Modulate Platelet Reactivity Research Articles

Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals

BackgroundAssessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing “one-size-fits-all” approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. ObjectivesOur aim was to compare well-used platelet reactivity assays. MethodsBlood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. ResultsA strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid–induced aggregation, and explained substantial variance (β = −1.735; P = 4.59 × 10−780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (β = −1.612; P = 6.75 × 10−27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. ConclusionCaution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

OC 61.2 MiR-146a-Mimic Therapy Modulates Platelet Reactivity Preventing Mortality Caused by Pulmonary Embolism Model

OC 61.2 MiR-146a-Mimic Therapy Modulates Platelet Reactivity Preventing Mortality Caused by Pulmonary Embolism Model

A regulatory node involving Gαq, PLCβ, and RGS proteins modulates platelet reactivity to critical agonists

BackgroundMost platelet agonists work through G protein-coupled receptors, activating pathways that involve members of the Gq, Gi, and G12/G13 families of heterotrimeric G proteins. Gq signaling has been shown to be critical for efficient platelet activation. Growing evidence suggests that regulatory mechanisms converge on G protein-coupled receptors and Gq to prevent overly robust platelet reactivity. ObjectivesTo identify and characterize mechanisms by which Gq signaling is regulated in platelets. MethodsBased on our prior experience with a Gαi2 variant that escapes regulation by regulator of G protein signaling (RGS) proteins, a Gαq variant was designed with glycine 188 replaced with serine (G188S) and then incorporated into a mouse line so that its effects on platelet activation and thrombus formation could be studied in vitro and in vivo. Results and ConclusionsAs predicted, the G188S substitution in Gαq disrupted its interaction with RGS18. Unexpectedly, it also uncoupled PLCβ-3 from activation by platelet agonists as evidenced by a loss rather than a gain of platelet function in vitro and in vivo. Binding studies showed that in addition to preventing the binding of RGS18 to Gαq, the G188S substitution also prevented the binding of PLCβ-3 to Gαq. Structural analysis revealed that G188 resides in the region that is also important for Gαq binding to PLCβ-3 in platelets. We conclude that the Gαq signaling node is more complex than that has been previously understood, suggesting that there is cross-talk between RGS proteins and PLCβ-3 in the context of Gαq signaling.

Deletion of Annexin A1 in Mice Upregulates the Expression of Its Receptor, Fpr2/3, and Reactivity to the AnxA1 Mimetic Peptide in Platelets.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

Abstract 156: Prenatal Exposure To Electronic Delivery Systems Modulates Platelet Reactivity, Epigenetics And Gene Expression In Mice

Tobacco smoking is a major public health threat and is associated with cardiovascular disease (CVD)-related morbidity and mortality. Being the single most preventable risk factor for CVD, a trend towards tobacco harm reduction started years ago. Thus, while tobacco usage has declined, that of Electronic Delivery Systems (ENDs) experienced widespread popularity, especially among pregnant women and women of childbearing age. This is alarming as pregnant women view them as safe(r) compared to cigarettes; which we now know not to be evidence-based. Thus, it is paramount to establish the health impact/CV safety of prenatal exposure to ENDs. This was addressed by employing a well-established ENDs inhalation system and performing whole-body exposures of 150 puffs/day on C57/BL6 mice, under prenatal/ in utero experimental settings throughout the gestational period. Our results show that platelets from the offspring of prenatally ENDs-exposed mice are hyperactive with enhanced: aggregation, dense and α granule secretion, activation of the αIIbβ3 integrin, clot retraction and phosphatidylserine expression, when compared to clean air exposed platelets, which suggests a prothrombotic state. Moreover, we observed that ENDs-exposed platelets are resistant to the inhibitory effects of prostacyclin, and that they exhibit increased activation of Akt and ERK. Furthermore, and consistent with these findings, ENDs were also found to shorten the thrombosis occlusion and bleeding times, relative to controls. Interestingly, we also observed alternations in the platelet miRNome, transcriptome and enrichment of platelet activation pathways, as follows: 1) miR-221 is differentially expressed, unlike mirR-146b; 2) Itgβ3, Src, and Rap2b transcripts are upregulated; and 3) oxidative phosphorylation, and platelet activation pathways are enriched. Taken together, our data demonstrate for the first time that prenatal exposure to ENDs modulates platelet reactivity and increase the risk of thrombogenesis, in part via impacting the platelet epigenetics and genetics. Thus, the negative health consequences of ENDs should not be underestimated, and warrant further investigation. These findings should also guide policy development for regulating exposure to ENDs.

