Canine Platelet-rich Plasma Research Articles

Literature review details and supports the application of platelet-rich plasma products in canine medicine, particularly as an orthobiologic agent for osteoarthritis.

Platelet-rich plasma (PRP) and other platelet-derived products represent a subset of regenerative medicine and have been researched in the veterinary community for the treatment of osteoarthritis, soft tissue wounds, tendinopathies, periodontitis, and fracture repairs. PRP is simple to produce, relatively affordable, safe, and can be delivered on site, making it an appealing therapeutic agent in veterinary medicine. As an orthobiologic for the treatment of osteoarthritis, it is one of few interventions with clinical study support that possess anabolic potential. Platelet product variability is wide ranging and often described in terms of cellular content or platelet enrichment. Growth factors associated with platelet activation and subsequent degranulation may mediate inflammation, modulate cellular immune response, and promote tissue repair. Product composition, dosage, and application likely influence treatment outcomes depending on the classification of the disease targeted. Sufficient canine data regarding the formulation and clinical application of canine PRP exist to warrant review. The aim of this narrative is to provide scientific background and clinical insight for veterinarians regarding platelet product content/formulation, mechanisms of action, considerations for use, and clinical application in dogs.

Lower centrifugation speed and time are positively associated with platelet concentration in a canine autologous conditioned plasma system.

To evaluate the effect of variable centrifugation protocols on the cellular composition of the final product of a canine autologous conditioned plasma double-syringe system. 30 client-owned healthy adult medium- to large-breed (17- to 45-kg) dogs. 35 mL of anticoagulated whole blood from each subject was aliquoted into 3 samples: a baseline and 2 double syringes. The syringes were processed for platelet-rich plasma (PRP). Each double syringe was randomly assigned to 1 of 5 groups, which varied in centrifugation settings between 580 and 1,304 X g and 5 and 10 minutes. CBC analysis was performed on each of the samples to determine cellular composition. A mixed-effect linear model was fit to the data. 60 PRP samples and 30 whole blood samples were analyzed. Manufacturer settings generated a platelet fold change > 1 but did not increase concentration to the extent expected. When comparing speed alone, increased centrifugation force was associated with lower platelet fold change. When comparing time alone, increased centrifugation time was also associated with lower platelet fold change and lower leukocyte concentration. Autologous conditioned plasma double syringes require a low volume of initial whole blood, making them preferable for canine PRP in clinical settings. This study aimed to evaluate the effect of the centrifugation protocol on the final product cellular composition in dogs and add to the available data on protocols to maximize platelet yield in PRP. Due to inherent individual variability, this study emphasized the importance of evaluating biological samples prior to administration to predict and improve patient outcomes.

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Evaluation of keeping quality of canine platelet rich plasma under different storage conditions

Platelet rich plasma (PRP) therapy is an integral part of regenerative medicine as the platelets possess a good healing capacity owing to the presence of a wide variety of growth factors in the platelet granules found in the cytoplasm of the platelet. Autologous PRP was prepared from the blood of the patient itself, without any preservatives. Storage of PRP was one of the main hurdles of the treatment modality. During storage, the platelet counts may get reduced, undergo activation or get contaminated with bacteria as no preservatives are used in the preparation of autologous PRP. Cytological changes and microbial quality of the PRP during storage at 4oC and -20oC for seven days were analysed in this study. Reduction in platelet count and the chance of microbial contamination were less when autologous PRP was stored at -20°C compared to 4°C.

Evaluation of Platelet and Leukocyte Counts in Canine Platelet-Rich Plasma Obtained After Successive Blood Collections From the Same Patient and the Effects of Freezing on the Concentration of Growth Factors Present in It.

ObjectiveThe purpose of this study was 2-fold: to evaluate whether the timing of collection influences the platelet counts and leukocyte counts of PRP samples, and to evaluate growth factor concentrations in canine PRP after freezing and storage without a preservation agent for 6 months of time.Materials and MethodsWhole blood collection was performed three times over the course of 4 weeks. All PRP samples were evaluated with a CBC analysis. The PRP samples were frozen and stored without a preservation agent for the duration of the 4-week study.ResultsThe platelet and leukocyte counts were not statistically significant between the timing of blood draws over the course of 4 weeks. All three growth factors were present in measurable quantities after freezing and storage for 6 months without a preservation agent.Clinical RelevancePDGF, TGF-β1, and VEGF were all present in measurable quantities. Furthermore, PDGF and TGF-β1 were correlated with platelet count of the final PRP. VEGF concentrations were able to be quantified. We correctly hypothesized growth factor concentrations would be present and measurable in canine PRP frozen and stored without a preservation agent for 6 months.

