Genetics of equine bleeding disorders.

Genetic bleeding disorders can have a profound impact on a horse's health and athletic career. As such, it is important to understand the mechanisms of these diseases and how they are diagnosed. These diseases include haemophilia A, von Willebrand disease, prekallikrein deficiency, Glanzmann's Thrombasthenia and Atypical Equine Thrombasthenia. Exercise-induced pulmonary haemorrhage also has a proposed genetic component. Genetic mutations have been identified for haemophilia A and Glanzmann's Thrombasthenia in the horse. Mutations are known for von Willebrand disease and prekallikrein deficiency in other species. In the absence of genetic tests, bleeding disorders are typically diagnosed by measuring platelet function, von Willebrand factor, and other coagulation protein levels and activities. For autosomal recessive diseases, genetic testing can prevent the breeding of two carriers.

Comparison of nonparametric and parametric methods for time-frequency heart rate variability analysis in a rodent model of cardiovascular disease.

The aim of time-varying heart rate variability spectral analysis is to detect and quantify changes in the heart rate variability spectrum components during nonstationary events. Of the methods available, the nonparametric short-time Fourier Transform and parametric time-varying autoregressive modeling are the most commonly employed. The current study (1) compares short-time Fourier Transform and autoregressive modeling methods influence on heart rate variability spectral characteristics over time and during an experimental ozone exposure in mature adult spontaneously hypertensive rats, (2) evaluates the agreement between short-time Fourier Transform and autoregressive modeling method results, and (3) describes the advantages and disadvantages of each method. Although similar trends were detected during ozone exposure, statistical comparisons identified significant differences between short-time Fourier Transform and autoregressive modeling analysis results. Significant differences were observed between methods for LF power (p ≤ 0.014); HF power (p ≤ 0.011); total power (p ≤ 0.027); and normalized HF power (p = 0.05). Furthermore, inconsistencies between exposure-related observations accentuated the lack of agreement between short-time Fourier Transform and autoregressive modeling overall. Thus, the short-time Fourier Transform and autoregressive modeling methods for time-varying heart rate variability analysis could not be considered interchangeable for evaluations with or without interventions that are known to affect cardio-autonomic activity.

Canine platelets express functional Toll-like receptor-4: lipopolysaccharide-triggered platelet activation is dependent on adenosine diphosphate and thromboxane A2 in dogs.

BACKGROUND: Functional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A2 (TxA2). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS). RESULTS: We discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B2 secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA2 pathway. CONCLUSION: Expression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA2 pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.

Pharmacokinetics and competitive pharmacodynamics of ADP-induced platelet activation after oral administration of clopidogrel to horses.

OBJECTIVE: To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS: Compared with results for preadministration samples, platelet aggregation in response to 12.5µM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25µM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20µM) and prostaglandin E1 (3.3µM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505-512).

Equine platelet concentrate preparation and validation.

BACKGROUND: Development of equine platelet concentrate (PC) would aid management of cases requiring transfused platelets (PLTs), where adminstration of whole-blood or platelet-rich plasma (PRP) might be contraindicated. OBJECTIVES: To test and validate a method for production of an equine PRP-PC product. ANIMALS: Six healthy Thoroughbred geldings from a research herd. METHODS: In this prospective experimental study, whole blood was collected and processed through multiple centrifugation steps to yield 120 mL of PC. The PC was stored at 22°C and gently and continuously agitated. Measurements of PLT count, pH, and concentrations of glucose, lactate, electrolytes, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST), as well as partial pressures of oxygen and carbon dioxide were performed on days 0, 1, 2, 3, 5, and 7. Platelet aggregometry and bacterial culture were also performed. RESULTS: The PC always had a PLT count of ≥550 × 103 cells/µL. Aggregometry graph amplitude (P < .0001) and area under the curve (P < .05) significantly decreased over time. Sodium, chloride, lactate (P < .0001), and oxygen (P < .01) concentrations significantly increased over time. pH (P < .001), glucose and bicarbonate concentrations (P < .0001) significantly decreased over time. There was no significant difference in potassium concentration, PLT count, LDH and AST activities and no bacterial growth from culture. CONCLUSIONS AND CLINICAL IMPORTANCE: The described technique yielded a PC that meets the standards of the American Association of Blood Banks for human PC.

A Comparative Review of Neutrophil Extracellular Traps in Sepsis.