The Antimicrobial Cathelicidin CRAMP Augments Platelet Activation during Psoriasis in Mice.

Platelet-associated complications including thrombosis, thrombocytopenia, and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis. Although several mechanisms that may contribute to the dysfunction of platelets during inflammatory diseases have been reported, knowledge on the primary molecules/mechanisms that underpin platelet-associated complications in such conditions is not fully established. Here, we report the significance of the mouse antimicrobial cathelicidin, mouse cathelicidin-related antimicrobial peptide (mCRAMP) (an orthologue of LL37 in humans), on the modulation of platelet reactivity during psoriasis using Imiquimod-induced psoriasis in mice as an inflammatory disease model for psoriasis vulgaris in humans. The activation of platelets during psoriasis is increased as evidenced by the elevated levels of fibrinogen binding and P-selectin exposure on the surface of platelets, and the level of soluble P-selectin in the plasma of psoriatic mice. The skin and plasma of psoriatic mice displayed increased levels of mCRAMP. Moreover, the plasma of psoriatic mice augmented the activation of platelets obtained from healthy mice. The effect of mCRAMP is partially mediated through formyl peptide receptor 2/3 (Fpr2/3, the orthologue to human FPR2/ALX) in platelets as a significant reduction in their activation was observed when FPR2/ALX-selective inhibitors such as WRW4 or Fpr2/3-deficient mouse platelets were used in these assays. Since the level of antimicrobial cathelicidin is increased in numerous inflammatory diseases such as psoriasis, atherosclerosis, and inflammatory bowel disease, the results of this study point towards a critical role for antimicrobial cathelicidin and FPR2/ALX in the development of platelet-related complications in such diseases.

The formyl peptide fMLF primes platelet activation and augments thrombus formation

Unlabelled Box BackgroundFormyl peptide receptors (FPRs) play pivotal roles in the regulation of innate immunity and host defense. The FPRs include three family members: FPR1, FPR2/ALX, and FPR3. The activation of FPR1 by its high‐affinity ligand, N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF) (a bacterial chemoattractant peptide), triggers intracellular signaling in immune cells such as neutrophils and exacerbates inflammatory responses to accelerate the clearance of microbial infection. Notably, fMLF has been demonstrated to induce intracellular calcium mobilization and chemotaxis in platelets that are known to play significant roles in the regulation of innate immunity and inflammatory responses. Despite a plethora of research focused on the roles of FPR1 and its ligands such as fMLF on the modulation of immune responses, their impact on the regulation of hemostasis and thrombosis remains unexplored. ObjectiveTo determine the effects of fMLF on the modulation of platelet reactivity, hemostasis, and thrombus formation. MethodsSelective inhibitors for FPR1 and Fpr1‐deficient mice were used to determine the effects of fMLF and FPR1 on platelets using various platelet functional assays. ResultsN‐formyl‐methionyl‐leucyl‐phenylalanine primes platelet activation through inducing distinctive functions and enhances thrombus formation under arterial flow conditions. Moreover, FPR1 regulates normal platelet function as its deficiency in mouse or blockade in human platelets using a pharmacological inhibitor resulted in diminished agonist‐induced platelet activation. ConclusionSince FPR1 plays critical roles in numerous disease conditions, its influence on the modulation of platelet activation and thrombus formation may provide insights into the mechanisms that control platelet‐mediated complications under diverse pathological settings.

Functional validation of microRNA-126-3p as a platelet reactivity regulator using human hematopoietic stem cells

Platelets are an abundant source of microRNAs (miRNAs), which may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, are pointed out as potential biomarkers of platelet reactivity and recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain and functional validation of these candidate miRNAs on human-derived cells is lacking. To functionally validate miR-126-3p as regulator of platelet reactivity in platelet-like structures derived from human hematopoietic stem cells. The functional impact of miRNAs on platelet-like structures derived from human hematopoietic stem cells was evaluated using a flow-based assay. CD34+ derived megakaryocytes were transfected with selected miRNA or siRNA and were differentiated in platelet-like structures. Platelet adhesion phenotype was assessed by perfusion of differentiated cells in a microfluidic system under a constant shear rate. Overexpression of miR-126-3p increased platelet-like structures adhesion compared to control. Moreover, miR-126-3p transfection was associated with downregulation of ADAM9, a validated target of miR-126-3p, and of PLXNB2, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results than miR-126-3p transfection. Reporter gene assay confirm that miR-126-3p directly targets PLXNB2 3′UTR sequence. Taken together, using a flow-based assay, we functionally validate miR-126-3p as a regulator of platelet reactivity on platelet-like structures derived from human hematopoietic stem cells. Moreover, PLXNB2 was identified as a platelet reactivity regulator supporting the hypotheses that miR-126-3p modulates platelet reactivity at least in part by downregulation of PLXNB2.

