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  • Author or Editor: James L. Catalfamo x
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Abstract

Objectives—To determine the molecular and genetic basis for thrombasthenic thrombopathia in Otterhounds and establish whether the defect would be best classified as type-I Glanzmann's thrombasthenia.

Animals—57 dogs, including 13 affected Otterhounds, 23 carrier Otterhounds, 17 unaffected Otterhounds, and 4 clinically normal unrelated dogs of other breeds.

Procedure—Functional (platelet aggregation, clot retraction, buccal mucosa bleeding time) and biochemical (electrophoresis, flow cytometry, fibrinogen content) analyses were conducted. In addition, firststrand cDNA synthesis from platelet total RNA was performed. Exons of the genes encoding for glycoproteins (GP) IIb and IIIa were amplified in overlapping fashion. The resulting products were excised from agarose gels and sequenced. The sequences obtained were compared with known cDNA sequences for canine GPIIb and GPIIIa.

Results—A single nucleotide change at position G1193 (1100) was detected in exon 12 of the gene encoding for platelet GPIIb in 2 affected Otterhounds. Carrier Otterhounds were heterozygous at this position, and 2 unaffected Otterhounds were unchanged. This nucleotide change would result in substitution of histidine for aspartic acid at position 398 (367) within the third calcium-binding domain of GPIIb.

Conclusions and Clinical Relevance—These studies suggest that thrombasthenic thrombopathia of Otterhounds is homologous phenotypically and has a similar molecular basis to type-I Glanzmann's thrombasthenia in humans. (Am J Vet Res 2001;62:1797–1804)

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in American Journal of Veterinary Research

Abstract

Objective—To assess the effect of desmopressin (DDAVP) administration in Doberman Pinschers with type 1 von Willebrand disease (vWD) on plasma von Willebrand factor (vWF) multimers through determination of vWF collagen binding activity (vWF:CBA; a functional vWF assay dependent on the presence of high–molecular-weight [HMW] multimers), comparison of vWF antigen concentration (vWF:Ag) to vWF:CBA, and vWF multimer size distribution.

Animals—16 Doberman Pinschers with type 1 vWD and 5 clinically normal control dogs.

Procedure—Plasma vWF:Ag and vWF:CBA assays and vWF multimer analysis were performed before and 1 hour after administration of DDAVP (1 µg/kg, SC).

Results—Following DDAVP administration, dogs with type 1 vWD had an increase in mean baseline values of plasma vWF:Ag and vWF:CBA from 10% to 17% for both variables. The mean vWF Ag:CBA ratio at baseline (0.95) was similar after DDAVP administration (0.97), indicating concordant increases in plasma vWF concentration and activity. In control dogs, mean plasma vWF:Ag and vWF:CBA increased from baseline values of 64% to 113% and 58% to 114%, respectively, and the vWF Ag:CBA ratios were unchanged (1.1 vs 1.0) after DDAVP administration. Plasma vWF multimer analysis revealed proportional increases in band intensity for all multimer sizes following DDAVP administration, in comparison to baseline for the control dogs and Doberman Pinschers with vWD, consistent with vWF Ag:CBA ratios of approximately 1.

Conclusions and Clinical Relevance—Beneficial effects of DDAVP on primary hemostasis in Doberman Pinschers with type 1 vWD cannot be explained by preferential increases in HMW vWF multimers. (Am J Vet Res 2005;66:861–867)

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in American Journal of Veterinary Research

Abstract

Objective—To develop an assay to measure canine von Willebrand factor (vWF):collagen-binding activity (CBA) to screen for type 2 von Willebrand disease (vWD) in dogs.

Sample Population—293 plasma samples submitted for analysis of canine vWF antigen (vWF:Ag) and 12 control plasma samples from dogs with inherited type 2 or 3 vWD.

Procedure—Bovine collagens were evaluated for suitability as binding substrate for vWF. Assay sensitivity to depletion, proteolytic degradation, or a genetic deficiency of high-molecular-weight vWF were determined. Amounts of vWF:Ag and vWF:CBA were measured. The ratio of vWF:Ag to vWF:CBA was used to discriminate between type 1 and type 2 vWD.

Results—An assay for canine vWF activity was developed by use of mixed collagen (types I and III). When vWF:Ag was used to subtype vWD, 48% of the dogs were classified as clinically normal, 9% as indeterminate, and 43% as type 1 vWD. Inclusion of vWF activity resulted in reclassification of 5% of those identified as type 1 to type 2 vWD. However, vWF:CBA of the reclassified dogs was not persistently abnormal, a finding compatible with acquired type 2 vWD. Some Doberman Pinschers had lower antigen-to-activity ratios than other breeds with type 1 vWD, suggesting that Doberman Pinschers have more functional circulating vWF.

