Baseline Platelet Activation Research Articles

A Western Diet Modifies the Activation Profile of Circulating Platelets

Background: While it is known that obesity increases the risk for cardiovascular disease (CVD), there has been no clear investigation into the effect an obesogenic diet exerts onto circulating platelets. As platelets are the critical actors in the formation of occlusive clots that manifest as a myocardial infarction or stroke, it is pertinent to establish any potential link between obesity and platelet activity. Methods: To examine the effect of a Western diet on baseline platelet activity, male C57BL6/J mice were fed either a control (10% fat, 7% sucrose, 0.3% salt Research Diets Inc. D12450J) or Western (40% fat, 30% sucrose, 8% salt Research Diets Inc. D06111701) diet ad libitum for 20 weeks beginning at the time of weaning. At 12, 16 and 20 weeks after beginning diet administration whole blood was collected via the retro-orbital sinus. Blood was used to generate platelet rich plasma (PRP) which was stimulated with collagen (20mg/ml) prior to staining for flow cytometry analysis. Flow cytometry staining utilized antibodies to recognize the platelet receptors GPIbα, P-Selectin and the activated form of integrin α IIb β 3 (JON/A). Additionally, body composition was likewise determined at these time points using Echo-MRI while bodyweight was measured weekly. Results: Western diet fed mice gained slightly more weight than control mice and had a higher percentage of body fat (measured by EchoMRI). Surprisingly, the two groups differed in GPIbα and activated α IIb β 3 expression on circulating platelets at all three observed time points. Specifically, platelets from control-fed mice displayed significantly higher GPIbα expression throughout the study while Western diet fed animals had higher levels of activated α IIb β 3 . There was no difference in platelet expression of P-Selectin between the groups at any time point. Conclusions: Platelet activation is not a binary process where platelets display a definitive “active” or “inactive” phenotype. Though terminal platelet activation elicits de-granulation and an increase in P-Selectin expression, the spectrum of platelet stimulation preceding this is marked by variable incidences of integrin activation and GPIbα shedding. Here we demonstrate proof of principle that dietary factors, such as the consumption of a Western diet, can facilitate early changes to the stimulation pattern of circulating platelets which is then maintained long-term. While the consequence of this altered platelet stimulation profile is unknown with regards to the pathways of hemostasis and thrombosis, further investigations of downstream signaling and amplification pathways are needed. Funding: Arkansas Children's Research Institute/Arkansas Bioscience Institute Postgraduate Fellowship: AWD-037176 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.

Evaluation of platelets activity and reactivity as risk factors for acute ischemic non-embolic stroke in young adults

BackgroundIschemic stroke (IS) constitutes a relevant health concern recently in younger population causing permanent cognitive and function-limiting disability and ranks as the 3rd cause of death in Egypt after cardiac and hepatic diseases. Platelet activation has a crucial mechanism in arterial thrombogenesis, thus in pathophysiology of IS. Surface expression of P-selectin (CD62P) reflects platelet activation and measured by flowcytometry. The purpose of the study is to evaluate whether platelet activity and reactivity are considered risk factors for IS so more restrict antiplatelet protocols could be implemented for management and recurrence prevention.ResultsStudy population was 60 IS patients and 60 apparently healthy age and gender-matched controls. Patients were subdivided into 37 patients without classical risk factors, aged 46.1 ± 8.2, and 23 patients with > 1 vascular risk factors, aged 52 ± 9.9. The percentage of platelets expressing CD62P reflecting ex vivo baseline activity was significantly higher in stroke patients to controls (p = 0.001), also platelet reactivity (CD62P expression after ADP provocation) was statistically significantly elevated in patients than in controls (p < 0.0001) and was significantly higher in IS patients with vascular risk factors compared to patients without risk factors (p = 0.02).ConclusionBoth baseline platelet activity and reactivity were significantly higher in IS patients, and were also higher in IS patients with other vascular risk factors than in cryptogenic stroke and considered risk factors for IS.