Sepsis is the leading cause of critical illness and mortality in human beings and animals. Neutrophils are the primary effector cells of innate immunity during sepsis. Besides degranulation and phagocytosis, neutrophils also release neutrophil extracellular traps (NETs), composed of cell-free DNA, histones, and antimicrobial proteins. Although NETs have protective roles in the initial stages of sepsis, excessive NET formation has been found to induce thrombosis and multiple organ failure in murine sepsis models. Since the discovery of NETs nearly a decade ago, many investigators have identified NETs in various species. However, many questions remain regarding the exact mechanisms and fate of neutrophils following NET formation. In humans and mice, platelet-neutrophil interactions via direct binding or soluble mediators seem to play an important role in mediating NET formation during sepsis. Preliminary data suggest that these interactions may be species dependent. Regardless of these differences, there is increasing evidence in human and veterinary medicine suggesting that NETs play a crucial role in the pathogenesis of intravascular thrombosis and multiple organ failure in sepsis. Because the outcome of sepsis is highly dependent on early recognition and intervention, detection of NETs or NET components can aid in the diagnosis of sepsis in humans and veterinary species. In addition, the use of novel therapies such as deoxyribonuclease and non-anticoagulant heparin to target NET components shows promising results in murine septic models. Much work is needed in translating these NET-targeting therapies to clinical practice.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy.

BACKGROUND: Canine neutrophils release neutrophil extracellular traps (NETs) in response to lipopolysaccharide but NETs from clinical septic dogs had not been identified. The primary aim is to describe the methodology of identifying and quantifying neutrophil extracellular traps (NETs) in cytology samples of septic foci in dogs with sepsis using immunofluorescence microscopy. Cytology samples including endotracheal tracheal wash (ETW), bronchoalveolar lavage (BAL), abdominal and pleural effusion collected from 5 dogs (3 septic, 2 non-septic) were fixed, permeabilized and stained for myeloperoxidase (MPO), citrullinated histone H3 (citH3) and cell-free DNA (cfDNA). Fluorescence microscopy was used to identify and quantify NETs in 10 random views at 40× magnification. NETs were identified based on co-localization of MPO, citH3 and cfDNA. NETs were quantified as a ratio (number of NETs: number of neutrophils). Neutrophils were identified based on cytoplasmic MPO, cellular diameter and nuclear morphology. RESULTS: NETs were identified and quantified in all cytology samples collected from septic dogs. A small number of NETs was documented in one dog with sterile chronic bronchitis. No NETs were found in sterile abdominal effusion collected from one dog with congestive heart failure. CONCLUSIONS: Immunofluorescence microscopy could be a useful tool for the study of NETs in dogs with clinical sepsis.

Ultrafine Particulate Matter Combined With Ozone Exacerbates Lung Injury in Mature Adult Rats With Cardiovascular Disease.

Particulate matter (PM) and ozone (O3) are dominant air pollutants that contribute to development and exacerbation of multiple cardiopulmonary diseases. Mature adults with cardiovascular disease (CVD) are particularly susceptible to air pollution-related cardiopulmonary morbidities and mortalities. The aim was to investigate the biologic potency of ultrafine particulate matter (UFPM) combined with O3 in the lungs of mature adult normotensive and spontaneously hypertensive (SH) Wistar-Kyoto rats. Conscious, mature adult male normal Wistar-Kyoto (NW) and SH rats were exposed to one of the following atmospheres: filtered air (FA); UFPM (∼ 250 µg/m3); O3 (1.0 ppm); or UFPM + O3 (∼ 250 µg/m3 + 1.0 ppm) combined for 6 h, followed by an 8 h FA recovery period. Lung sections were evaluated for lesions in the large airways, terminal bronchiolar/alveolar duct regions, alveolar parenchyma, and vasculature. NW and SH rats were similarly affected by the combined-pollutant exposure, displaying severe injury in both large and small airways. SH rats were particularly susceptible to O3 exposure, exhibiting increased injury scores in terminal bronchioles and epithelial degeneration in large airways. UFPM-exposure groups had minimal histologic changes. The chemical composition of UFPM was altered by the addition of O3, indicating that ozonolysis promoted compound degradation. O3 increased the biologic potency of UFPM, resulting in greater lung injury following exposure. Pathologic manifestations of CVD may confer susceptibility to air pollution by impairing normal lung defenses and responses to exposure.

Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase.

Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×106 cells/ml) from healthy dogs were isolated and treated with 100µg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200µM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.

Comparison of equine platelet function and survival in whole blood collected in acid-citrate-dextrose solution or citrate-phosphate-dextrose-adenine solution.

BACKGROUND: Equine whole blood collection and storage methods have been evaluated to assess red blood cell viability; however, platelet (PLT) viability has not been comprehensively assessed. OBJECTIVES: The purpose of the study was to compare viability of PLTs collected in whole blood into 2 different anticoagulants. METHODS: Whole blood from 6 healthy adult Thoroughbred horses was collected into citrate-phosphate-dextrose-adenine (CPDA) or acid-citrate-dextrose (ACD). Platelet count, pH, and concentrations of glucose, lactate, carbon dioxide, oxygen, bicarbonate, sodium, potassium, and chloride were measured within 10 minutes of collection and then again one hour later at which time PLT aggregometry was performed to assess PLT function. RESULTS: Aggregometry mean amplitudes were significantly higher in CPDA compared to ACD. Blood glucose, pH, bicarbonate, sodium, and lactate concentrations were significantly higher in CPDA compared to ACD. Lactate concentration was higher following one hour in either anticoagulant. Potassium, oxygen, and carbon dioxide concentrations were significantly higher in ACD compared to CPDA at collection. CONCLUSIONS: Platelet aggregometry results suggest that CPDA is superior to ACD for maintaining PLT viability following whole blood collection. This may be associated with the higher, more neutral pH as well as an increase in glucose available for metabolism. Although lactate was increased in the CPDA samples it was not high enough to decrease pH and therefore may not have been high enough to cause morphologic lesions and loss of PLT viability.

Early Life Wildfire Smoke Exposure Is Associated with Immune Dysregulation and Lung Function Decrements in Adolescence.

The long-term health effects of wildfire smoke exposure in pediatric populations are not known. The objectives of this study were to determine if early life exposure to wildfire smoke can affect parameters of immunity and airway physiology that are detectable with maturity. We studied a mixed-sex cohort of rhesus macaque monkeys that were exposed as infants to ambient wood smoke from a series of Northern California wildfires in the summer of 2008. Peripheral blood mononuclear cells (PBMCs) and pulmonary function measures were obtained when animals were approximately 3 years of age. PBMCs were cultured with either LPS or flagellin, followed by measurement of secreted IL-8 and IL-6 protein. PBMCs from a subset of female animals were also evaluated by Toll-like receptor (TLR) pathway mRNA analysis. Induction of IL-8 protein synthesis with either LPS or flagellin was significantly reduced in PBMC cultures from wildfire smoke-exposed female monkeys. In contrast, LPS- or flagellin-induced IL-6 protein synthesis was significantly reduced in PBMC cultures from wildfire smoke-exposed male monkeys. Baseline and TLR ligand-induced expression of the transcription factor, RelB, was globally modulated in PBMCs from wildfire smoke-exposed monkeys, with additional TLR pathway genes affected in a ligand-dependent manner. Wildfire smoke-exposed monkeys displayed significantly reduced inspiratory capacity, residual volume, vital capacity, functional residual capacity, and total lung capacity per unit of body weight relative to control animals. Our findings suggest that ambient wildfire smoke exposure during infancy results in sex-dependent attenuation of systemic TLR responses and reduced lung volume in adolescence.

TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist.

Biological dose response to PM2.5: effect of particle extraction method on platelet and lung responses.

Particulate matter (PM) exposure contributes to respiratory diseases and cardiopulmonary mortality. PM toxicity is related to sources and composition, such as abundance of polycyclic aromatic hydrocarbons (PAHs). We exposed adult male BALB/c mice, via oropharyngeal aspiration, to a range of doses of PM2.5 collected during the winter in downtown Sacramento near a major freeway interchange (SacPM). Two preparation methods (spin-down and multi-solvent extraction) were tested to remove particles from collection filters. Three doses were analyzed 24 h after treatment for (1) leukocytes and total protein in bronchoalveolar lavage fluid (BALF), (2) airway-specific and whole lobe expression of PAH-sensitive genes (CYP1B1 and CYP1A1) and IL-1 b, (3) lung histology, and (4) platelet function. Both extraction methods stimulated biological responses, but the spin-down method was more robust at producing IL-1 b and CYP1B1 gene responses and the multi-solvent extraction induced whole lung CYP1A1. Neutrophils in the BALF were increased 5- to 10-fold at the mid and high dose for both preparations. Histopathology scores indicated dose-dependent responses and increased pathology associated with spin-down-derived PM exposure. In microdissected airways, spin-down PM increased CYP1B1 gene expression significantly, but multi-solvent extracted PM did not. Platelet responses to the physiological agonist thrombin were approximately twice as potent in the spin-down preparation as in the multi-solvent extract. We conclude (1) the method of filter extraction can influence the degree of biological response, (2) for SacPM the minimal effective dose is 27.5-50 µg based on neutrophil recruitment, and (3) P450s are upregulated differently in airways and lung parenchyma in response to PAH-containing PM.