Conclusions and Clinical Relevance—Analysis of canine vWF activity should be included among the vWF-specific assays used to confirm type 2 vWD. The prevalence of inherited forms of type 2 vWD in screened dogs is lower than acquired forms that can result secondary to underlying disease.

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in American Journal of Veterinary Research

Abstract

Objective—To compare effects of 3.8% sodium citrate and anticoagulant citrate dextrose solution National Institutes of Health formula A (ACD-A) on pH, extracellular ionized calcium (iCa) concentration, and platelet aggregation in canine platelet-rich plasma (PRP).

Sample Population—Samples from 12 dogs.

Procedures—Blood samples were collected into 3.8% sodium citrate (dilution, 1:9) and ACD-A (dilution, 1:5). Platelet function, pH, and iCa concentration were evaluated in PRP. Platelet agonists were ADP, γ-thrombin, and convulxin; final concentrations of each were 20μm, 100nM, and 20nM, respectively. Washed platelets were used to evaluate effects of varying the pH and iCa concentration.

Results—Mean pH and iCa concentration were significantly greater in 3.8% sodium citrate PRP than ACD-A PRP. Platelet aggregation induced by ADP and γ-thrombin was markedly diminished in ACD-A PRP, compared with results for 3.8% sodium citrate PRP. Anticoagulant had no effect on amplitude of convulxin-induced platelet aggregation. In washed platelet suspensions (pH, 7.4), there were no differences in amplitude of platelet aggregation induced by convulxin or γ-thrombin at various iCa concentrations. Varying the pH had no effect on amplitude of aggregation induced by convulxin or γ-thrombin, but the aggregation rate increased with increasing pH for both agonists.

Conclusions and Clinical Relevance—Aggregation of canine platelets induced by ADP and γ-thrombin was negligible in ACD-A PRP, which suggested an increase in extraplatelet hydrogen ion concentration inhibits signaling triggered by these agonists but not by convulxin. Choice of anticoagulant may influence results of in vitro evaluation of platelet function, which can lead to erroneous conclusions.

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in American Journal of Veterinary Research

Abstract

Objective—To measure platelet membrane–derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.

Animals—31 clinically normal dogs (19 males and 12 females).

Procedures—Citrate-anticoagulated blood samples obtained from each dog were centrifuged at 2,500 × g to isolate platelet-poor plasma (PPP), then PPP was centrifuged at 21,000 × g to isolate microparticle-free plasma (MPF) and microparticle-enriched plasma (MPEP). Whole blood and paired samples of fresh and frozen-thawed PPP, MPF, and MPEP were dual labeled for flow cytometric detection of membrane CD61 (constitutive platelet antigen) and annexin V (indicating phosphatidylserine externalization). Platelets and PMPs were enumerated with fluorescent, size-calibrated beads. Thrombin generation in fresh and frozen-thawed PPP, MPF, and MPEP was measured via kinetic fluorometric assays configured with low tissue factor and low phospholipid concentrations.

Results—Initial centrifugation yielded PPP with < 0.5% the platelets of whole blood, with median counts of 413 PMPs/μL for males and 711 PMPs/μL for females. Sequential centrifugation resulted in a 10-fold concentration of PMPs in MPEP and virtually depleted PMPs from MPF. Thrombin generation depended on PMP content, with median endogenous thrombin potential of 0, 893, and 3,650 nmol•min for MPF, PPP, and MPEP, respectively. Freeze-thaw cycling caused significant increases in PMP counts and phosphatidylserine externalization.

Conclusions and Clinical Relevance—Canine PMPs were major determinants of thrombin-generating capacity; preanalytic variables influenced plasma PMP content. Processing conditions described here may provide a basis for characterization of PMPs in clinical studies of thrombosis in dogs.

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in American Journal of Veterinary Research

Abstract

OBJECTIVE To measure thrombin generation by high and low tissue factor (TF)–expressing canine cancer cell lines.

SAMPLE Canine cell lines CMT25 (high TF–expressing mammary gland tumor cell line) and HMPOS (low TF–expressing osteosarcoma cell line).

PROCEDURES Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation.

RESULTS CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604nM thrombin•min and 636 ± 440nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation.

CONCLUSIONS AND CLINICAL RELEVANCE High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

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in American Journal of Veterinary Research