Nitrite in paraffin-stimulated saliva correlates with blood nitrite

Nitrite anion (NO2−) is a circulating nitric oxide (NO) metabolite considered an endothelial function marker. Nitrite can be produced from nitrate (NO3−) secreted from plasma into saliva. The nitrate reductase of oral bacteria converts salivary nitrate to nitrite, which is swallowed and absorbed into circulation. In this study, we aimed to examine the relevance between these species' salivary and blood levels. We collected three whole saliva samples (unstimulated, paraffin-stimulated, and post-chlorhexidine mouthwash stimulated saliva) and blood from 75 healthy volunteers. We measured the nitrite and nitrate by the chemiluminescence method. The nitrite levels in stimulated saliva and post-mouthwash stimulated saliva exhibited weak correlations with blood nitrite. There was no correlation between nitrite in unstimulated saliva with blood nitrite. The baseline platelet activity, determined as P-selectin expression, negatively correlated with nitrite in plasma and post-mouthwash stimulated saliva. The salivary nitrate in all saliva samples showed correlations with its plasma levels. We conclude that nitrite in stimulated saliva correlates with blood nitrite.

Platelet hyperactivity in COVID-19: Can the tomato extract Fruitflow® be used as an antiplatelet regime?

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus is now considered a global public health threat. The primary focus has been on reducing the viral spread and treating respiratory symptoms; as time goes on, the impact of COVID-19 on neurological and haemostatic systems becomes more evident. The clinical data suggest that platelet hyperactivity plays a role in the pathology of COVID-19 from its onset and that platelets may serve critical functions during COVID‐19 progression. Hyperactivation of blood platelets and the coagulation system are emerging as important drivers of inflammation and may be linked to the severity of the ‘cytokine storm’ induced in severe cases of COVID-19, in which disseminated intravascular coagulation, and platelet hyperactivity are associated with poor prognosis and increased risk of mortality.We propose that targeting platelet hyperactivity in the early stages of COVID-19 infection may reduce the immunothrombotic complications of COVID-19 and subdue the systemic inflammatory response. Lowering baseline platelet activity may be of particular importance for higher-risk groups. As an alternative to antiplatelet drugs, an inappropriate intervention in public health, we propose that the dietary antiplatelet agent Fruitflow®, derived from tomatoes, may be considered a suitable therapy. Fruitflow® contains antiplatelet and anti-inflammatory compounds that target the mechanisms of platelet activation specific to COVID-19 and can be considered a safe and natural antiplatelet regime.

Abstract 15888: Platelet Activation is Associated With All-cause Mortality in Patients With Covid-19

Background: COVID-19 is a global pandemic with patients at increased risk for all-cause mortality. Virus-platelet interactions are linked to viral pathogenesis and increased risk of adverse events. Aim: To investigate the relationship between in vivo platelet activity markers and all-cause mortality in hospitalized patients with COVID-19. Method: Plasma samples were collected from 100 hospitalized patients on the day of PCR-confirmed COVID-19 diagnosis. Thromboxane B 2 (TxB 2 ), P-selectin, and soluble CD40 ligand (sCD40L) were measured in plasma, and mean platelet volume (MPV) assessed. Subjects were followed until discharge or death. Results: Among 100 patients, the median age was 65 years (IQR: 55, 75), 39% were female, and 32 died or experienced a thrombotic event. The baseline platelet activation markers, P-selectin (p=0.02), sCD40L (p=0.03) and MPV (p=0.005) were higher in patients who died. After adjustment for age, sex, race/ethnicity, platelet count, antiplatelet therapy, and chronic obstructive pulmonary disease, TxB 2 (p=0.036), P-selectin (p=0.007), sCD40L (p=0.02) and MPV (p=0.01) were each independently associated with death after multivariable adjustment ( Table 1 ). Conclusions: Biomarkers of platelet activation are significantly associated with death or thrombosis in patients hospitalized with COVID-19. Our findings suggest multiple platelet activation mechanisms may contribute to adverse events. Further investigation into the mechanistic role of platelets in COVID-19 pathogenesis and the potential role of antiplatelet therapy is warranted. Table 1. Multivariable regression models of all-cause mortality. Odds ratios (OR) from logistic regression analysis per SD increase for biomarker levels adjusted for age, sex, race, antiplatelet therapy, platelet count, and chronic obstructive pulmonary disease (COPD).

Hemoglobin-bound platelets correlate with the increased platelet activity in hemoglobin E/β-thalassemia.

