Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.

Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.

Clopidogrel resistance is common in patients undergoing vascular and coronary interventions.

OBJECTIVES: "Clopidogrel resistance," also defined as heightened platelet reactivity (HPR) while on clopidogrel therapy, may lead to a sub-optimal antiplatelet effect and a potential thrombotic event. There is limited literature addressing the prevalence of HPR in a large cohort of patients receiving either coronary or endovascular interventions. METHODS: In a large integrated healthcare system, patients with a P2Y12 reaction units (PRU) test were identified. HPR was defined as a PRU ≥ 200 during clopidogrel therapy. Vascular and coronary interventions were identified utilizing CPT codes, HPR prevalence was calculated, and Fischer's exact test was used to determine significance. RESULTS: From an initial cohort of 2,405,957 patients (October 2014 to January 2020), we identified 3301 patients with PRU tests administered. Of these, 1789 tests had a PRU ≥ 200 (HPR overall prevalence, 54%). We then identified 1195 patients who underwent either an endovascular or coronary procedure and had a PRU measurement. This corresponded to 935 coronary and 260 endovascular interventions. In the coronary cohort, the HPR prevalence was 54% (503/935). In the vascular cohort, the HPR prevalence was 53% (137/260); there was no difference between cohorts in HPR prevalence (p = 0.78). CONCLUSION: "Clopidogrel resistance" or HPR was found to be present in nearly half of patients with cardiovascular disease undergoing intervention. Our data suggest HPR is more common in the cardiovascular patient population than previously appreciated. Evaluating patients for HPR is both inexpensive ($25) and rapid (< 10 min). Future randomized studies are warranted to determine whether HPR has a clinically detectable effect on revascularization outcomes.

Impacts of Commonly Used Edible Plants on the Modulation of Platelet Function.

Cardiovascular diseases (CVDs) are a primary cause of deaths worldwide. Thrombotic diseases, specifically stroke and coronary heart diseases, account for around 85% of CVDs-induced deaths. Platelets (small circulating blood cells) are responsible for the prevention of excessive bleeding upon vascular injury, through blood clotting (haemostasis). However, unnecessary activation of platelets under pathological conditions, such as upon the rupture of atherosclerotic plaques, results in thrombus formation (thrombosis), which can cause life threatening conditions such as stroke or heart attack. Therefore, antiplatelet medications are usually prescribed for people who are at a high risk of thrombotic diseases. The currently used antiplatelet drugs are associated with major side effects such as excessive bleeding, and some patients are resistant to these drugs. Therefore, numerous studies have been conducted to develop new antiplatelet agents and notably, to establish the relationship between edible plants, specifically fruits, vegetables and spices, and cardiovascular health. Indeed, healthy and balanced diets have proven to be effective for the prevention of CVDs in diverse settings. A high intake of fruits and vegetables in regular diet is associated with lower risks for stroke and coronary heart diseases because of their plethora of phytochemical constituents. In this review, we discuss the impacts of commonly used selected edible plants (specifically vegetables, fruits and spices) and/or their isolated compounds on the modulation of platelet function, haemostasis and thrombosis.

Assessment of Platelet Reactivity and Inflammatory Markers in Coronary Artery Bypass Graft Patients Treated with Acetylsalicylic Acid with Flavonoid Supplementation.

The recommended pharmacological therapy for patients with coronary artery disease (CAD) treated by coronary artery bypass grafting (CABG) is acetylsalicylic acid (ASA). To improve the antiplatelet effect, supplementation with flavonoids is also recommended. The aim of this study was to estimate anti-aggregation properties of diosmin, in combination with ASA, pre- and postoperatively and assess the relationship of this therapy with inflammatory processes in CAD patients undergoing CABG. The study patients (n = 26) took diosmin (1000 mg/day); the control patients (n = 27) took a placebo. The therapeutic period for taking diosmin was from at least 30 days before to 30 days after CABG. All patients also took 75 mg/day ASA. Platelet aggregation and IL-6, CRP, and fibrinogen concentrations were determined before and 30 days after surgery. Results showed that diosmin did not enhance the anti-aggregation effect of ASA at any assessment time. However, there was a stronger anti-aggregation effect 30 days after surgery that was diosmin independent and was associated with acute-phase markers in the postoperative period. Increased levels of inflammatory markers in the late phase of the postoperative period may provide an unfavorable prognostic factor in long-term follow-up, which should prompt the use of stronger antiplatelet therapy in patients after CABG.