The endogenous antimicrobial cathelicidin LL37 induces platelet activation and augments thrombus formation

AbstractPlatelet-associated complications including thrombosis, thrombocytopenia, and hemorrhage are commonly observed during various inflammatory diseases such as sepsis, inflammatory bowel disease, and psoriasis. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX, in platelets and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide, but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, hemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilizing a pharmacological inhibitor and Fpr2/3 (an ortholog of human FPR2/ALX)–deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Because the level of LL37 is increased in numerous inflammatory diseases, these results point toward a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases.

Thrombotic Regulation From the Endothelial Cell Perspectives.

Thrombosis, the localized clotting of blood that affects arterial or venous circulation, is one of the leading causes of death worldwide.1 Currently used antithrombotic drugs have increased bleeding risk.2,3 Exploring new antithrombotic strategies that preserve physiological hemostasis is under intensive investigation. Classical triad of causative factors leading to thrombosis includes alterations in blood content, alterations in blood flow, and alterations in the vessel wall. Endothelium, the inner most single layer of cells lining the blood vessels, provides a surface for thrombosis formation and critically regulates blood fluidity and homeostasis. As barrier, endothelium separates blood clotting factors from exposure to subendothelial prothrombotic extracellular matrix components. Endothelium also secretes or expresses vasoactive factors that modulate platelet reactivity, coagulation, fibrinolysis, and vascular contractility, all of which contribute to thrombotic formation. Such factors include nitric oxide, prostacyclin, Von Willebrand factor (VWF), thrombomodulin, endothelin, etc. Accumulating evidences show that endothelial cells (ECs) play a pivotal role in modulating thrombosis, highlighting ECs as a potential target for thrombosis control. In this Recent Highlights, we reviewed recent publications in Arteriosclerosis, Thrombosis, and Vascular Biology and other leading journals that are focused on the mechanisms of endothelial regulation of thrombosis and discussed their merits in therapeutic discovery. Arterial thrombosis is commonly initiated by vascular endothelial erosions and ruptures of an atherosclerotic plaque3,4 while venous thrombosis mainly stems from blood stasis.5 Despite these differences, platelet adhesion/activation, and fibrin deposition as the result of coagulation constitute the fundamental processes of thrombus formation.6,7 Platelet activation occurs when they interact with activated ECs (with increased VWF release and selectin expression) via glycoprotein Ib-V-IX complex binding to VWF, or when they expose to subendothelial extracellular matrix components, such as collagen (via glycoprotein VI receptor), in the cases of endothelial injury or plaques …