An increase in platelet activity is a contributing factor to vascular complications in hemoglobin E/β-thalassemia (HbE/β-thal). Plasma-free hemoglobin (Hb) increases in HbE/β-thal patients and correlates with platelet activation, but the levels of Hb-bound platelets have never been reported. In this study, we aimed to investigate the levels of Hb-bound platelets and its association with platelet activity in HbE/β-thal patients. Hb-bound platelets were measured by flow cytometry in 22 healthy subjects and 26 HbE/β-thal patients (16 nonsplenectomized and 10 splenectomized HbE/β-thal patients). Plasma Hb was measured by the chemiluminescence method based on the consumption of nitric oxide (NO) by Hb. Expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on platelets was measured by flow cytometry as a marker of platelet activity. Both nonsplenectomized and splenectomized HbE/β-thal patients had higher levels of Hb-bound platelets and plasma Hb than healthy subjects. In vitro incubation of dialyzed Hb from patients with platelets of healthy subjects caused an increase in Hb-bound platelets, which was partially inhibited by anti-GPIbα antibody. Plasma Hb positively correlated with Hb-bound platelets. Platelet P-selectin expression at baseline and in response to adenosine diphosphate (ADP, 1µM) stimulation was higher in nonsplenectomized and splenectomized HbE/β-thal patients than healthy subjects. The ADP-induced aGPIIb/IIIa expression on platelets was also higher in HbE/β-thal patients than healthy subjects. Hb-bound platelets correlated with baseline P-selectin expression and ADP-induced P-selectin expression. HbE/β-thal patients have increased Hb-bound platelets, which is associated with increased baseline platelet activation and reactivity.

The Mitochondrion: A Link Between Hemolysis and Platelet Activation

Patients with Sickle Cell Disease (SCD) demonstrate characteristics of chronic hemostatic activation including elevated baseline platelet activation. While it is well established that platelet activation is positively correlated with the magnitude of erythrocytic hemolysis in these patients, the mechanisms linking hemolysis to platelet activation remain unclear. In this study, we investigate the role of the platelet mitochondrion as the molecular link between hemolysis and downstream platelet activation. Using extracellular flux analysis, we demonstrate that platelets isolated from patients with SCD show a distinct alteration in mitochondrial function characterized by a decrease in the activity of the mitochondrial ATP Synthase (electron transport chain Complex V), which leads to enhanced mitochondrial membrane potential and elevated reactive oxygen species (ROS) production by the electron transport chain. Notably, levels of platelet mitochondrial ROS production was significantly correlated with markers of hemolysis (lactate dehydrogenase and plasma free hemoglobin concentration) as well as platelet activation in this cohort. Moreover, the mitochondrial dysfunction observed in platelets from SCD patients was recapitulated by exposing healthy platelets to cell-free hemoglobin ex vivo. Importantly, augmented mitochondrial ROS generation appears to initiate platelet activation, as scavenging of mitochondrial ROS inhibited cell free hemoglobin-induced activation of healthy platelets. Studies in the Berkley (BERK) transgenic murine model of SCD supported ex vivo data demonstrating that hemolysis-induced mitochondrial ROS production stimulates platelet activation. Homozygous BERK mice showed significantly inhibited platelet mitochondrial complex V activity and elevated mitochondrial ROS production, concomitant with enhanced baseline platelet activation compared to hemizygote mice. When subjected to a ferric chloride-induced vascular injury model of thrombosis, BERK homozygous mice showed a significantly shorter time to vascular occlusion due to thrombosis compared to hemizygote mice. Treatment of homozygous BERK mice with the mitochondrial ROS scavenger MitoTEMPO (supplemented in drinking water) significantly increased time to vascular occlusion in the ferric chloride thrombosis model. These ex vivo human platelet data and in vivo studies in murine models demonstrate that hemolysis induces platelet mitochondrial ROS production, which stimulates downstream platelet activation. Ongoing studies are focused on determining the molecular mechanisms by which cell free hemoglobin elicits an inhibition of platelet mitochondrial Complex V and implicate a role for activation of the platelet surface toll like receptor 4 (TLR4). Collectively, these data identify the platelet mitochondrion as an important signaling hub that links hemolysis to platelet activation in SCD patients. The potential of therapeutically modulating platelet mitochondrial ROS production to attenuate hemolysis induced thrombotic activation will be discussed. Disclosures No relevant conflicts of interest to declare.

Mesenchymal stromal cells lower platelet activation and assist in platelet formation in vitro.

The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.

