You are looking at 1 - 9 of 9 items for
- Author or Editor: Fern Tablin x
- Refine by Access: All Content x
Objective—To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb).
Sample Population—Platelets obtained from 11 specific-pathogen-free cats.
Procedure—Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation.
Results—Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 µg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton.
Conclusions and Clinical Relevance—Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis. (Am J Vet Res 2001;62:195–201)
Objective—To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP).
Sample Population—PRP obtained from 6 horses.
Procedures—PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 μg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor β, isoform 1 (TGFβ1); and insulin-like growth factor, isoform 1 (IGF-1) were quantified by use of ELISAs. Statistical analysis was conducted via repeated-measures ANOVA.
Results—Platelet numbers were significantly higher in tube-prepared PRP than in automated-prepared PRP Growth factor concentrations did not differ significantly between preparation methods. Mean PDGF-BB concentration ranged from 134 to 7,157 pg/mL, mean TGFβ1 concentration ranged from 1,153 to 22,677 pg/mL, and mean IGF-1 concentration ranged from 150 to 280 ng/mL. Shear force did not affect growth factor concentrations. Dose-dependent increases in PDGF-BB and TGFβ1 were detected in response to collagen, but equalled only 10% of the estimated total platelet content. Concentrations of IGF-1 were not significantly different among treatments and negative or positive control treatments. Serum concentrations of PDGF-BB and TGFβ1 exceeded concentrations in PRP for most treatment conditions.
Conclusions and Clinical Relevance—Release of growth factors from equine PRP was negligible as a result of the injection process alone. Investigation of platelet-activation protocols is warranted to potentially enhance PRP treatment efficacy in horses.
Objective—To assess platelet count, mean platelet volume (MPV), metabolic characteristics, and platelet function in a dimethyl sulfoxide (DMSO)–stabilized canine frozen platelet concentrate (PC).
Sample Population—11 units of a commercial frozen PC in 6% DMSO and fresh plateletrich plasma from 6 healthy control dogs.
Procedures—PCs were thawed, and the following data were collected: thaw time, platelet count, MPV, pH, PCO 2, and PO2 and HCO3 −, glucose, and lactate content. Phosphatidylserine translocation was determined by use of flow cytometry. Fresh platelet-rich plasma from healthy dogs served as a source of control platelets for flow cytometric analysis.
Results—At thaw, the platelet count in the frozen PC ranged from 243,000 to 742,000 platelets/μL. Median platelet count of paired samples was 680,000 platelets/μL and decreased significantly to 509,000 platelets/μL at 2 hours after thaw. Median MPV at thaw was 11.15 femtoliters and was stable after 2 hours. Compared with fresh platelets, frozen PC had increased amounts of phosphatidylserine in the outer leaflet of the platelet membrane in the resting (ie, not treated with thrombin) state (19% vs 99%, respectively) and alterations in cellular morphology, all of which were consistent with platelet activation.
Conclusions and Clinical Relevance—Results of this in vitro study indicated that there was a decrease in platelet quantity and function as well as an increase in platelet activation during the freeze-and-thaw process in DMSO-stabilized canine frozen PC. In vivo effects on PC remain to be determined.
Objective—To evaluate the effect of 2 hydroxyethyl starch (HES) preparations (ie, HES solution with a molecular weight of 600 kd and a degree of substitution of 0.7 [HES 600/0.7] and a calcium-containing polyionic HES solution with a molecular weight of 670 kd and a degree of substitution of 0.75 [HES 670/0.75]) on canine platelet function.
Sample Population—Blood samples from 10 healthy adult dogs.
Procedures—Dilution of citrated whole blood was performed with saline (0.9% NaCl) solution, HES 600/0.7, and HES 670/0.75 at ratios of 1:9 (ie, 1 part saline solution or colloid to 9 parts whole blood) and 1:3. Measurements of time to platelet plug formation in a capillary tube (ie, closure time) were made by use of a bench-top platelet function analyzer with collagen and ADP platelet agonists.
Results—Mean baseline closure time was 68.0 ± 15.3 seconds. A 1:3 dilution of whole blood with saline solution, HES 600/0.7, and HES 670/0.75 resulted in mean closure times of 85.8 ± 15.7 seconds, 100.6 ± 18.6 seconds, and 101.6 ± 16.2 seconds, respectively. Closure time following 1:3 dilution of whole blood with saline solution was significantly different from baseline and from 1:9 dilution with saline solution. Closure time following 1:3 dilution of whole blood with HES 670/0.75 was significantly different from baseline, 1:3 and 1:9 dilutions with saline solution, and 1:9 dilutions with HES 600/0.7 or HES 670/0.75.
Conclusions and Clinical Relevance—Saline solution, HES 600/0.7, and HES 670/0.75 affect canine platelet function by prolonging closure times; HES solutions prolonged closure time to a greater extent than saline solution.
Objective—To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm.
Animals—444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males.
Procedures—Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen.
Results—Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL).
Conclusions and Clinical Relevance—Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of αllb-β3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.
Objective—To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay.
Animals—6 clinically normal adult horses.
Procedures—Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor β1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS).
Results—Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor–stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS.
Conclusions and Clinical Relevance—The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.
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-β1 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-β1 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.
To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses.
6 healthy adult horses.
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.
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)
Objective—To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days.
Animals—13 healthy adult Thoroughbreds.
Procedures—Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4°C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined.
Results—Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 μg of streptavidin-PE/1 × 106 RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively.
Conclusions and Clinical Relevance—Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.