The Antithrombotic Effects of 12‐LOX Derived Metabolites of DPA, ω‐6

IntroductionPlatelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. A number of anti‐platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti‐platelet therapies, uncontrolled thrombosis or bleeding complications still persist, warranting the development of novel anti‐thrombotic strategies. Previously, we had presented that 12‐HETrE, an oxylipin generated by 12‐lipoxygenase (12‐LOX) oxidation of ω‐6 polyunsaturated fatty acid (PUFA), dihomo‐g‐linolenic acid (DGLA), could be an approach to modulate platelet reactivity through the prostacyclin receptor. Here, we demonstrate that the elongated form of DGLA, docosapentaenoic acid (DPA; 22:5n‐6) can also be endogenously oxidized by platelet 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA, to regulate platelet reactivity.ObjectiveTo determine the efficacy of and the mechanistic target of 12‐LOX derived metabolites of DPA 11‐ and 14‐oxy‐DPA, ω‐6 in the regulation of platelet function.ResultsBoth 11‐ and 14‐oxy‐DPA potently inhibited platelet aggregation in an aggregometer measuring optical density. Treatment of whole blood with 11‐ and 14‐oxy‐DPA, ω‐6 also significantly reduced platelet adhesion as well as thrombus growth on an ex vivo collagen‐coated perfusion chamber under arterial shear. In support of these findings, using flow cytometry, calcium mobilization was attenuated in platelets treated with 11‐ and 14‐oxy‐DPA, ω‐6. Interestingly, both 11‐ and 14‐oxy‐DPA inhibited platelet activation in a Gas independent manner. Instead, 11‐oxy‐DPA and 14‐oxy‐DPA exerted their anti‐platelet effects through a peroxisome proliferated‐activated receptor (PPAR), by which pharmacological antagonism of the PPARα isoform reversed the ability of the metabolites to inhibit platelet aggregation. In supplement of these ex vivo findings, ongoing experiments are being performed to measure laser‐induced thrombus formation (platelet and fibrin) in the cremaster vessel of wild‐type mice intravenously administered with either 11‐ and 14‐oxy‐DPA.ConclusionThis is the first study to demonstrate the novel metabolites of DPA, ω‐6 generated by 12‐LOX also have a role in regulating platelet activation through the PPAR pathway. Our findings suggest that 11‐ and 14‐oxy‐DPA, ω‐6 shows great promise as a potential anti‐platelet therapeutic for the treatment for cardiovascular‐related diseases.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Randomised, double-blind, placebo-controlled study investigating the effects of inorganic nitrate on vascular function, platelet reactivity and restenosis in stable angina: protocol of the NITRATE-OCT study

IntroductionThe mainstay treatment for reducing the symptoms of angina and long-term risk of heart attacks in patients with heart disease is stent implantation in the diseased coronary artery. While this...

New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach.

Platelet reactivity (PR) is variable between individuals and modulates clinical outcome in cardiovascular (CV) patients treated with antiplatelet drugs. Although several data point to a genetic control of platelet reactivity, the genes contributing to the modulation of this phenotype are not clearly identified. Integration of data derived from high-throughput technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study is to identify candidate genes modulating platelet reactivity in aspirin-treated CV patients using an integrative network-based approach. Patients with extreme high (n = 6) or low PR (n = 6) were selected and data derived from quantitative proteomic of platelets and platelet sub-cellular fractions, as well as from transcriptomic analysis were integrated with a network biology approach. Two modules within the network containing 123 and 182 genes were identified. We then specifically assessed the level of miRNAs in these two groups of patients. Among the 12 miRNAs differentially expressed, 2 (miR-135a-5p and miR-204-5p) correlated with PR. The predicted targets of these miRNAs were mapped onto the network, allowing the identification of seven overlapping genes (THBS1, CDC42, CORO1C, SPTBN1, TPM3, GTPBP2, and MAPRE2), suggesting a synergistic effect of these two miRNAs on these predicted targets. Integration of several omics data sets allowed the identification of 2 candidate miRNAs and 7 candidate genes regulating platelet reactivity in aspirin-treated CV patients.

Expression of the PlA2 allele of glycoprotein IIIa and its impact on platelet function.

BackgroundThe platelet fibrinogen receptor represents the final common pathway of platelet activation, and is formed from two glycoprotein (GP) subunits (GPIIb/IIIa). Carriage of the mutant PlA2 allele of GPIIIa has been shown to confer an increased risk of cardiovascular events, but published studies have disagreed as to the mechanism for this association.ObjectivesTo assess whether carriage of the PlA2 allele conforms to Mendelian patterns of expression and to identify whether carriage of the mutant allele modulates platelet function.MethodsExpression of the PlA2 allele was assessed in both healthy subjects (n = 25) and patients with known coronary artery disease (n = 90) through the development and validation of a liquid chromatography, tandem mass spectrometry (LC-MS/MS) assay. Platelet function was assessed in the patient cohort in response to multiple agonists, and these data were analysed in the context of the proteomic data.ResultsExpression of the wild-type PlA1 allele and mutant PlA2 alleles was readily quantifiable and conformed to Mendelian patterns in both healthy and patient cohorts. Patients who were homozygous for the mutant PlA2 allele had an increased aggregatory response to adenosine diphosphate, collagen, adrenaline, ristocetin, thrombin receptor-activating peptide 6 and U46619, when assessed using agonist-concentration response curves.ConclusionsThese findings support the hypothesis that carriage of the mutant PlA2 allele mediates an increased risk of cardiovascular events through the modulation of platelet reactivity.