Comparison of the platelet-rich plasma and buffy coat protocols for preparation of canine platelet concentrates.

BACKGROUND: Platelet (PLT) concentrates (PC) can be produced via the buffy coat (BC) or platelet-rich plasma (PRP) protocols. The 2 methods have not been compared with canine blood. OBJECTIVES: The aims of the study were to compare the PLT, WBC, and RBC concentrations, in vitro PLT function, and markers of platelet storage lesion (PSL) in canine PC generated by 2 different protocols, and determine microbial growth throughout storage. METHODS: PC from 8 healthy donor dogs were produced using 2 standard protocols, PRP and BC. PLT, WBC, and RBC counts, optical aggregometry assays, and PSL markers (pH, pCO2 , HCO3 , lactate and glucose concentrations, and LDH activity) were determined on storage days 0, 1, 3, 5, and 7. Aerobic and anaerobic bacterial cultures were also performed. RESULTS: Mean PLT counts were comparable between protocols and remained stable throughout storage up to day 7, while median WBC and RBC counts on day 0 were significantly higher in the BC-PC group (17,800 WBCs/µL; 195,000 RBCs/µL) than in the PRP-PC group (200 WBCs/µL; 10,000 RBCs/µL) (P = .012). In PRP-PC aggregometry, the median slope and amplitude in response to γ-thrombin and convulxin (+ ADP) were significantly decreased, and virtually absent in BC-PC during storage. PSL markers (lactate, LDH activity) were higher in BC-PC. Aerobic bacterial growth was observed in 2 PRP-PC and 1 BC-PC. CONCLUSIONS: This in vitro study suggests that PRP-PC had lesser WBC and RBC contamination and superior PLT function compared with BC-PC. In vivo studies are required to address safety and efficacy of PRP-PC.

Ultrastructure and growth factor content of equine platelet-rich fibrin gels.

OBJECTIVE: To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). SAMPLE: PRFGs and conventional FGs prepared from the blood of 10 healthy horses. PROCEDURES: Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/µL, 250 × 10³ platelets/µL, 500 × 10³ platelets/µL, and 1,000 × 10³ platelets/µL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-ß1 concentrations were quantified with ELISAs. RESULTS: Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-ß1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/µL. CONCLUSIONS AND CLINICAL RELEVANCE: The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

Synovial fluid growth factor and cytokine concentrations after intra-articular injection of a platelet-rich product in horses.

Platelet-rich plasma (PRP) products may be useful for treatment of joint disease in horses, but may contain undesirable pro-inflammatory cytokines in addition to growth factors. This study investigated whether autologous PRP increases synovial fluid growth factor and cytokine concentrations when injected into normal equine metacarpophalangeal and metatarsophalangeal (fetlock) joints. Fetlock joints of seven healthy horses received one of four treatments: saline, resting PRP, CaCl2-activated PRP or thrombin-activated PRP. Synovial fluid was sampled prior to injection and at 6, 24, 48 and 96 h post-injection. Platelet-derived growth factor (PDGF-BB), transforming growth factor ß1 (TGFß1), interleukin (IL)-6 and tumor necrosis factor α (TNFα) concentrations in synovial fluid and PRP were measured by ELISA. Synovial fluid PDGF-BB, TGFß1, IL-6, TNFα and IL-1 concentrations were also measured in vitro after incubation for 6h with resting PRP only. Growth factor concentrations, but not cytokine concentrations, were significantly higher in activated PRP than in resting PRP samples. After intra-articular injection with resting or thrombin-activated PRP, synovial TGFß1 increased significantly compared to baseline levels. TNFα and IL-6 were significantly increased in synovial fluid after thrombin-activated PRP injection. In vitro, growth factor concentrations increased significantly in synovial fluid after mixing with PRP, indicating that exogenous activation of PRP for intra-articular injection may be unnecessary, whereas cytokine levels did not. In conclusion, thrombin-activated PRP induced an inflammatory cytokine response in joints, whereas resting or CaCl2-activated PRP did not. Synovial growth factor levels were low overall; the reported benefits of intra-articular PRP may not be attributable to changes in local PDGF or TGFß1 concentrations.