Antibacterial Properties of Canine Platelet-Rich Plasma and Other Non-Transfusional Hemo-Components: An in vitro Study.

This in vitro study was carried out to evaluate the potential antibacterial properties of canine non-transfusional hemo-components. Therapeutic formulations commonly used for regenerative medicine purposes (platelet-rich plasma, platelet gel, platelet lysate, fibrin glue), considering both leukocyte-rich and leukocyte-poor formulations, but also platelet-poor plasma and activating substances (thrombin, calcium gluconate), were tested to detect elements with potential antimicrobial properties. The antibacterial effect was tested on different bacterial strains (Staphylococcus aureus subspecies aureus, Staphylococcus cohnii subspecies cohnii, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae subspecies pneumoniae) isolated from canine wounds and classified as susceptible, multidrug-, extensively, and pandrug-resistant bacteria toward a known panel of human and veterinary antibiotics. The evaluation was carried out by agar gel diffusion method (Kirby–Bauer) and micro-inhibition in broth using microplates and spectrophotometer reading. The study findings confirmed the hypothesized antibacterial properties of canine non-transfusional hemo-components. A more effective bacteriostatic effect was found against Gram-negative bacteria, drug-resistant too. The presence of leukocytes or platelets does not appear to be essential for the antibacterial effect. Further studies should be conducted to evaluate the exact mechanism of action of the antimicrobial activity. However, non-transfusional hemo-components could be a useful natural aid in controlling bacterial infections in dogs.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

Rheological Properties and Growth Factors Content of Platelet-Rich Plasma: Relevance in Veterinary Biomedical Treatments.

Platelet-rich plasma (PRP) is a nontransfusional hemocomponent, considered as a powerful concentrate of growth factors (GFs) therapeutically used to stimulate tissue regeneration. The use of autologous PRP, as the patient’s own biological material, for therapeutic purposes represents a safe and effective alternative to conventional treatments in both human and veterinary medicine. The aim of this study was the characterization of canine PRP from rheological and biological points of view. Thus, a characterization of the viscoelastic properties of the PRP systems was performed in order to clarify the influence of different calcium concentrations, in the presence of autologous thrombin-rich solution, on the PRP gels’ mechanical properties, from which the applicability of these systems in biomedical treatments is strongly dependent. Then, an evaluation of the content of GFs in PRP, activated or not with thrombin, and stored at different temperatures (37 °C and −20 °C) was performed over time, outlining, for the first time, the importance of the effect of physiological temperature (37 °C) on the production of GFs. A clinical case study conducted in a dog with a complete rupture of the common calcaneal tendon (Achilles tendon) confirmed the relevance of this hemocomponent in the daily veterinary clinical activity and the potential translational value for human health.

Platelet Function and Therapeutic Applications in Dogs: Current Status and Future Prospects.

Simple SummaryPlatelets, small blood cells produced from megakaryocytes mainly in the bone marrow, are implicated not only in hemostasis, but also in different physiological and pathophysiological processes. This paper reviews canine platelet structure and functions, including an accurate description of the numerous surface receptors involved in the mechanisms of platelet adhesion and aggregation. In addition, we describe the most important canine platelet disorders that can affect platelet numbers (thrombocytopenias and thrombocytosis) or platelet function (thrombopathias). Platelet-related tests are vital in the analysis of primary hemostatic disorders and for this reason, we also discuss the efficacy of various tests in the diagnosis of canine platelet disorders. Finally, innovative therapeutic approaches based on the use of platelets and their derivatives for the treatment of various canine diseases such as inflammatory conditions (tendinitis and osteoarthritis) and for wound healing and tissue repair are reported.Significant progress has been made in the functional characterization of canine platelets in the last two decades. The role of canine platelets in hemostasis includes their adhesion to the subendothelium, activation, and aggregation, leading to primary clot formation at the site of injury. Studies on canine platelet function and advancements in laboratory testing have improved the diagnosis and understanding of platelet-related disorders as well as the knowledge of the mechanisms behind these diseases. This review focuses on the most recent discoveries in canine platelet structure, function, and disorders; and discusses the efficacy of various tests in the diagnosis of platelet-related disorders. With the relatively recent discovery of angiogenetic and reparative effects of growth factors found in platelets, this review also summarizes the use of canine platelet-rich plasma (PRP) alone or in association with stem cells in regenerative therapy. The characterization of proteomic and lipidomic profiles and development of platelet gene therapy in veterinary species are areas of future study with potential for major therapeutic benefits.