[Platelet function tests and Chinese medicines with antiplatelet function:a review].

Platelet function tests have been increasingly used to assist in the diagnosis of platelet disorders and prethrombotic state, monitoring of the efficacy of antiplatelet therapies, and personalized treatment. On the basis of light transmission aggregometry, new methods for platelet function test have been developed successively. At present, the research and development of platelet function detector is in its infancy in China. The active constituents of antiplatelet Chinese medicines can be classified into terpenoids, flavonoids, saponins, organic acids, lignans, diketones, volatile oils, and stilbenes. The results of dose-antiplatelet effect relationship of Chinese medicines and the active constituents showed that the effective concentration of the extracts or monomers of Chinese medicines was at micromolar level(µmol·L~(-1)), among which salvianolic acid B and ginkgolide K, ginkgolide B, and ginkgolide A had the strongest antiplatelet effect. These results suggest that the antiplatelet effect of Chinese medicine may be weaker than that of chemical drugs and biological products. Therefore, it is necessary to explore the structure-activity relationship of the active constituents in existing Chinese medicines and further improve their efficacy through structure modification. The antiplatelet effect of Chinese medicines and the constituents involves multiple pathways and multiple targets. These research results provide a reference for clinical application of them. However, there is still a lack of large-scale multi-center clinical trials to confirm the efficacy and safety of them. The regularity of the relationship between the structures of various constituents and their corresponding functions is still unknown and the relevant signal transduction pathways and structure-activity relationship need to be further studied. This paper summarized and analyzed the determination methods of platelet functions and the research results of antiplatelet Chinese medicines, which is of reference value for the research of effective and safe antiplatelet Chinese medicines.

Investigation of platelet function in patients with chronic kidney disease stages IV-V.

INTRODUCTION: Patients with renal failure are at increased risk of both bleeding and thrombosis. Further descriptions of laboratory investigations in these patients are required. METHODS: Investigation of 24 patients with chronic kidney disease (CKD) stages IV-V with light transmission aggregometry, platelet secretion assays and platelet nucleotide analysis. Patients were in a nonbleeding state and not on antiplatelet medication. Results were compared with our local reference range used within the clinical haematology service. RESULTS: Of the 24 patients, two had decreased responses to arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activator peptide-6 and one had decreased responses to high dose ristocetin, and one had increased response to low dose ristocetin. 11 and 13 out of 24 had abnormal platelet secretion release to collagen and thrombin, respectively. Platelet nucleotide analysis in patients was normal with the exception of a reduction in ADP content in one patient and ATP/ADP ratio in one patient. CONCLUSIONS: In our collection of patients with CKD investigated for platelet function in the nonbleeding state, they generally had normal light transmission aggregometry and nucleotide analysis but around 50% had decreased platelet secretion assays. These results could be important in determining the significance of platelet function tests in patients with bleeding symptoms and renal failure. Further characterization of platelet function tests in future will help characterize haemostasis in renal failure further.

Dietary Choline Supplements, but Not Eggs, Raise Fasting TMAO Levels in Participants with Normal Renal Function: A Randomized Clinical Trial.