12-HETrE, a Novel 12-LOX Oxylipin, Prevents Platelet Activation in a Gαs-like Manner

Platelet-mediated thrombosis is the primary underlying mechanism leading to cardiovascular life-threatening clinical events. Control of excessive platelet responses is an essential aspect of antithrombotic therapy. A number of anti-platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti-platelet therapies, uncontrolled thrombosis or bleeding complications still persist. We had proposed a potential novel therapeutic approach by which oxylipins generated by 12-lipoxygenase (12-LOX) oxidation of ω-6 could modulate platelet reactivity. We observed 12-hydroxyeicosatrienoic acid (12-HETrE), a 12-LOX oxidized oxylipin of ω-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), significantly attenuated human platelet activation. We then verified that DGLA oxidation to 12-HETrE depended on functional platelet 12-lipoxygenase (12-LOX) in our transgenic mouse model deficient in 12-LOX enzyme in the platelets (12-LOX-/-). To determine whether 12-HETrE could be inducing its inhibitory regulation in a GPCR-like manner by which it could potentially be behaving similarly to prostacyclin to activate adenylyl cyclase and increase cAMP through the Gs pathway, we measured cAMP level in the presence of 12-HETrE. We observed 12-HETrE significantly increased cAMP levels. We investigated a downstream effector of cAMP, such as VASP 157 phopshorylation, which is a PKA substrate. Also observed both Rap1 and GPIIbIIa activation to be attenuated in the presence 12-HETrE, confirming our aggregation result. This the first study to show the signaling mechanism of 12-HETrE which is dependent on active 12-LOX oxidation of DGLA to regulate platelet reactivity in a Gs-like manner. DisclosuresNo relevant conflicts of interest to declare.

Comparison of the effect of CYP2C19 polymorphism on clinical outcome between acute coronary syndrome and stable angina

AimCYP2C19 polymorphism modulates platelet reactivity in coronary artery disease patients with stent implants. However, the impact of the CYP2C19 genotype on clopidogrel response and clinical outcome has not been fully understood to date. MethodsWe enrolled 518 consecutive patients with acute coronary syndrome (ACS) (n=214) and stable angina (SA) (n=304). All patients received stent implants followed by dual antiplatelet therapy of aspirin and clopidogrel. We determined CYP2C19 phenotype, measured platelet reactivity, and assessed the risk of cardiovascular events. ResultsDuring a median follow-up of 894 days, the rate of cardiovascular events was higher in patients of the ACS group than the SA group (ACS: 20.1%, SA: 12.5%, p=0.015). The mean platelet reactivity was significantly higher in the CYP2C19 loss-of-function allele carriers of the two groups (ACS, non-carriers: 3909±1836AUmin, carriers: 4854±1594AUmin, respectively, p<0.01; SA, 3606±1579AUmin, 4381±1373AUmin, ±SD, p<0.01). In the ACS group, cardiovascular events were higher in the loss-of-function allele carriers (24.6%) versus non-carriers (11.1%, p<0.05), but no such difference was noted in the SA group (carriers: 14.8%; non-carriers: 7.9%, p=0.078). Furthermore, landmark analysis from 30 days did not show differences in ACS group (carriers: 14.8%, non-carriers: 11.1%, p=0.315). Multivariate Cox proportional hazards analysis identified the presence of loss-of-function allele as an independent predictor of cardiovascular events (hazard ratio, 2.1, 95% CI, 1.194–3.587, p=0.010). ConclusionsThe impact of CYP2C19 loss-of-function gene on clinical outcome is more powerful in early phase of ACS compared with SA.

Impact of aspirin dose on adenosine diphosphate-mediated platelet activities

Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators.