Comparison of the protocols for obtaining platelet-rich plasma in dogs: a cellular study

ABSTRACT: This study aimed to evaluate two protocols (PA and PB) that are used to obtain canine platelet-rich plasma (PRP) for cellularity. Twenty healthy dogs were used. Blood samples were collected and placed in two tubes of 3.2% sodium citrate. PA used double centrifugation at 210 x g, and 370 x g and PB used double centrifugation with 140 x g and 330 x g. The PRP samples from the protocols were examined in terms of their platelet, erythrocyte, and leukocyte count in the Neubauer chamber, differential leukocyte count and platelet morphological observation in blood smears. Data (mean and standard deviation) were analyzed with the 95% probability t-test (P <0.05) using Pearson’s correlation to test the relationship between platelets and erythrocytes, platelets, and leukocytes, and the leukocyte count versus the erythrocytes. Very weak negative correlation between platelets and leukocytes (p= -0.03), weak negative correlation between platelets and erythrocytes (p= -0.3) and a strong positive correlation between leukocytes and erythrocytes (ρ = 0.75) were noted. Although, BP did not reach the desired mean of one million platelets (979300 ± 79631 cells / μL), both protocols, A and B (4.42 ± 1.61 and 3.85 ± 1.55 times more platelets than total blood, respectively) (p <0.05) were efficient in concentrating platelets. Platelet activation was present in 26.55 ± 6.72% of the PA platelets and 26.25 ± 7.03% in PB (p> 0.05). PA and PB presented low erythrocyte concentration (p> 0.05), and PA had more leukocytes (p <0.05) than PB, with higher concentrations of basophils that were segmented, and lymphocytes.

Multicenter in vitro thromboelastography and thromboelastometry standardization.

To establish and compare the repeatability and reproducibility of activated thromboelastography (TEG) and thromboelastometry (ROTEM) assays. Multicenter in vitro test standardization. Veterinary academic centers. Test samples were obtained from normal, healthy dogs. Sixty identical 5mL aliquots of canine platelet-rich plasma collected by apheresis, frozen in 6% dimethyl sulfoxide, were tested initially. Sixty identical 6mL aliquots of canine fresh frozen plasma with admixed cryoprecipitate were subsequently evaluated. None. Frozen study samples, quality controls, reagents, and consumables were distributed to participating centers (7 TEG and 3 ROTEM). TEG centers analyzed study samples with kaolin and tissue factor activated assays; ROTEM centers ran proprietary ellagic acid activated and tissue factor activated assays. All machines underwent quality control prior to sample analysis. Within- and between-center coefficients of variation (CVs) were calculated and compared using Mann-Whitney tests and calculation of intraclass correlation coefficients. Within and between centers, individual parameters for both TEG and ROTEM assays were comparable. Both within-center and between-center CVs varied markedly (0.7-120.5% and 1.4-116.5%, respectively) with assay type, instrument, and parameter. CVs for equivalent parameters were not significantly different between the 2 platforms. Intraclass correlation coefficients suggested moderate agreement between centers. In general, individual parameter CVs for platelet-rich plasma samples were lower in TEG centers, while CVs for canine fresh frozen plasma with admixed cryoprecipitate samples were lower in ROTEM centers. More variation within and between centers was identified than anticipated, but some parameters such as alpha angle were repeatable and reproducible. Sample types for future multicenter standardization efforts will require further optimization and may need to be adapted separately to each platform. Individual centers using viscoelastic tests for evaluation and management of clinical patients should take steps to minimize preanalytical and analytical sources of variation.

Use of a Cyclooxygenase-2 Inhibitor Does Not Inhibit Platelet Activation or Growth Factor Release From Platelet-Rich Plasma

