Agonists Adenosine Diphosphate Research Articles

Assessment of Aspirin and Clopidogrel Resistance in Patients Undergoing Cardiovascular Surgery: A Single-Center Cross-Sectional Study

We aimed to investigate antiplatelet drug resistance utilizing light transmission-lumiaggregometry (LT-LA) and the Platelet Function Analyzer-100 (PFA-100) in patients undergoing cardiovascular surgery. The study included 60 patients diagnosed with stable coronary artery disease and peripheral vascular diseases that required surgery. Participants were divided into three groups: patients receiving aspirin (ASA) (n=21), patients receiving clopidogrel (CLO) (n=19), and patients receiving dual therapy (ASA+CLO) (n=20). Aggregation and secretion tests by LT-LA and closure time by the PFA-100 were used to measure antiplatelet drug resistance. Based on the adenosine diphosphate (ADP)-induced aggregation test, 43% of patients were resistant to ASA, 22% to CLO, and 15% to dual therapy. Diabetes, hypertension, and hyperlipidemia were the most commonly identified comorbid disorders. In patients with comorbid risk factors, the median value of platelet aggregation response to ADP was significantly higher in the ASA group than in the CLO and dual therapy groups (p=0.0001). In patients receiving ASA monotherapy, the maximum amplitude of aggregation response to platelet agonists was ≥70% in 43% of patients for ADP and 28% for collagen by LT-LA. Elevated ADP (≥0.29 nmol) and collagen (≥0.41 nmol)-induced adenosine triphosphate release were found by LT-LA in 66% of patients utilizing an ADP agonist and 80% of patients using a collagen agonist undergoing ASA therapy. Closure times obtained with the PFA-100 were normal in 28% of patients using collagen-ADP cartridges and 62% of patients using collagen-epinephrine (CEPI) cartridges who received ASA. Recurrent thrombosis and bleeding were observed in 12 (20%) patients with cardiovascular disease. Three of these individuals (25%) showed ASA resistance with normal responses to ADP-induced aggregation (≥70%) and secretion (≥0.29 nmol), as well as normal CEPI closure times. Our findings suggest that antiplatelet drug monitoring by LT-LA and PFA-100 may be useful for high-risk and complicated cardiovascular patients.

Abstract 15758: Platelet Aggregation is Elevated in Psoriasis and Associated With Cardiovascular Risk

Introduction: Psoriasis is associated with increased CV risk the mechanisms and prevention of which are not fully known. We evaluated the platelet phenotype (implicated in atherothrombosis) and in vitro response to aspirin in patients with psoriasis and its association with vascular stiffness and coronary atherosclerosis. Methods: Patients with psoriasis (n = 64, age 47.9 ± 15 years, 65% male), percent body surface area (BSA) of psoriasis 3%, were compared to controls (n = 40, age 40.8 ± 16 years, 45% male). Percent platelet aggregation in response to agonists adenosine diphosphate (ADP), collagen (Col), and arachidonic acid (AA - with and without aspirin) was measured in freshly isolated platelet-rich plasma via light transmission aggregometry (LTA). Vascular stiffness (via pulse wave velocity [PWV]) and CT coronary angiography were each performed as readouts of CV risk. Results: Compared to controls, psoriasis patients were older, of Caucasian ethnicity (Figure 1A), and overweight, but otherwise similar. Platelet aggregation after treatment with Col (p=0.01), ADP (p < 0.01), and AA (p = 0.05) were all higher in psoriasis vs. control (Figure 1B-D). Platelet aggregation after activation with high dose AA was similar between groups (Figure 1E) however co-incubation with high dose AA plus aspirin displayed incomplete platelet inhibition of psoriasis platelets vs. controls (Figure 1F). Platelet aggregation to low dose AA and high dose AA plus aspirin co-incubation were each higher in those with enhanced vascular stiffness (figure 1G/1H) and the presence of coronary plaque (Figure 1I/1J). Conclusion: Platelet aggregation is consistently elevated in psoriasis across a variety of agonists, incompletely inhibited by aspirin, and associated with biomarkers of CV risk such as vascular stiffness and the presence of coronary plaque. These findings highlight the need to evaluate various anti-platelet strategies to reduce atherothrombotic risk in patients with psoriasis.