PB2424 AUGMENTED AND SYNERGISTIC EFFECT OF FACTOR V LEIDEN (FVL) AND MTHFR GENES POLYMORPHISMS AND PLATELET ACTIVITY AND REACTIVITY AS RISK FACTORS FOR ISCHEMIC CEREBROVASCULAR STROKE (IS)

Background:Ischemic cerebrovascular stroke (IS) is a polygenic multifactorial disease; effect of thrombotic gene polymorphism, platelet activation and reactivation synergistic risk effect are plausible postulation.Aims:To assess synergistic effect of FVL and MTHFR genes polymorphisms and platelet activity and reactivity as risk factors for ischemic cerebrovascular strokeMethods:A hospital‐based case‐control study; approvered by Suez Canal University (SCU) ethical committee, after obtaining an informed consent. 120 individuals were included: 60 acute IS patients, subdivided into 2 subgroups regarding the vascular risk factors recruited from neurology department, SCU Hospital; and 60 matched healthy individuals. FVL G1691A, FVLA4070G, FVL A5279G, MTHFRC677T, and MTHFR A1298C were assessed using multiplex real‐time PCR assay (AnyplexTM II Thrombosis SNP Panel Assay, seegene); platelet ex vivo activity &amp; reactivity (CD62P; after ADP provocation) were assessed by four‐colors flow cytometry, FACS Caliber, BD‐Biosciences).Results:Both FVLG1691A &amp; FVL A4070G (OR 19.6; 95 % CI = 2.5 to 154.4; p = 0.0001), (OR 7.6; 95 % CI = 1.13 to 178; p = 0.012), and MTHFRC677T (OR 10; 95 % CI = 1.16to 85.8; p = 0.03) and MTHFRA1298C (OR 11.6; 95 % CI = 1.35to 100.7; p = 0.03) &amp; the presence of &gt;1 mutation (p &lt; 0.0001) showed a statistically significant association with IS. Multivariate logistic regression models showed that studied SNPs add synergistic effect on the vascular risk factors. A statistically significant difference was detected in platelet CD62p % after adding ADP (IS patients: 72.95 ± 14.8 ‐ controls: 62.2 ± 11.39), MFI IS patients: 248 ± 101‐controls 215 ± 75.8). both FVLG1691A and MTHFRC677T were significantly associated with higher baseline platelet activation in the co‐dominant and recessive models for FVLG1691A and in the dominant model for MTHFRC677TSummary/Conclusion:FVLG1691A, FVL14070G, MTHFRC677T &amp; MTHFRA1298C were independent risk factors for IS especially in younger patients &amp; existence of &gt;1 mutation significantly increased IS risk, platelet activity and reactivity and genetic polymorphism play a potential synergistic role increasing the risk of IS.image

HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease

Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD.Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests.Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12).Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle).Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares).Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A trial of type 12 purinergic (P2Y12) receptor inhibition with prasugrel identifies a potentially distinct endotype of patients with aspirin-exacerbated respiratory disease

Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, recurrent nasal polyposis, and respiratory reactions on ingestion of COX-1 inhibitors. Increased numbers of platelet-leukocyte aggregates are present in the sinus tissue and blood of patients with AERD compared with that of aspirin-tolerant patients, and platelet activation can contribute to aspirin-induced reactions. We sought to determine whether treatment with prasugrel, which inhibits platelet activation by blocking the type 12 purinergic (P2Y12) receptor, would attenuate the severity of sinonasal and respiratory symptoms induced during aspirin challenge in patients with AERD. Forty patients with AERD completed a 10-week, double-blind, placebo-controlled crossover trial of prasugrel. All patients underwent oral aspirin challenges after 4weeks of prasugrel and after 4weeks of placebo. The primary outcome was a change in the provocative dose of aspirin that would elicit an increase in Total Nasal Symptom Score (TNSS) of 2 points. Changes in lung function, urinary eicosanoids, plasma tryptase, platelet-leukocyte aggregates, and platelet activation were also recorded. Prasugrel did not significantly change the mean increase in TNSS of 2 points (79±15 for patients receiving placebo and 139±32 for patients receiving prasugrel, P=.10), platelet-leukocyte aggregates, or increases in urinary leukotriene E4 and prostaglandin D2 metabolite levels during aspirin-induced reactions in the study population as a whole. Five subjects (responders) reacted to aspirin while receiving placebo but did not have any reaction to aspirin challenge after the prasugrel arm. In contrast to prasugrel nonresponders (35 subjects), the prasugrel responders had smaller reaction-induced increases in TNSS; did not have significant aspirin-induced increases in urinary leukotriene E4, prostaglandin D2 metabolite, or thromboxane B2 levels; and did not display increases in serum tryptase levels during aspirin reactions on the placebo arm, all of which were observed in the nonresponders. In the overall study population, prasugrel did not attenuate aspirin-induced symptoms, possibly because it failed to decrease the frequencies of platelet-adherent leukocytes or to diminish aspirin-induced mast cell activation. In a small subset of patients with AERD who had greater baseline platelet activation and milder upper respiratory symptoms during aspirin-induced reactions, P2Y12 receptor antagonism with prasugrel completely inhibited all aspirin-induced reactionsymptoms, suggesting a contribution from P2Y12 receptor signaling in this subset.