Background: It remains unestablished whether use of cyclooxygenase (COX)–2 inhibitors impairs platelet activation and anabolic growth factor release from platelets in platelet-rich plasma (PRP). Purpose: The purpose of this study was to assess the effects of a COX-2 inhibitor on platelet activation and anabolic growth factor release from canine PRP when using a clinically applicable PRP activator and to determine whether a 3-day washout would be sufficient to abrogate any COX-2 inhibitor-related impairment on platelet function. Study Design: Controlled laboratory study. Methods: Ten healthy dogs underwent blood collection and PRP preparation. Dogs were then administered a COX-2 inhibitor for 7 days, after which PRP preparation was repeated. The COX-2 inhibitor was continued for 4 more days and PRP preparation performed a third time, 3 days after discontinuation of the COX-2 inhibitor. Immediately after PRP preparation, the PRP was divided into 4 aliquots: 2 unactivated and 2 activated using human γ-thrombin (HGT). One activated and 1 unactivated sample were assessed using flow cytometry for platelet expression of CD62P and platelet-bound fibrinogen using the canine activated platelet-1 (CAP1) antibody. The 2 remaining samples were centrifuged and the supernatant assayed for transforming growth factor–β1 (TGF-β1), platelet-derived growth factor–BB (PDGF-BB), and thromboxane B2 (TXB2) concentrations. Differences in platelet activation and TGF-β1, PDGF-BB, and TXB2 concentrations over the 3 study weeks were evaluated using a 1-way repeated-measures ANOVA, and comparisons between activated and unactivated samples within a study week were assessed with paired t tests. Results: There were no statistically significant (P > .05) effects of the COX-2 inhibitor on percentage of platelets positive for CD62P or CAP1 or on concentrations of TGF-β1, PDGF-BB, or TXB2. All unactivated samples had low levels of activation or growth factor concentrations and significantly (P < .05) greater activation and growth factor concentrations in HGT-activated samples. Conclusion: This COX-2 inhibitor did not impair platelet activation, growth factor release, or TXB2 production in this canine PRP when using HGT as an activator. Studies are warranted to determine whether COX-2 inhibitors affect platelet activation and growth factor release from human PRPs. Clinical Relevance: These results suggest that there is no need to withhold a COX-2 inhibitor before PRP preparation, particularly if thrombin is going to be used to activate the PRP. This is clinically relevant information because many patients who are candidates for PRP therapy for treatment of musculoskeletal injury are also using COX-2 inhibitors.

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma

BackgroundPlatelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method.MethodsWhole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl2-activated platelet fractions.ResultsThe highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions.ConclusionsWe have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

Canine Platelet-Rich Plasma Systems: A Prospective Analysis.

To quantitate key parameters of the platelet-rich plasma (PRP) product from five commercial canine PRP systems in healthy, adult canines. A prospective study was performed from January 2013 to April 2014. Five commercial systems were analyzed using 10 healthy dogs per system.(-) Blood was obtained according to the manufacturer's protocol for each system. The mean baseline whole blood platelet, RBC, WBC, neutrophil, monocyte, and lymphocyte concentrations were determined for each PRP system. All blood samples were processed according to the manufacturer's protocols. The mean PRP product platelet, RBC, WBC, neutrophil, monocyte, and lymphocyte concentrations were determined for each PRP system. These values were then compared to the mean baseline values. Comparisons of mean whole blood and mean PRP product parameters were calculated using a paired t-test with significance established at p = 0.05. Platelet concentration was significantly increased for System 1 (p = 0.0088) and System 3 (p < 0.0001), and was significantly decreased for System 2 (p < 0.0001). All five systems significantly decreased the red blood cell concentration (p < 0.0001 for each system comparison). Neutrophil concentration was significantly decreased for System 2, System 3, and System 4 (p < 0.0001 for each system comparison). Neutrophil concentration was significantly increased for System 5 (p = 0.0089). The systems with the highest platelet yield were System 1 and System 3. System 3 increased platelet concentration while significantly reducing the RBC and neutrophil concentrations. Further study is indicated to assess the efficacy of PRP therapy in canines, the efficacy of canine PRP systems, and the clinical applications for PRP therapy in dogs.

Characteristics of canine platelet-rich plasma prepared with five commercially available systems.

To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma.

The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC), which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP) as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.

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.

Chrono-lume® and Magnesium Potentiate Aggregation of Canine but not Human Platelets in Citrated Platelet-Rich Plasma

The effects of Chrono-lume (CL) and magnesium sulfate (Mg2+), a component of this luciferin-luciferase reagent, on platelet aggregation were studied in platelet-rich plasma (PRP) obtained from blood anticoagulated with sodium citrate from humans, dogs, cats, horses, and cows. The final added Mg2+ concentration of both solutions ranged from 0.75-3.7 mM. CL and Mg2+ had no effect on maximum aggregation of platelets from humans induced by sub-threshold concentrations of collagen and ADP. In contrast, addition of CL or Mg2+ to canine PRP resulted in a dose-dependent and equal potentiation of platelet aggregation in response to sub-threshold concentrations of collagen, ADP, and thrombin in normal and thrombopathic dogs. The effect of CL on platelet aggregation induced by sub-threshold concentrations of agonists was less pronounced and varied in other species according to the agonist. The reason for the marked difference in sensitivity of human and canine platelets to CL or Mg2+ is not clear, although a difference in releasable cation pools of the platelets from these two species has been recognized. Platelet aggregation studies of animals with suspected thrombopathias should be performed without CL to prevent masking of a platelet function defect.