Abstract 18621: Platelet Aggregation is Heightened in Those With Metabolic Syndrome and Less Effectively Mediated by Aspirin Therapy

Background: Patients with metabolic syndrome (MetS) are at a higher risk of developing cardiovascular disease (CVD); however, interventions to mitigate this risk are not fully known. Platelets play a significant role in atherothrombosis, and aspirin is an important, yet controversial therapy used for the primary prevention of CVD. In this study, we examined platelet phenotype in MetS and degree of inhibition from ex vivo aspirin administration. Methods: In a study examining the mechanisms of atherothrombosis in pro-inflammatory conditions, patients without clinical CVD were enrolled and underwent phenotyping. Patients with MetS (n = 34, age 53.3 ± 13 years, 59% male) were compared to patients with no MetS (n = 63, age 41.1 ± 16 years, 52% male). Platelet aggregation of freshly isolated platelets was measured via light transmission aggregometry (LTA) in resting and stimulated conditions, in response to agonists adenosine diphosphate (ADP), high and low dose of arachidonic acid (AA), and after ex vivo aspirin (ASA) administration. Results: MetS patients were older and similar in sex and race (Figure 1A). As expected, patients with MetS had higher individual components of MetS, yet similar LDL-C (Figure 1A). Platelet aggregation to low dose ADP (p=0.09) and AA (p=0.04) was significantly increased in MetS (Figure 1B). Platelet aggregation in response to AA and coincubation with ASA was significantly lower in the no MetS cohort (Figure 1C). The efficacy of aspirin therapy was significantly lower in MetS (Figure 1D). In response to high dose AA after ASA treatment, platelet aggregation increased as number of MetS parameters also increased (Figure 1E). However, no individual metabolic syndrome parameter appeared to be driving these findings (Figure 1E). Conclusion: Among MetS patients, platelet aggregation is heightened and less inhibited by aspirin than those with no MetS. Additional research is warranted to explore possible anti-platelet strategies in patients with MetS.

Cystic fibrosis transmembrane conductance regulator potentiators attenuate platelet activation and aggregation in blood of healthy donors and COVID-19 patients

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl - channel and ABC transporter; its mutations cause the clinical picture of cystic fibrosis (CF). Of late, CFTR has emerged as an important regulator of platelet function, as CFTR dysfunction causes agonist-induced platelet hyperactivation. These findings are reminiscent of platelets from SARS-CoV-2 infected patients since thromboembolic complications represent hallmarks of severe COVID-19 that may critically contribute to morbidity and mortality. CFTR modulators have recently been introduced as a treatment for patients with various CFTR mutations, but have also been reported by us and others to enhance channel function of wild type CFTR. We therefore postulated that CFTR modulators may exert anti-coagulant effects on platelets of healthy donors (HD) and COVID-19 patients.We recruited 36 COVID-19 patients with moderate, and 34 COVID-19 patients with severe disease course (all w/o anti-platelet drugs), and 38 HDs. In line with our hypothesis, we observed significant reductions in platelet agonists adenosine diphosphate (ADP)- or thrombin receptor activating protein-6 (TRAP6)-induced CD62p/CD63 expression, Ca 2+ -mobilization, aggregation, and adhesion of platelets from HDs by pre-treatment with ivacaftor. In blood from COVID-19 patients, platelet activation correlates with disease severity, as demonstrated by a 5-fold and 8-fold increase in the proportion of CD62p + platelets from patients with moderate and severe disease, respectively, relative to HDs. Similarly, the proportion of CD63 + platelets in patients with severe COVID-19 was 2-fold higher than in HDs. Retrospective analysis of clinical data from a total of 4,050 CF patients with COVID-19 receiving single or combination therapy of ivacaftor, lumacaftor, tezacaftor, or elexacaftor in comparison to an untreated cohort revealed that CF therapy reduced the relative risk to suffer thromboembolism-associated cardiovascular events such as heart attack or deep vein thrombosis by 50.0% or 61.1%, respectively, suggesting an anti-thrombotic effect of CFTR modulators in CF COVID-19 patients. In line with this observation, ex vivo pre-treatment of platelets from acute COVID-19 patients with ivacaftor reduced Ca 2+ mobilization, adhesion, and aggregation of platelets .Our results demonstrate an anticoagulant effect of CFTR potentiators on platelets from HDs and severe COVID-19 patients and thus, suggest CFTR potentiators as a promising strategy to reduce the risk of thrombotic events in the clinical management of COVID-19 and similar pro-thrombotic disease states. F. Behrens received funding from the Berlin Institute of Health (BIH). L. Michalick reports grants from the BIH and the German Centre for Cardiovascular Research (DZHK). A. Haghikia is participant in the BIH-Charité Advanced Clinician Scientist Pilotprogram funded by the Charité - Universitätsmedizin Berlin and the BIH and reports a research grant within the BIH & MDC Focus Area Translational Vascular Biomedicine. R. Preissner reports partial funding of this work by the German Research Foundation (KFO339, TRR295). M. Witzenrath reports grants from the German Research Foundation (SFB-TR84 C06 and C09, SFB 1449 B02), and from the German Ministry of Education and Research (BMBF) in the framework of CAPSyS (01ZX1604B, 01ZX1304B), SYMPATH (01ZX1906A), PROVID (01KI20160A), Phage4Cure (16GW0141), MAPVAP (16GW0247) and NUM-NAPKON. W. M. Kuebler reports grants from the German Research Foundation (SFB-TR84 A2 and C9, SFB 1449 B1, SFB 1470 A4, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the BMBF in the framework of SYMPATH (01ZX1906A) and PROVID (01KI20160A), and the DZHK. S. Simmons reports grants from the DZHK and the German Foundation for Heart Research (F-09-19). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In vitro Investigation of Platelet Dysfunction Induced by Osmotic Pressure Variations

