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. METHODS: Platelets were isolated from 10 healthy dogs and stimulated with 50 nmol/L of γ-thrombin or saline. Proteins were in-solution trypsin-digested and analyzed by nano-liquid chromatography-tandem spectrometry. Core releasate proteins were defined as those present in 10 of 10 dogs, and CAPS defined as releasate proteins with a significantly higher abundance in stimulated versus saline controls (corrected P < .05). RESULTS: A total of 2865 proteins were identified; 1126 releasate proteins were present in all dogs, 650 were defined as CAPS. Among the differences from human platelets were a canine lack of platelet factor 4 and vascular endothelial growth factor C, and a 10- to 20-fold lower concentration of proteins such as haptoglobin, alpha-2 macroglobulin, von Willebrand factor, and amyloid-beta A4. Twenty-eight CAPS proteins, including cytokines, adhesion molecules, granule proteins, and calcium regulatory proteins have not previously been attributed to human platelets. CONCLUSIONS: CAPS proteins represent a robust characterization of a large animal platelet secretome and a novel tool to model platelet physiology, pathophysiology, and to identify translational biomarkers of platelet-mediated disease.

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. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.

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. 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 ± 604 nM thrombin•min and 636 ± 440 nM 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.

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. Camel FVIII can be concentrated 2 to 3 times in cryoprecipitate. There was a significant loss of camel FVIII when comparing levels of FVIII in camel plasma after 1 h of incubation at 37°C (533%), 40°C (364%), and 50°C (223%). Thrombin generation of camel plasma is comparable to that of human plasma. It was concluded that camel plasma contains very elevated levels of FVIII:C, approaching 8 times the levels in human plasma, and that these elevated levels could not be attributed to excessive thrombin generation. Unlike human FVIII:C, camel FVIII:C is remarkably heat stable. Taken together, these unique features of camel FVIII could be part of the physiological adaptation of hemostasis of the Arabian camel in order to survive in the hot desert environment.

L'objectif de la présente étude était de caractériser les niveaux très élevés du facteur de coagulation VIII (FVIII) dans le plasma de chameau. Du sang entier a été prélevé de chameaux en santé et des épreuves d'activité de coagulation du facteur VIII (FVIII:C) ont été effectuées en utilisant des techniques chromogéniques et de coagulation. L'anticoagulant citrate phosphate dextrose adénine (CPDA) a permis la récolte la plus élevée de FVIII:C, le niveau plasmatique de facteur VIII, comparativement aux anticoagulants héparine:saline et héparine CPDA. Le FVIII de chameau peut être concentré 2 à 3 fois dans des cryoprécipités. Il y avait une perte significative de FVIII de chameau lorsque l'on comparait les niveaux de FVIII dans le plasma de chameau après 1 h d'incubation à 37 °C (533 %), 40 °C (364 %), et 50 °C (223 %). La génération de thrombine dans le plasma de chameau est comparable à celle dans le plasma humain. Il a été conclu que le plasma de chameau contient des niveaux très élevés de FVIII:C, atteignant près de 8 fois le niveau dans le plasma humain, et que ces niveaux élevés ne pouvaient pas être attribué à une génération excessive de thrombine. Comparativement au FVIII:C humain, le FVIII:C de chameau est très stable à la chaleur. Prises dans leur ensemble, ces caractéristiques uniques du FVIII de chameau pourraient faire partie de l'adaptation physiologique de l'hémostase du chameau arabe afin de lui permettre de survivre dans l'environnement chaud du désert.(Traduit par Docteur Serge Messier).

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). 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.

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. 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, gamma-thrombin, and convulxin; final concentrations of each were 20microm, 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 gamma-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 gamma-thrombin at various iCa concentrations. Varying the pH had no effect on amplitude of aggregation induced by convulxin or gamma-thrombin, but the aggregation rate increased with increasing pH for both agonists. CONCLUSIONS AND CLINICAL RELEVANCE-Aggregation of canine platelets induced by ADP and gamma-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.

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). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.

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. Platelet number, von Willebrand factor, clot retraction, and coagulation screening assays were normal, whereas bleeding time tests were prolonged. Platelet aggregation and release responses to all agonists, except thrombin, were markedly impaired. Platelet membranes had normal concentrations of integrin alphaIIb-beta3; however, ADP-induced fibrinogen binding by activated platelets was markedly impaired. Forskolin-stimulated platelets exhibited a marked increase in intraplatelet cAMP associated with impaired phosphodiesterase (PDE) activity, whereas levels of extractable phosphoinositides were 1.5-fold to 2-fold higher in thrombin-stimulated affected platelets. DNA analysis of the CalDAG-GEFI gene in affected dogs documented the existence of 3 distinct mutations within portions of the CalDAG-GEFI gene encoding for structurally conserved regions within the catalytic domain of the protein. The mutations are predicted to result in either lack of synthesis, enhanced degradation, or marked impairment of protein function. The dysfunctional profile of canine platelets observed in mutant dogs putatively links CalDAG-GEFI and its target Rap1 or other Ras family member, for the first time, to a role in pathways that regulate cAMP PDE activity and thrombin-stimulated phosphoinositide anchoring or metabolism. The finding of distinct functional mutations in 3 dog breeds suggests that mutations in the CalDAG-GEFI gene may be implicated in similar defects in human patients with congenital platelet disorders having primary secretion defects of unknown etiology.

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. 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.

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). 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.

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. Affected cat 1 had a single nucleotide change in exon 8 at the 1st nucleotide position of the codon encoding an arginine (CGA to TGA) at amino acid position 338. This mutation would be predicted to result in the appearance of a premature stop codon in the portion of the gene encoding much of the catalytic domain of the protein. Affected cat 2 had a single nucleotide change in exon 4 at the 2nd nucleotide position of the codon encoding amino acid 82 (TGT to TAT), which would be predicted to result in the substitution of a tyrosine for a cysteine. This substitution would likely result in disruption of a disulfide bond crucial to normal protein structure and function. This study represents the 1st time hemophilia B has been characterized at the molecular level in cats.

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. Fresh whole blood was transfused to control frequent bleeding episodes throughout the monkey's life. Although vWD is the most common inherited bleeding disorder in humans and dogs, this is the first report of vWD in a nonhuman primate.

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. Washed platelets from the affected dogs demonstrated approximately twice normal clotting times in a platelet factor 3 availability assay and, in a prothrombinase assay, generated only background levels of thrombin in response to calcium ionophore, thrombin, or combined thrombin plus collagen stimulation. While platelet phospholipid content was normal, flow cytometric analyses revealed diminished phosphatidylserine exposure and a failure of microvesiculation in response to calcium ionophore, thrombin, and collagen stimulation. Pedigree studies indicate a likely homozygous recessive inheritance pattern of the defect. These findings confirm the importance of platelet procoagulant activity for in vivo hemostasis and provide a large animal model for studying agonist-induced signal transduction, calcium mobilization, and effector pathways involved in the late platelet response of transmembrane phospholipid movement and membrane vesiculation.