Intra-articular use of a platelet-rich product in normal horses: clinical signs and cytologic responses.

OBJECTIVES: (1) To report the clinical and synovial effects of a platelet-rich product (PRPr) in normal equine joints, (2) to assess the persistence of platelets within synovial fluid after intra-articular injection, (3) to compare responses to different preparations of that product, and (4) to evaluate a gravity filtration system for PRPr preparation in horses. STUDY DESIGN: Experimental. METHODS: A platelet-rich saline product (PRPr) was prepared from 7 normal horses using a proprietary preparation device and was divided into 3 treatments: resting, CaCl2 -activated (23 mM, final), and bovine thrombin-activated (10 U/mL, final). Each horse had 3 concurrent randomly assigned intra-articular PRPr treatments administered in their metacarpophalangeal/metatarsophalangeal joints; the fourth limb was injected with saline (0.9% NaCl) solution as a control. Clinical assessments, cytologic analysis of synovial fluid and hemograms were performed at 6, 24, 48, and 96 hours after injection. PRPr composition and growth factor content were analyzed. RESULTS: The gravity filtration system produced a moderately concentrated PRPr. At 6 and 24 hours, when compared to control values, all PRPr treatments caused a significant increase in synovial WBC concentration (P < .0059) and neutrophil percentage (P < .0005). Bovine thrombin-activated PRPr injection consistently caused increased effusion scores and periarticular signs. At all time points, the synovial WBC concentration after thrombin-activated PRPr was significantly greater (P < .001) than for the control, CaCl2 -activated or resting PRPr. Intact platelets could be observed in synovial fluid for up to 5 days after intra-articular PRPr injection. CONCLUSIONS: Resting and CaCl2 -activated PRPr may be safely used to treat equine joints, but bovine thrombin activation is not recommended at 10 U/mL. A PRPr can be prepared using a gravity filtration system, eliminating the need for centrifugation.

Seasonal influences on CAPs exposures: differential responses in platelet activation, serum cytokines and xenobiotic gene expression.

Increasing evidence suggests a role for a systemic pro-coagulant state in the pathogenesis of cardiac dysfunction subsequent to inhalation of airborne particulate matter (PM). We evaluated platelet activation, systemic cytokines and pulmonary gene expression in mice exposed to concentrated ambient particulate matter (CAPs) in the summer of 2008 (S08) and winter of 2009 (W09) from the San Joaquin Valley of California, a region with severe PM pollution episodes. Additionally, we characterized the PM from both exposures including organic compounds, metals, and polycyclic aromatic hydrocarbons. Mice were exposed to an average of 39.01 µg/m(3) of CAPs in the winter and 21.7 µg/m3 CAPs in the summer, in a size range less than 2.5 µm for 6 h/day for 5 days per week for 2 weeks. Platelets were analyzed by flow cytometry for relative size, shape, CD41, P-selectin and lysosomal associated membrane protein-1 (LAMP-1) expression. Platelets from W09 CAPs-exposed animals had a greater response to thrombin stimulation than platelets from S08 CAPs-exposed animals. Serum cytokines were analyzed by bead based immunologic assays. W09 CAPs-exposed mice had elevations in IL-2, MIP-1α, and TNFα. Laser capture microdissection (LCM) of pulmonary vasculature, parenchyma and airways all showed increases in CYP1a1 gene expression. Pulmonary vasculature showed increased expression of ICAM-1 and Nox-2. Our findings demonstrate that W09 CAPs exposure generated a greater systemic pro-inflammatory and pro-coagulant response to inhalation of environmentally derived fine and ultrafine PM. Changes in platelet responsiveness to agonists, seen in both exposures, strongly suggests a role for platelet activation in the cardiovascular and respiratory effects of particulate air pollution.