Background: Severe variations in osmotic pressure are significant contributors to critical patient morbidity and mortality and might also affect platelet volume. We aimed to investigate possible osmotic-induced changes in mean platelet volume (MPV) and their possible effects on platelet aggregation activity (PLAG). Methods: We induced experimental variations of serum osmolality in blood samples from healthy volunteers (heparinized whole blood [WB]) and isolated platelets (platelet-rich plasma [PRP]) by adding isotonic, hypertonic, and hypotonic solutions of saline/water (pH = 7.2–7.4). PLAG was tested in WB samples with impedance aggregometry (IA) and in PRP samples with light transmission aggregometry (LTA) using three agonists: adenosine diphosphate (ADP, 10 μ<sc>m</sc>), thrombin receptor activating peptide (TRAP-6, 10 μ<sc>m</sc>), and arachidonic acid (500 μ<sc>m</sc>). Osmolality was either calculated using a formula or measured directly. Results: We found almost identical osmolalities in WB and PRP preparations. Osmotic stress did not produce significant changes in MPV. In IA testing, the hypotonic challenge of WB preparations produced significant reductions at 50% (p = 0.056) (95% CI: 11.2–2.4, in Ohms) of ADP and at 31% (p = 0.017) (95% CI: 13.4–8.6, in Ohms) of TRAP-6-induced PLAG, respectively. In PRP, we did not observe any variations in PLAG with LTA. Conclusions: We conclude that in vitro hypotonic stress of WB samples has an inhibitory effect on the PAR-1 (TRAP-6-induced) pathway and on the P2Y₁₂ (ADP-induced) pathway and reflects a distinct in vivo effect of hypoosmotic stress on WB human platelet preparations.

Development of the whole tomato and olive-based food supplement enriched with anti-platelet aggregating nutrients.

Platelet dysfunctions are shared by cardiovascular diseases and a wide range of inflammatory diseases. To determine the ability of a new whole tomato-based food supplement (WTBFS) containing carotenoid and olive polyphenols to inhibit platelet aggregation. Aggregation was evaluated in platelet-rich plasma using microtiter plates and a plate reader. Platelets treated with WTBFS showed a >70% reduction of 5 µM adenosine diphosphate (ADP)-induced platelet aggregation; at 10 µM of ADP, the inhibitory effect of WTBFS was reduced of about 50%. Similarly, 78% and 48% reduction were obtained using 5 µg/mL and 10 µg /mL of collagen as an agonist. Since the compounds in WTBFS share the ability to inhibit STAT3, the inhibition of its signaling pathway may represent the mechanism underlying the antiplatelet activities. The activity of a lipophilic solution prepared from WTBS was in vitro tested on the platelet aggregation in response to ADP agonists and Collagen.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency.