Clopidogrel pharmacokinetics and pharmacodynamics in out-of-hospital cardiac arrest patients with acute coronary syndrome undergoing target temperature management

BackgroundTarget temperature management (TTM) after cardiac arrest (CA) improves outcome in patients with acute coronary syndrome (ACS). Previous data point to an interaction between hypothermia and drug metabolism, potentially impacting on platelet function in patients on antiplatelet therapy. PurposeTo compare clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with TTM (33°C, n=15) and in ACS patients (troponin positive) without TTM (n=18). MethodsPlatelet function was measured by multiple electrode platelet aggregometry (MEA), light transmittance aggregometry (LTA) and VASP analysis before and after administration of a 600mg clopidogrel loading dose. Plasma levels of clopidogrel and its metabolites were measured. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. TTM was carried out for 24h at a target temperature of 33°C using a computer feedback surface cooling device in cardiac arrest patients. ResultsPlasma concentration of clopidogrel and metabolites was lower in the TTM group after 2 and 4h, respectively (all p<0.005 vs. controls), and platelet function tests revealed an attenuated response to clopidogrel with respect to baseline platelet activity in the TTM group. This was significant for MEA, LTA and VASP analysis (all p<0.05). Moreover, there was no significant difference in genotype and platelet function determined ex vivo at 33 or 37°C, respectively. ConclusionInhibition of platelet function is significantly lessened in TTM at 33°C, likely due to reduced clopidogrel absorption. Patients with TTM might thus have a higher risk for further cardiovascular events despite antiplatelet therapy with clopidogrel.

Thromboinflammatory response and predictors of outcomes in patients undergoing transcatheter aortic valve replacement.

Transcatheter aortic valve replacement (TAVR) has been increasingly used to treat patients with symptomatic aortic stenosis. Despite improvements in valve deployment, patients that have undergone TAVR are at high risk for major adverse events following the procedure. Blood cell numbers, platelet function, and biomarkers of systemic inflammation were analyzed in 58 patients undergoing TAVR with the Edward's SAPIEN valve. Following valve deployment, platelet count and agonist-induced platelet activity declined and plasma markers of systemic inflammation (interleukin-6 and S100A8/A9) increased. Baseline platelet activity prior to TAVR correlated with perioperative changes plasma interleukin-6 levels. Moreover, perioperative changes in plasma inflammatory markers predicted the decline in platelet count in the days following the TAVR procedure. Additionally, a significant effect of gender on platelet count following TAVR and was observed. Finally, post-procedural mortality was associated with sustained thrombocytopenia after TAVR. Our findings suggest that TAVR elicits a thromboinflammatory state that may contribute to post-procedural thrombocytopenia. Importantly, our results add to the growing body of literature that suggests the thromboinflammatory changes that occur early after TAVR may predict long-term outcomes.

A Study of Platelet Inhibition, Using a 'Point of Care' Platelet Function Test, following Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction [PINPOINT-PPCI

BackgroundRapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure (‘door-to-end’) time and baseline platelet activity on platelet inhibition within 24hours post-STEMI.Methods and Findings108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8–109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40–70] and infusion duration 30minutes [20–42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention.ConclusionsBaseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity.

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15 µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.

