significantly less than the number in samples containing sodium citrate (187.3 ± 30.3 × 103 cells/µl). 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.

Conclusion 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. (Am J Vet Res 2001; 62:547–554)

Full access
in American Journal of Veterinary Research