BackgroundPatients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (δ-SPD). δ-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet δ-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients with δ-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with δ-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with δ-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies.MethodsOur study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL).ResultsSeventy-seven percent of our patients were found to have δ-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have δ-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL).ConclusionThese data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet δ-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal.

Rapid Release of Interleukin-1β from Human Platelets Is Independent of NLRP3 and Caspase.

Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1β (IL-1β), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1β) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1β in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1β. Platelets release IL-1β within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1β in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1β were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1β by a mechanism which is independent of caspase-1 and NLRP3.

Anticoagulant, fibrinogenolytic and anti-platelet aggregation activities of Lablab purpureus (L.) Sweet seed radicle aqueous extract

The current study explores the anticoagulant, fibrinogenolytic and anti-platelet aggregation activities of Lablab purpureus (L.) Sweet seed radicle extract (LPRE). It is firstly reported that LPRE has protein at a concentration of 1.5 mg/ml and further evaluated for protein in gel electrophoresis. The proteolytic activity of LPRE was evaluated using casein and gelatin as substrate. LPRE showed a specific activity of 0.35 U. LPRE increased the plasma clotting time significantly showing its anticoagulant property. LPRE hydrolyzed the A?, B? and ? chains of fibrinogen. Furthermore, LPRE significantly inhibited the platelet aggregation induced by agonist adenosine diphosphate (ADP).

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbβ3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen.

The commensal microbiota is a recognized enhancer of arterial thrombus growth. While several studies have demonstrated the prothrombotic role of the gut microbiota, the molecular mechanisms promoting arterial thrombus growth are still under debate. Here, we demonstrate that germ-free (GF) mice, which from birth lack colonization with a gut microbiota, show diminished static deposition of washed platelets to type I collagen compared with their conventionally raised (CONV-R) counterparts. Flow cytometry experiments revealed that platelets from GF mice show diminished activation of the integrin αIIbβ3 (glycoprotein IIbIIIa) when activated by the platelet agonist adenosine diphosphate (ADP). Furthermore, washed platelets from Toll-like receptor-2 (Tlr2)-deficient mice likewise showed impaired static deposition to the subendothelial matrix component type I collagen compared with wild-type (WT) controls, a process that was unaffected by GPIbα-blockade but influenced by von Willebrand factor (VWF) plasma levels. Collectively, our results indicate that microbiota-triggered steady-state activation of innate immune pathways via TLR2 enhances platelet deposition to subendothelial matrix molecules. Our results link host colonization status with the ADP-triggered activation of integrin αIIbβ3, a pathway promoting platelet deposition to the growing thrombus.

Functional fermented cherimoya (Annona cherimola Mill.) juice using autochthonous lactic acid bacteria

The biochemical and functional properties of fermented Annona cherimola Mill. (cherimoya) juice using five lactic acid bacteria (LAB) isolated from autochthonous fruits from Northwestern Argentina were studied in this work. Fermentation was carried out at 30 °C for 48 h followed by a 21 day-storage period at 4 °C. The assayed LAB grew well during fermentation (final count of 108 CFU/mL, ΔpH ca. 1 U) and survived after the storage period. All strains consumed fructose and glucose present in cherimoya juice as energy sources, with the consequent synthesis of lactic and/or acetic acids as final metabolic products. However, only two of the five evaluated strains were capable to produce fermented cherimoya juices with a perceptible color change. Due to lactic acid fermentation, a moderate reduction in the total phenolic content (between 13% and 43%) was observed in the majority of the samples, although no change in the antioxidant capacity was detected. The fermented cherimoya juices showed a weak antiplatelet activity when adenosine diphosphate agonist was used. The findings of this study evidenced the potential use of Annona cherimola Mill. fermented juice as a novel matrix for the formulation of stable functional beverages with appealing nutritional and functional properties.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

AbstractArterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists

Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbβ3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbβ3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.