Abstract 17314: Impact of Mild Therapeutic Hypothermia on Platelet Inhibition in Acute Coronary Syndrome

Background: Mild therapeutic hypothermia (MTH) improves outcome for patients with acute coronary syndrome (ACS) after cardiac arrest (CA). Previous data point to an interaction between hypothermia and drug metabolism, thus potentially impacting on platelet function under antiplatelet therapy. Purpose: The aim of the study is to determine clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with mild hypothermia (33°C, n=12) compared with ACS patients (troponin positive) with normal body temperature (n=14). Methods: Platelet function was measured by light transmittance aggregometry (LTA), multiple electrode platelet aggregometry (MEA) and VASP phosphorylation analysis before, 2h, 4h and 24h after administration of a 600 mg clopidogrel loading dose. Furthermore, plasma concentrations of clopidogrel, the active thiol metabolite and the inactive carboxyl metabolite were determined. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. Mild hypothermia was carried out according to current guidelines for 24 hours at a target temperature of 33°C. Results: Plasma concentration of clopidogrel and metabolites were lower in the MTH group after 2h and 4h, respectively (all p&lt;0.005). All platelet function tests showed an attenuated response to clopidogrel with respect to baseline platelet activity in the MTH group. This was significant for VASP analysis and LTA (p&lt;0.05). Moreover, there was no difference in genotype and platelet function determined ex vivo with 33°C and 37°C, respectively. Conclusion: Inhibition of platelet function is decreased under MTH, presumably due to a diminished clopidogrel absorption and metabolization. Thus, these patients might have a higher risk for cardiovascular events despite antiplatelet therapy.

Chronic kidney disease is associated with increased platelet activation and poor response to antiplatelet therapy

Chronic kidney disease (CKD) is a common co-morbidity of patients with atherosclerotic vascular disease, and may influence the response to antiplatelet therapy. We, therefore, sought to investigate its effect on platelet activation and on-treatment residual platelet reactivity. We assessed platelet activation and the response to clopidogrel and aspirin in 316 patients after percutaneous intervention with stent implantation. CKD was defined as a glomerular filtration rate <60 mL/min/1.73 m(2) according to the Modification of Diet in Renal Disease formula. Surface expression of activated glycoprotein IIb/IIIa without the addition of agonists was determined to assess baseline platelet activation. GPIIb/IIIa in response to adenosine diphosphate (ADP) and arachidonic acid (AA), as well as the VerifyNow assays and light transmission aggregometry (LTA) were used to measure residual platelet reactivity. Baseline platelet activation was significantly increased in CKD patients compared with patients without renal insufficiency [3.1 versus 2.7 mean fluorescence intensity (MFI), P = 0.001]. Moreover, patients with CKD exhibited a more pronounced expression of GPIIb/IIIa in response to ADP (13 versus 9.6 MFI) and AA (6 versus 5.1 MFI; both P≤ 0.02) than patients without CKD. In the VerifyNow assays, CKD patients showed significantly higher platelet reactivity than patients without CKD (P2Y12 assay: 239 versus 182 P2Y12 Reaction Units; aspirin assay: 415 versus 399 Aspirin Reaction Units; both P≤ 0.03). Further, patients with CKD had significantly higher platelet reactivity by LTA in response to ADP (49.9 versus 43.2%, P = 0.01). Finally, high on-treatment residual ADP-inducible platelet reactivity by the VerifyNow P2Y12 assay and by LTA occurred significantly more frequent in patients with CKD (VerifyNow: 52.2 versus 26.2%, P < 0.001; LTA: 23.3 versus 12.1%, P = 0.01). Patients with CKD exhibit increased platelet activation, and an attenuated response to dual antiplatelet therapy compared with patients without renal insufficiency.

Altered platelet contents in survivors of early ischemic ventricular fibrillation: Preliminary findings

Early ischemic ventricular fibrillation (VF) in the setting of an acute myocardial infarction (AMI) due to thrombotic coronary occlusion remains a major health problem. Several animal studies have shown that platelet-dense granule contents released during thrombus formation can induce arrhythmias. We hypothesize that the platelet release reaction is involved in the predisposition to early ischemic VF. A case-control study was performed in patients who survived VF during a first AMI (“cases,” n = 26) and in patients with one previous AMI without arrhythmias (“controls,” n = 24). All patients were on aspirin 100 mg OD. Baseline platelet activation was assessed with flow cytometry. Response to activation was assessed with aggregometry, flow cytometry and PFA-100 analysis. Differences in platelet contents and content release were assessed by labeling platelet-dense granules with mepacrine and by measuring serotonin and ADP/ATP content. Patient and infarct characteristics and baseline platelet function tests were similar between groups. The mean time from event was 4.9 (±3.2) years among cases and 4.7 (±2.7) years among controls. Dense granule release was similar in cases versus controls. Platelet serotonin content in cases was higher than in controls (611 ± 118 ng/10E9 platelets vs. 536 ± 141 ng/109, p = 0.048). Even years after the event, elevations in the platelet dense granule contents between VF survivors and controls may be detected. These preliminary findings shed new light on the pathophysiological mechanisms underlying ischemic VF, as platelet-dense granules may contain mediators of early ischemic VF risk.

