The canine activated platelet secretome (CAPS): A translational model of thrombin-evoked platelet activation response.

Background: Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label-free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS.

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data.

Role of tissue factor expression in thrombin generation by canine tumor cells.

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.

Current diagnostic trends in coagulation disorders among dogs and cats.

The diagnostic workup to differentiate hemorrhage caused by vascular injury from a systemic hemostatic imbalance typically involves a combination of broad screening tests and specific assays. The characterization of 3 overlapping phases of primary hemostasis, secondary hemostasis, and fibrinolysis provides a simple diagnostic framework for evaluating patients with clinical signs of hemorrhage. New techniques such as flow cytometry, thrombin-generation assays, thrombelastography, and anticoagulant drug monitoring are under investigation for veterinary patients; however, their ability to improve diagnosis or treatment requires further study in clinical trials.

Clotting factor VIII (FVIII) and thrombin generation in camel plasma: A comparative study with humans.

The objective of this study was to characterize the highly elevated levels of clotting factor VIII (FVIII) in camel plasma. Whole blood was collected from healthy camels and factor VIII clotting activity (FVIII:C) assays were conducted using both the clotting and the chromogenic techniques. The anticoagulant citrate phosphate dextrose adenine (CPDA) produced the highest harvest of FVIII:C, the level of plasma factor VIII, compared to heparin:saline and heparin:CPDA anticoagulants.

Flow cytometric detection and procoagulant activity of circulating canine platelet-derived microparticles.

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).

Comparative hemostasis: animal models and new hemostasis tests.

This article provides an overview of animal model systems to include their strengths and limitations in the study of hemostasis. Specific examples of spontaneous and engineered animal models are described in the context of cell-based hemostasis. The article concludes with a review of the comparative aspects of 3 laboratory assays of cell-based hemostasis: thromboelastography, thrombin generation, and flow cytometric assessment of platelet activation.

Effects of anticoagulant on pH, ionized calcium concentration, and agonist-induced platelet aggregation in canine platelet-rich plasma.

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.

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits.

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg).

Calcium-diacylglycerol guanine nucleotide exchange factor I gene mutations associated with loss of function in canine platelets.

Calcium-Diacylglycerol Guanine Nucleotide Exchange Factor I (CalDAG-GEFI) has been implicated in platelet aggregation signaling in CalDAG-GEFI knockouts. Functional mutations were identified in the gene encoding for CalDAG-GEFI in 3 dog breeds. Affected dogs experienced epistaxis, gingival bleeding, and petechiation.

Development of a collagen-binding activity assay as a screening test for type II von Willebrand disease in dogs.

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.

Effect of desmopressin on von Willebrand factor multimers in Doberman Pinschers with type 1 von Willebrand disease.

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 [HMWI 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 microg/kg, SC).

Characterization of the mutations causing hemophilia B in 2 domestic cats.

The purpose of the present study was to determine the normal sequence for the gene encoding factor IX in cats and to characterize the genetic basis for hemophilia B in 2 unrelated male, domestic, mixed-breed cats. Genomic DNA sequence for the entire coding region of the factor IX gene was determined in the affected cats and compared to the sequence obtained from a healthy cat. The factor IX gene in cats encodes a mature protein consisting of 420 amino acids, unlike genes in humans and dogs that encode 415 and 413 amino acid proteins, respectively.

Type-3 von willebrand's disease in a rhesus monkey (Macaca mulatta).

Severe type-3 von Willebrand's disease (vWD) was diagnosed in a young male rhesus monkey that had excessive bleeding from minor wounds. Plasma samples from the monkey had no detectable quantitative or functional von Willebrand factor (vWF), low Factor-VIII coagulant activity, and moderate prolongation of activated partial thromboplastin time. Testing of the affected monkey's extended family revealed a likely hereditary basis for the vWD, in that the sire and a paternal half-sister had markedly reduced plasma vWF concentration.

A hereditary bleeding disorder of dogs caused by a lack of platelet procoagulant activity.

We have discovered a novel canine hereditary bleeding disorder with the characteristic features of Scott syndrome, a rare defect of platelet procoagulant activity. Affected dogs were from a single, inbred colony and experienced clinical signs of epistaxis, hyphema, intramuscular hematoma, and prolonged bleeding with cutaneous bruising after surgery. The hemostatic abnormalities identified were restricted to tests of platelet procoagulant activity, whereas platelet count, platelet morphology under light microscopy, bleeding time, clot retraction, and platelet aggregation and secretion in response to thrombin, collagen, and adenosine diphosphate stimulation were all within normal limits.