BACKGROUND: Choline is a dietary precursor to the gut microbial generation of the prothrombotic and proatherogenic metabolite trimethylamine-N-oxide (TMAO). Eggs are rich in choline, yet the impact of habitual egg consumption on TMAO levels and platelet function in human subjects remains unclear. METHODS: Healthy volunteers (41% male, 81% Caucasian, median age 28 years) with normal renal function (estimated glomerular filtration rate >60) were recruited and assigned to 1 of 5 daily interventions for 4 weeks: 1) hardboiled eggs (n = 18); 2) choline bitartrate supplements (n = 20); 3) hardboiled eggs + choline bitartrate supplements (n = 16); 4) egg whites + choline bitartrate supplements (n = 18); 5) phosphatidylcholine supplements (n = 10). Fasting blood and urine samples were collected for quantification of TMAO, its precursors, and platelet aggregometry. RESULTS: Participants' plasma TMAO levels increased significantly in all 3 intervention arms containing choline bitartrate (all P < .0001), but daily ingestion of 4 large eggs (P = .28) or phosphatidylcholine supplements (P = .27) failed to increase plasma TMAO levels. Platelet reactivity also significantly increased in the 3 intervention arms containing choline bitartrate (all P < .01), but not with eggs (P = .10) or phosphatidylcholine supplements (P = .79). CONCLUSIONS: Despite high choline content in egg yolks, healthy participants consuming 4 eggs daily showed no significant increase in TMAO or platelet reactivity. However, choline bitartrate supplements providing comparable total choline raised both TMAO and platelet reactivity, demonstrating that the form and source of dietary choline differentially contributes to systemic TMAO levels and platelet responsiveness.

Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline.

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

Extracts from Uncaria tomentosa as antiplatelet agents and thrombin inhibitors - The in vitro and in silico study.

ETHNOPHARMACOLOGICAL RELEVANCE: A wide range of traditional medicine applications of Uncaria tomentosa (Willd. ex Schult.) DC., commonly known as 'vilcacora' or 'cat's claw', includes blood purification, its anticoagulant properties and its use in haemorrhage therapy. AIM OF THE STUDY: Our work is devoted to the effects of ethanol and aqueous extracts (1-50 µg/ml) from U. tomentosa leaves and bark on the haemostatic system. The study is based on two main questions: Can these extracts influence the coagulation cascade of blood plasma or the activation of blood platelets? Do they feature any anticoagulant properties? MATERIALS AND METHODS: Blood platelet aggregation was measured in human platelet-rich plasma; the anticoagulant tests were based on the thrombin, prothrombin and the activated partial thromboplastin time. For the thrombin (TH)-inhibitory activity evaluation, the chromogenic substrate S-2238 and fibrinogen, i.e. physiological substrate for this enzyme, were used. In silico studies included the interactions of TH and the main components of the extracts. RESULTS: The examined extracts demonstrated slight antiplatelet activity. The thrombin time was slightly prolonged. The most efficient TH inhibitor was the ethanolic fraction from leaves (IC50 = 5.86 and 12.48 µg/ml, for the amidolytic and proteolytic assay, respectively). The plant ingredients interacted with TH within and outside the active site, dependently on the compound. The higher binding affinity was found for procyanidins B2 and C1. CONCLUSIONS: The examined extracts demonstrated slight antiplatelet effects; however, they may be promising candidates for the natural inhibitors of TH, which is critical for the formation of fibrin clot.

Zinc: an endogenous and exogenous regulator of platelet function during hemostasis and thrombosis.

Zinc (Zn2+) is an essential micronutrient and the second most abundant trace metal in the human body. The important role that Zn2+ plays in hemostasis is exemplified by platelet-related bleeding phenotypes coinciding with dietary Zn2+ deficiency. These phenotypes are rectified upon Zn2+ supplementation. Labile (unbound) Zn2+ is present in the plasma at micromolar levels, but is also detected in atherosclerotic plaques, and released from platelet α granules. Therefore, it is likely that localized Zn2+ concentrations are higher at sites of thrombosis and hemostasis. Exogenous Zn2+ is a regulator of the hemostatic responses, with roles during coagulation and platelet activation. Extracellular Zn2+ gains access to the platelet cytosol and induces full platelet activation at high concentrations, and potentiates platelets to activation by conventional agonists at lower concentrations. Zn2+-induced platelet activation is dependent on PKC and integrin αIIbß3, and is associated with tyrosine phosphorylation of platelet proteins. Agonist evoked platelet activation results in intracellular Zn2+ ([Zn2+]i) fluctuations that are sensitive to the platelet redox state. Increases in [Zn2+]i correlate with activation responses, including shape change, granule release, αIIbß3 activation and phosphatidyl-serine exposure, consistent with a role as a second messenger. This review provides insight into the numerous demonstrated and potential roles for Zn2+ in platelet function during thrombosis and hemostasis, highlighting its increasing acceptance as an intracellular and extracellular platelet regulatory agent.