Our First Experience with Magnetic Separation of Platelets for Analyses of Platelet MicroRNA in Patients with Sticky Platelet Syndrome

Abstract Introduction: Sticky platelet syndrome (SPS) is referred to as a platelet hyperaggregability triggered by low concentrations of platelet agonists adenosine diphosphate (ADP) and/or epinephrine (EPI). Platelet aggregation with other inducers (collagen, arachidonic acid, ristocetin, and thrombin) remains within a normal range. MicroRNAs (miRNAs) are small, non-coding RNA molecules that play an important role in post-transcriptional regulation of protein expression. More recently, several studies show that the platelets are an abundant source of miRNAs and that the miRNA expression profiles within platelets correlate with the platelet reactivity. Aim: The principle objective of this article is to describe the method which we developed for the preparation of the pure platelet samples and report the results of this method. These final pure platelet samples are intended to be the first step for the platelet miRNA testing. Methods: The blood samples from 50 subjects were examined in the study. Then, the platelet rich plasma (PRP) samples obtained by centrifugation of the patient blood samples were used for our experiments. Subsequently, the erythrocytes and leucocytes remaining in PRP sample were magnetically labelled by CD45 Microbeads and CD235a Microbeads. After incubation the PRP sample passed through the magnetic separation system and the magnetically labelled cells (erythrocytes and leucocytes) were retained within the column of separator. The number of cells in the final PRP samples was measured by the blood cell analyser. Results and conclusion: We successfully developed and optimized the effective and reproducible method for magnetic separation of platelets, resulting in the leukocyte-depleted and erythrocyte-depleted platelet samples, which can be used for further genetic analyses.

Platelet consumption and hyperreactivity coexist in experimental traumatic hemorrhagic model

Introduction Platelets are critical for hemostasis, and a low platelet count predicts mortality in trauma. The role of platelet dysfunction in severe traumatic hemorrhage and coagulopathy needs to be further defined. The aim of this study was to evaluate the platelet function in a new model of experimental traumatic hemorrhage. Material and methods New Zealand white rabbits (n = 10) were subjected to tracheostomy and trauma laparotomy, and then bilateral femur fractures with 40% hemorrhage of their estimated blood volume. Arterial blood gases, standard coagulation tests, mean platelet volume, platelet aggregation using impedance aggregometry with agonist collagen, arachidonic acid (ASPI), and adenosine diphosphate (ADP), rotational thromboelastometry, and fibrinogen binding of platelets were analyzed using flow cytometry. Results After traumatic hemorrhage, there was a significant physiological response with a rise in lactate (P < .001) and a decrease in base excess (P < .001) and temperature (P < .001). Platelet count decreased from a mean of 244x109/L to 94 x109/L (P = .004) and the mean platelet volume increased from 5.1fL to 6.1fL (P = .002). Impedance aggregometry with the agonist collagen, ASPI, and ADP was all significantly decreased after hemorrhage (P = .007). However, there was an increased fibrinogen binding of ADP-activated platelets after traumatic hemorrhage analyzed by flow cytometry (P < .05). Conclusions This traumatic hemorrhage model presents two parallel pathophysiological responses of platelets; platelet consumption as evidenced by a significant decrease in platelet count and aggregation, and platelet hyperreactivity as shown by a higher mean platelet volume and enhanced platelet fibrinogen binding. Further studies are needed to characterize these different aspects of platelet function in severe traumatic hemorrhage.