The in vitro pharmacological profile of SK&F 106760, a novel GPIIB/IIIA antagonist

The properties of SK&F 106760 [N α-acetyl-cyclo(S,S)-cysteinyl-N α-methylarginylglycyl-aspartyl-penicillamine-amide] as a GPIIb/IIIa antagonist have been studied in vitro and compared with those of the parent molecule, Ac-RGDS-NH 2. Ac-RGDS-NH 2 inhibited biotinylated fibrinogen binding to purified human GPIIb/IIIa immobilized on plastic microtitre plates with a Ki of 530 ± 73 nM. In canine platelet rich plasma Ac-RGDS-NH 2 produced a concentration related inhibition of adenosine diphosphate-induced platelet aggregation following preincubation for 3 min with an IC50 of 91 ± 1 μM. However, incubation in platelet rich plasma for 3 hr abolished the activity of Ac-RGDS-NH 2. SK&F 106760 inhibited biotinylated fibrinogen binding to purified human GPIIb/IIIa immobilized on plastic microtitre plates with a K i of 477 ± 57 pM. SK&F 106760 inhibited adenosine diphosphate-induced platelet aggregation in human platelet rich plasma with an IC50 of 230 ± 60 nM but did not inhibit the von Willebrand Factor receptor (GPIb/IX)-mediated platelet agglutination produced by ristocetin. In canine platelet rich plasma SK&F 106760 inhibited aggregation produced by adenosine diphosphate, collagen and epinephrine/U-46619 with IC 50 values of 355 ± 35, 260 ± 20, and 490 ± 90 nM, respectively and in gel filtered platelets inhibited thrombin-mediated aggregation with an IC50 of 188 ± 10 nM. Preincubation of SK&F 106760 in platelet rich plasma for three hours had no significant effect on its ability to inhibit adenosine diphosphate-induced platelet aggregation. SK&F 106760 produced insurmountable inhibition of adenosine diphosphate-induced platelet aggregation in the presence of constant fibrinogen concentrations, but produced competitive inhibition of the concentration-response curve to fibrinogen in adenosine diphosphate-activated platelets with a Kb of 8.0 ± 1.0 nM. Thus, SK&F 106760 is a potent, stable competitive GPIIb/IIIa antagonist with no detectable activity at the von Willebrand Factor receptor (GPIb/IX).

Prevention of Canine Coronary Artery Thrombosis with Echistatin, a Potent Inhibitor of Platelet Aggregation from the Venom of the Viper, Echis carmatus

A model of acute, platelet-dependent canine coronary artery thrombosis was utilized to assess the antithrombotic effect of a synthetic, RGD-containing 49-residue protein termed echistatin. This protein is derived from the venom of the viper, Echis carinatus. In vitro, echistatin inhibited ADP (10 microM)-induced platelet aggregation with IC50 values in human and canine platelet-rich plasma of 101 +/- 4 and 127 +/- 32 nM, respectively. In vivo, in the dog, infusion of echistatin for 30 min at 20 micrograms kg-1 min-1 or 2.6 nM kg-1 min-1 resulted in total abolition of acute platelet-dependent coronary thrombus formation in all dogs tested (n = 5). Infusion of a lower dose (10 micrograms kg-1 min-1) was not effective in prevention of thrombus formation. Blood samples were taken before and after infusion of echistatin in order to determine ex vivo platelet aggregatory responses. Echistatin (20 micrograms kg-1 min-1, i.v.) attenuated ex vivo platelet aggregation elicited by ADP, U-46619 and collagen and increased bleeding time by 2.9 +/- 0.5-fold over control. Thus, in the dog, echistatin is an effective antithrombotic agent inhibiting both platelet aggregation in vivo in the coronary artery as well as ex vivo with a concomitant increase in bleeding time. Furthermore, the effects of echistatin on platelet aggregation and bleeding time are reversible with restoration to control levels occurring 30-60 min after termination of the infusion.