Platelet function tests and Chinese medicines with antiplatelet function:a review / 中国中药杂志

Platelet function tests have been increasingly used to assist in the diagnosis of platelet disorders and prethrombotic state, monitoring of the efficacy of antiplatelet therapies, and personalized treatment. On the basis of light transmission aggregometry, new methods for platelet function test have been developed successively. At present, the research and development of platelet function detector is in its infancy in China. The active constituents of antiplatelet Chinese medicines can be classified into terpenoids, flavonoids, saponins, organic acids, lignans, diketones, volatile oils, and stilbenes. The results of dose-antiplatelet effect relationship of Chinese medicines and the active constituents showed that the effective concentration of the extracts or monomers of Chinese medicines was at micromolar level(μmol·L~(-1)), among which salvianolic acid B and ginkgolide K, ginkgolide B, and ginkgolide A had the strongest antiplatelet effect. These results suggest that the antiplatelet effect of Chinese medicine may be weaker than that of chemical drugs and biological products. Therefore, it is necessary to explore the structure-activity relationship of the active constituents in existing Chinese medicines and further improve their efficacy through structure modification. The antiplatelet effect of Chinese medicines and the constituents involves multiple pathways and multiple targets. These research results provide a reference for clinical application of them. However, there is still a lack of large-scale multi-center clinical trials to confirm the efficacy and safety of them. The regularity of the relationship between the structures of various constituents and their corresponding functions is still unknown and the relevant signal transduction pathways and structure-activity relationship need to be further studied. This paper summarized and analyzed the determination methods of platelet functions and the research results of antiplatelet Chinese medicines, which is of reference value for the research of effective and safe antiplatelet Chinese medicines.

Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release.

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

Effects of Rivaroxaban on Platelet Aggregation.

Rivaroxaban is a direct oral anti-factor Xa anticoagulant. It has recently been suggested that rivaroxaban may affect platelet function in vitro; however, little is known about the clinical impact of this likely antiplatelet effect and whether this probable phenomenon is dose-dependent. Our aim was to determine whether rivaroxaban at 4 different doses inhibits direct platelet aggregation. We included adult patients of both sexes and who were allocated to one of the following groups depending on the prescribed daily dose of rivaroxaban: 5, 10, 15, and 20 mg. In 80 patients (20 patients/group), the percentage of platelet aggregation was determined by means of platelet aggregometry tests before and after rivaroxaban use. Basal samples were obtained before starting rivaroxaban and 1 month after treatment, both 2 and 24 hours after the last dose of the drug (12 hours after in the case of rivaroxaban 5 mg). We used 5 platelet agonists: adenosine diphosphate, epinephrine, arachidonic acid, collagen, and thrombin. There were no significant changes in the percentage of platelet aggregation before and after rivaroxaban use independently of the dose administered and the agonist used. Our results have clearly shown that rivaroxaban, even at a high dose, does not directly affect platelet aggregation.

Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice.

The exploration of thrombotic mechanisms relies on the application of blood collection methods from laboratory mice with a minimal pre-activation of platelets and the clotting system. So far, very little is known on how the blood collection method and the anticoagulant used influence pre-activation of mouse platelets and coagulation. To determine the most suitable blood collection method, we systematically compared blood collection by heart puncture, Vena cava puncture, and puncture of the retro-orbital vein plexus and the use of citrate, heparin, and EDTA as frequently used anticoagulants with regard to platelet activation and whole blood clotting parameters. The activation of platelet-rich plasma diluted in Tyrode's buffer was analyzed by flow cytometry, analyzing the exposure of P-selectin and activated integrin αIIbß3. Clotting of whole blood was profiled by thrombelastometry. Puncture of the retro-orbital vein plexus by plastic capillaries is not superior in terms of blood volume and platelet pre-activation, whereas heart puncture and Vena cava puncture resulted in similarly high blood volumes. Cardiac puncture and Vena cava puncture did not result in pre-activated platelets with citrate as an anticoagulant, but the use of EDTA resulted in increased levels of integrin αIIbß3 activation. Puncture of the retro-orbital vein plexus by plastic capillaries resulted in increased platelet integrin αIIbß3 activation, which could be prevented by soaking with citrate or coating with heparin. Further, activation of coagulation in citrated whole blood by puncture of the retro-orbital vein plexus using a blunt plastic capillary was observed by thromboelastometry. The use of citrate is the optimal anticoagulant in mouse platelet assays. Blood collections from the heart or Vena cava represent reliable alternatives to retro-orbital puncture of the vein plexus to avoid pre-activation of platelets and coagulation.