Chronic dysfunction of the endothelium is associated with mortality in acute coronary syndrome patients

IntroductionPlatelet activation and endothelium dysfunction are determinants of atherothrombosis in acute coronary syndrome (ACS) patients. The aim of this study was to investigate the relationship between platelet and endothelial cell activation markers and mortality in patients presenting with ACS. Materials and MethodsPlasma levels of RANTES, Neutrophil Activating Protein-2 (NAP-2), Thrombospondin-1 (TSP-1), Von Willebrand Factor (VWF), Von Willebrand Factor Propeptide (VWF:pp) and Osteoprotegerin (OPG) were measured in a cohort study of 339 consecutive ACS patients who underwent percutaneous coronary interevention (PCI). The primary endpoint was 4-year mortality. ResultsThere were 46 deaths during the follow up. Median values of VWF (12.2μg/mL versus 7.86μg/mL, P=0.001) and VWF:pp (7.34nM versus 6.17nM, P=0.011) were higher in non-survivors compared to survivors. High levels of OPG were found in 37 patients: 27 of them were survivors (9.2%) and 10 were non-survivors (21.7%, P=0.011). Kaplan-Meier estimates of mortality for VWF were 7.5% in the first quartile (n=6 deaths), 12.2% in the second quartile (n=10 deaths), 11.2% in the third quartile (n=9 deaths) and 25% in the fourth quartile (n=21 deaths) of VWF (P=0.004). There was a 27.8% of probability of mortality when high OPG was measured versus 12.4% when low OPG was measured (P=0.007). No relationship between baseline platelet activation markers and mortality was found. ConclusionIn patients with ACS undergoing PCI, increased chronic endothelial cell activation and dysfunction is associated with an increased risk of long-term mortality.

Abstract 368: Increased Platelet Activity in HIV-Infected Subjects on Antiretroviral Therapy Is Inhibited with Low-Dose Aspirin

Introduction: HIV-infected adults are at increased risk for cardiovascular events and increased platelet activity may contribute to this risk. Hypothesis: Platelet activity measurements are increased in subjects with HIV on fully suppressive antiretroviral therapy (ART) and low-dose aspirin attenuates this increase in platelet activity. Methods: We studied platelet aggregation in 25 HIV infected subjects on ART with undetectable plasma HIV-1 RNA and median CD4 521 cells/mm 3 and 29 healthy HIV seronegative controls in response to submaximal adenosine diphosphate (ADP, 0.4uM), collagen (0.05ug/ml), epinephrine (0.1uM ), arachidonic acid (AA, 0.15mM) and without agonist (spontaneous platelet aggregation [SPA]). The effects of a 1-week course of aspirin 81mg daily on platelet activity were also investigated. Results are presented as median (interquartile range) and analyzed using the Mann-Whitney U test. Results: Compared to controls, subjects with HIV had increased platelet aggregation in response to ADP (13.5% [6.8, 81.6] vs 6.4% [2.8, 10.7], p&lt;0.01), collagen (13.5% [6.8, 81.6] vs 6.4% [2.8, 10.7], p=0.01), AA (81.9% [9.4, 90.2] vs 9.6% [2.8, 77], p&lt;0.01), and SPA, 8.2% [4.7, 13.2] vs 4.5% [2.2, 8.9], p=0.04). There was no difference in baseline aggregation in response to epinephrine. Following aspirin therapy, percent aggregation in response to AA decreased more in subjects with HIV (-43% vs -20%, P=0.04), yet remained significantly higher in subjects with HIV versus controls (9.8% [5.8, 16.6] vs 7.4 [4.4, 9.8], P=0.02). In response to ADP, collagen, epinephrine, and SPA, subjects with HIV experienced numerically greater platelet inhibition than controls and residual platelet aggregation was not significantly different between groups. Conclusions: Baseline platelet activity is increased in subjects with HIV infection virologically suppressed on ART which may contribute to the excess cardiovascular risk commonly observed in this population. Subjects with HIV experienced greater decreases in platelet activity in response to aspirin, retaining modestly more platelet activity compared to controls. Cardiovascular prevention studies of aspirin in subjects with HIV are warranted.