Periprocedural variations of platelet reactivity during elective percutaneous coronary intervention

Percutaneous coronary intervention (PCI) modulates platelet reactivity (PR). To assess: (i) the impact of coronary interventions on periprocedural variations (Δ) of PR; (ii) whether ΔPR correlates with periprocedural myocardial infarction (PMI); and (iii) the mechanisms of these variations in vitro. We enrolled 65 patients on aspirin (80-100 mg day(-1)) and clopidogrel (600 mg, 12 h before PCI): 15 with coronary angiography (CA group), 40 with PCI (PCI group), and 10 with rotational atherectomy plus PCI (RA group). PR was assessed by ADP, high-sensitivity ADP and thrombin receptor activator peptide 6 tests prior to, immediately after and 24 h after the procedure. E-selectin and ICAM-1 were assessed prior to and immediately after the procedure. In vitro, PR was measured during pulsatile blood flow at baseline, after balloon inflation and after stent implantation in six porcine carotid arteries and five plastic tubes. PR declined in the CA group, but significantly increased in the PCI and RA groups immediately postprocedure, and decreased to baseline at 24 h. ΔPR increased across the three groups (P < 0.0001). In the PCI group, ΔPR was directly related to total inflation time (r = 0.435, P = 0.005) and total stent length (r = 0.586, P < 0.001). The change in E-selectin significantly and inversely correlated with ΔPR (P < 0.001). No correlation was found with sICAM-1. PR increased significantly more in patients with PMI than in patients without PMI (P = 0.013). In vitro, platelet activation was observed in the presence of carotid arteries but not in the presence of plastic tubes. Despite dual antiplatelet therapy, PCI affected platelet function proportionally to procedural complexity and the extent of vascular damage.

Nitrite Inhibits Platelet Reactivity in Presence of Deoxygenated Red Blood Cells As Measured by Flow Cytometry

AbstractAbstract 5146Nitric oxide (NO), a small gas molecule with multiple functions in the body, has been shown to have several precursors in body, the most abundant of which is nitrite. In the last decade several studies show that nitrite is reduced to NO by various enzymatic and non-enzymatic reductive processes, especially at low oxygenation conditions. These pathways are alternative mammalian sources of NO as compared to its enzymatic production by a family of nitric oxide synthase enzymes (NOS). NO has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of this has not been well clarified. We recently showed that addition of nitrite into plasma in the presence of red blood cells inhibits platelet aggregation and that this effect, as measured by cGMP production is enhanced by deoxygenation of erythrocytes. In order to study these effects more thoroughly on the molecular level, we have been developing flow-cytometric assays to study molecular processes on the platelet cell membrane known to correlate with platelet aggregation. We focused on P-selectin (CD62P) and activated GpIIb/IIIa (PAC-1) levels in platelets exposed to three activators, ADP, collagen and thrombin, in the presence of various concentrations of red blood cells and at variable levels of oxygen. We found that nitrite at physiological concentration (0. 1 to 1. 0 uM) significantly inhibited exposure of both of these surface markers on the outer membrane of platelets at levels of hematocrit above 23% and mean oxygen levels lower than 49 mmHg. The effect increased with increasing hematocrit and decreasing oxygen saturation into the hypoxic range. Addition of an NO scavenger prevented nitrite-related platelet inhibition, but addition of an inhibitor of NOS or of xanthine oxidoreductase had no effect. These results indicate that physiological levels of nitrite ions in the blood can modulate platelet reactivity in the presence of partially deoxygenated erythrocytes by the reduction to NO and that this process can modulate platelet activation and aggregation levels and confirm and extend our previous report (Srihirun et al, PLoS One 7, e30380, 2012), done primarily using aggregation assays. Our flow cytometry assays not only allows a more detailed and systematic study of the kinetics and equilibrium aspects of the aggregation process, but also offers a possibility to identify the individual molecular processes involved in these effects. The results may also explain certain differences between arterial and venous thrombosis. Disclosures:Schechter:NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.

Effect of alpha lipoic acid on cardiac autonomic dysfunction and platelet reactivity in type 1 diabetes: Rationale and design of the AUTOnomic function and platelet REACTivity trial (AUTO-REACT protocol)

Background The aim of this study is to assess the relationship between cardiac autonomic dysfunction and increased platelet reactivity in patients with type 1 diabetes and whether alpha lipoic acid therapy (ALA) might improve both abnormalities in these patients. Methods and materials Cardiac autonomic function will be assessed by heart rate variability (HRV) analysis. Platelet reactivity will be investigated by both measuring the aggregation time on the PFA-100 method and flow cytometry. HRV and platelet reactivity will be re-assessed in the patients with depressed HRV after 4 weeks from randomization to ALA or placebo. Discussion The cardiovascular autonomic system might play a role in the modulation of platelet reactivity which is increased in diabetes. Within this framework, ALA might be found to exercise antithrombotic effects in addition to its known antioxidants properties. Trial registration Australian Clinical Trials Registry ACTRN 12610000291088.