Cellular and Molecular Mechanisms of Antithrombogenic Plants: A Narrative Review.

Heart attack, stroke, and deep vein thrombosis are among the conditions that alter blood coagulation and are modulated by antithrombogenic drugs. Natural products are an important source of antithrombogenic agents and have been considered remarkable alternatives with greater efficacy and usually with fewer side effects. However, the efficacy and toxicity of many of these plants that are used in traditional medicine must be scientifically tested. Despite a large number of published articles that report that plants or plant-derived components may act as antithrombogenic agents, few studies have investigated the mechanism of action of medicinal plants. This review presents the current knowledge about the major cellular and molecular mechanisms of antithrombogenic plants and their main components. Many well-established mechanisms (e.g., platelet aggregation, coagulation factors, and thrombolysis) are related to the antithrombogenic activity of many natural products. However, the central pathways that are responsible for their activity remain unclear. Further studies are needed to clarify the central role of each of these pathways in the pleiotropic response to these agents.

Isorhapontigenin, a resveratrol analogue selectively inhibits ADP-stimulated platelet activation.

Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85 µM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100 µM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50 > 100 µM) but inhibited collagen induced platelet aggregation at 50 µM and 100 µM. Isorhapontigenin also inhibited integrin αIIbß3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100 µM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85 µM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor.

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis.

Discrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis.

Influence of different supplementation on platelet aggregation in patients with rheumatoid arthritis.

INTRODUCTION: Long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs; eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) have been reported to reduce platelet aggregation. Our aim was to prospectively assess the potential influence of different supplementation omega-3 PUFA on the antiplatelet effects in rheumatoid arthritis (RA) patients. METHODS: The study included 60 patients with RA at the Department of Rheumatology, Clinical Center Kragujevac. Patients were divided into three groups depending on who used concentrated fish oil only or concentrated fish oil in combination with evening primrose oil or control group without supplementation in a period of 3 months. Platelet aggregation was measured using the multiplate analyzer and expressed through the value of adenosine diphosphate (ADP) test, aranchidonic acid-induced aggregation (ASPI) test, thrombin receptor-activating peptide (TRAP) test (to assess baseline platelet aggregation), and the ratio of ADP/TRAP and ASPI/TRAP representing the degree of inhibition of platelet aggregation compared to the basal value. The platelet function analysis in whole blood was performed 18-24 h before starting supplementation and after 90 days. Considerations were taken in the representation of demographic, clinical characteristics, and laboratory parameters between the groups. RESULTS: Patients who used concentrated fish oil only had a significantly lower value of the ratio of ADP/TRAP (0.68 ± 0.20) compared to patients without supplementation (0.83 ± 0.12; p = 0.008), while there was no statistically significant difference in values of other laboratory parameters of platelet function between other groups. CONCLUSIONS: Co-administration of supplementation-concentrated fish oil may reduce platelet aggregation in adults with RA. KEY POINTS: • Omega-3 PUFAs are essential for health and are known to possess anti-inflammatory properties, improving cardiovascular health as well as benefiting inflammatory diseases.. • In this paper, we report on anti-aggregation effects n-3 PUFAs and ɤ-linolenic acid in RA. • The risk of cardiovascular morbidity and mortality is increased in RA, and dietary supplementation of n-3 PUFA may have preventive potential for the cardiovascular management in rheumatoid arthritis.