Correlation of serum level of albumin with platelet count and platelet aggregation assessed by adenosine diphosphate agonist in children with nephrotic syndrome

Background Nephrotic syndrome (NS) is the most common renal disease among children, and contributes to life-threatening complications such as thromboembolic disease. Platelets are considered to be important agents in thrombotic events among NS patients. The gold standard assessment for platelet aggregation is the use of adenosine diphosphate (ADP) agonist, however, it is expensive and not accessible in smaller health facilities. Thus, other thrombosis parameters are needed. Previous studies suggested that low albumin increased the risk of thrombosis in NS patients.&#x0D; Objective To assess for a possible correlation between albumin level and platelet count as well as platelet aggregation.&#x0D; Methods This cross-sectional study was conducted in children with nephrotic syndrome who were admitted to the Pediatric Nephrology Department in Dr. Hasan Sadikin General Hospital, Bandung, West Java, from November 2017 to March 2018. Subjects were selected by consecutive sampling. Serum albumin, platelet count, and platelet aggregation were measured. Statistical analysis was conducted by Spearman’s test.&#x0D; Results A total of 32 patients participated in the study, with mean age of 109 (SD 7.4) months. Most subjects were male (56%). Subjects’ mean serum albumin level was 2.06 (SD 1.23) g/dL; mean platelet count was 453,062.5 (SD 187,443.90)/mm3; and mean platelet aggregation values for ADP agonist concentrations of 10, 5, 2.5, and 1 mM were 86.8 (SD 8.63)%, 82.4 (SD 15.33)%, 66.6 (SD 24.90)%, 34.95 (SD 31.69)%, respectively. Partial correlation analysis revealed significant negative associations between albumin and platelet count as well as platelet aggregation assessed with 1 mM of ADP concentration (P&lt;0.001), with Spearman correlation coefficients of -0.641 and -0.634, respectively.&#x0D; Conclusion Serum albumin level had a moderately negative correlation with platelet count and platelet aggregation value.

Investigation of Solubility Enhancement Approaches of Ticagrelor

ABSTRACT&#x0D; Ticagrelor is an orally administered antiplatelet medicine, direct-acting P2Y12-receptor antagonist. Ticagrelor binds reversibly and noncompetitively to the P2Y12 receptor at a site distinct from that of the endogenous agonist adenosine diphosphate (ADP). Inhibition of platelet aggregation stimulated by ADP is a commonly used pharmacodynamic parameter for P2Y12-receptor antagonists.&#x0D; Ticagrelor is a crystalline powder with an aqueous solubility of approximately 10?g/mL at room temperature. Ticagrelor exhibits no pKa value within the physiological range. Ticagrelor does not exhibit pH-dependent solubility and is defined as ‘low solubility low permeability’ under the Biopharmaceutics Classification System (Class IV). The mean absolute bioavailability of ticagrelor in healthy volunteers is 36 %&#x0D; Nanoparticles preparation and complexation is one of the recently used approaches to enhance the solubility of drugs. The aim of the present work was to improve the solubility and dissolution of ticagrelor by preparing nanoparticles and cyclodextrin inclusion complex of ticagrelor and then incorporated in to tablet dosage form. Fifteen formulas of nanoparticles were prepared by antisolvent precipitation method (solvent displacement method) utilizing one of the three polymers (PVP, Poloxamer, and HPMC) at three different drugs: polymer and solvent: anti-solvent ratios and nine formulas of cyclodextrin inclusion complex with HP?CD by three preparation methods, physical trituration, kneading and solvent evaporation, which increase the solubility and dissolution rate of ticagrelor via formation of inclusion complex with HP?CD.&#x0D; The prepared formulas were characterized regarding the saturated solubility, polydispersity index, particle size by nano laser particle size analyzer, % yield, entrapment efficiency, and flowability, FTIR, DSC, and SEM. The selected formulas were prepared as tablets.&#x0D; The prepared tablets were evaluated for drug content, weight variation, hardness, and friability. In vitro dissolution data of the prepared tablets were analyzed using similarity factor (f2) and dissolution efficiency (DE). &#x0D; Among all the prepared nanoparticles formulas, formula (F12) which contain HPMC as a polymer at polymer: drug ratio of (1:1) and solvent: antisolvent ratio of (1:1) was considered as the optimum formula which shows good evaluation parameters in addition to the increment in the solubility to about 9 times than that of the pure drug. The nanoparticle of the selected formula (F12) incorporated tablets showed an acceptable tablet properties in addition to a considerable increase in the dissolution efficiency to (DE=92 % and 88 % in PH 1.2 and PH 6.8 respectively) in comparison to that of the marketed tablet (DE=89% and 85% in PH 1.2 and PH 6.8 respectively). Moreover, the analysis by DSC and SEM of the nanoparticles of the selected formula (F12) indicate a reduction in the crystallinity and amorphization of the drug. It can be concluded that the selected formula is a promising formula for the preparation of ticagrelor nanoparticles the incorporation in a tablet dosage form.&#x0D; Regarding ticagrelor inclusion complex with HP?CD Solvent evaporation method was the most effective method regarding ticagrelor solubilization and optimum formula of inclusion complex (F23) show increment in saturated solubility about ten times that of pure drug.&#x0D; The ticagrelor inclusion complex of the selected formula (F23) incorporated tablets showed an acceptable tablet properties in addition to a considerable increase in the dissolution efficiency to (DE=92 % and 90 % in PH 1.2 and PH 6.8 respectively) in comparison to that of the marketed tablet (DE=89% and 85% in PH 1.2 and PH 6.8 respectively). &#x0D;

The E3 Ubiquitin Ligase WWP2 Regulates Endosomal Trafficking of the Purinergic Receptor P2Y1

P2Y1 is a G protein‐coupled receptor (GPCR) for adenosine diphosphate (ADP) that signals to produce pro‐inflammatory effects in blood vessels. P2Y1 signaling contributes to vascular endothelial barrier disruption, leading to cardiovascular diseases such as atherosclerosis. When bound to ADP, P2Y1 internalizes and is either recycled for reuse at the cell surface or sorted to and degraded by lysosomes to attenuate signaling. P2Y1 lysosomal sorting is mediated by binding to the adaptor protein ALIX. Since ALIX activity is regulated by ubiquitination by the WW domain‐containing E3 ubiquitin protein ligase 2 (WWP2), we hypothesized that WWP2 is required for P2Y1 degradation. Here, we show that P2Y1 colocalizes with WWP2 at late endosomes upon ADP agonist stimulation. In addition, we demonstrate that depletion of endogenous WWP2 inhibits P2Y1 sorting into the lumen of late endosomes. We are currently examining how receptor signaling regulates WWP2 translocation to late endosomes and interaction with ALIX. Our evidence suggests that WWP2 is required at endosomes for P2Y1 degradation in response to agonist stimulation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diagnosis of platelet function disorders: A standardized, rational, and modular flow cytometric approach

A high proportion of patients with mucocutaneous bleeding diathesis and suspected inherited or acquired platelet disorder remain without diagnosis even after comprehensive laboratory testing. Since flow cytometry allows investigation of resting and activated platelets on the single cell level by requiring only minimal amounts of blood, this method has become an important assay within the diagnostic algorithm, especially in pediatrics. We therefore developed a standardized and modular flow cytometric approach that contributes to clarify impaired platelet function in a rational step-by-step manner.Due to simultaneous analysis of four fluorophores in a basic panel design, we are able to readily detect the most common and clinically significant platelet disorders: Glanzmann thrombasthenia or Glanzmann-like diseases (fibrinogen receptor GPIIb-IIIa), Bernard–Soulier syndrome (von Willebrand-factor receptor complex GPIb-IX-V) and less well characterized β1-integrins that serve as the collagen, laminin or fibronectin receptor (CD29-CD49b, e and f, respectively). Platelet reactivity was investigated in response to the agonists adenosine diphosphate (ADP) and thrombin receptor activator peptide 6 (TRAP6) in suboptimal and optimal concentrations by quantifying surface expression of activation markers CD62P and CD63 as well as binding of PAC-1 antibody to the high affinity conformation of the fibrinogen receptor. For advanced diagnostic questions, several further modules were implemented: (i) calcium mobilization for evaluation of early signal transduction, (ii) a kinetically resolved mepacrine assay for estimation of delta-granule content and release, and (iii) a module to determine platelet reactivity upon additional agonists like the thromboxane A2-analogue U46619 or collagen. Blood withdrawn from a healthy control cohort allowed generating preliminary standard values for all parameters. The modules were validated by analysis of patients with known or suspected platelet defects (leukocyte-adhesion deficiency type III, Wiskott–Aldrich syndrome, acute myeloid leukemia, sickle cell disease and chronic immune thrombocytopenia).