Abstract 600: Novel Assays to Determine in vivo Platelet Activation

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Iberia R Sosa ◽  
Irene Zagol-Ikapitte ◽  
Manju Bala ◽  
Olivier Boutaud ◽  
John Oates
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Internal Standard ◽  
Antiplatelet Agents ◽  
High Rate ◽  
Thrombotic Risk ◽  
Thromboxane Synthase ◽  
Coronary Syndrome ◽  
Increased Risk

In vivo platelet activation is associated with several pathologic entities, including acute coronary syndrome, essential thrombocythemia, anti-phospholipid antibody syndrome, diabetes and metabolic syndrome. The need for more robust biomarkers with which to assess platelet activation in vivo in human diseases is well recognized. Our lab has developed two novel assays to study in vivo platelet activation and rate of turnover. During platelet activation, malondialdehyde (MDA) is produced by the thromboxane synthase in amounts equimolar to thromboxane A2 and also non-enzymatically by lipid peroxidation resulting from the oxidative processes that accompany activation. MDA is a reactive dicarbonyl that reacts with amines, notably lysines on proteins, yielding covalent modifications of the proteins that then accumulate over the lifetime of the platelet. We developed an LC/MS/MS method for quantification of the most stable of three MDA adducts, the dilysyl-MDA crosslink, employing a [13C12] labeled internal standard. We found that activation of platelets with arachidonic acid leads to an increase in the levels of dilysyl-MDA crosslinks in platelets, which is inhibited by the thromboxane synthase inhibitor, ozagrel, by the cyclooxygenase inhibitor, aspirin, and by scavengers of reactive carbonyls, 3- methoxysalicylamine and Salicylamine. High platelet turnover has been associated with increased risk for thrombosis and failure of antiplatelet agents. We propose a novel approach to studying platelet turnover by labeling platelets in vivo by oral administration of aspirin containing a deuterium labeled acetyl group (d3-aspirin). We can measure the clearance of d3-labeled platelets by using LC/MS/MS to measure the tryptic peptide (SLK) of COX-1 labeled with d3. This approach avoids exposure to radioactivity and the artifact resulting from manipulation of platelets labeled ex vivo. The results of this study provide two novel assays with the potential to serve as markers of in vivo platelet activation and turnover, which may be useful in predicting thrombotic risk and efficacy of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity and high rate of platelet turnover.

Quantification Of Malondialdehyde Adducts In Platelet Activation As An Indicator Of Proinflammatory and Prothombotic State

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4735-4735
Author(s):  
Irene Zagol-Ikapite ◽  
Iberia Romina Sosa ◽  
Audra M Judd ◽  
Olivier Boutaud ◽  
John A Oates
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Primary Amines ◽  
Specific Marker ◽  
Medical Conditions ◽  
Thrombotic Risk ◽  
Thromboxane Synthase ◽  
Reticulated Platelets ◽  
Platelet Proteins

Background The formation of malondialdehyde (MDA) has been previously described as a product of the thromboxane synthase. However, the reported approaches for its quantification have not been reliable, stymieing its use in research. As a reactive di-carbonyl, MDA reacts with primary amines, notably lysines on proteins, to form covalent adducts of several types. Three of the products of the reaction of MDA with lysine are an N-propenal adduct, a dihydropyridine ring adduct (N-lysyl-4-methyl-2, 6-dihydropyridine-3, 5-dicarbaldehyde), and a lysyl-MDA crosslink. Measurement of platelet protein modifications, such as MDA adducts, could provide a specific marker of in vivo activation of platelets, since these modifications accumulate over the lifespan of the platelet. Methods and Results To investigate thromboxane synthase-dependent formation of MDA adducts on platelet proteins, we developed an LC/MS/MS method for analysis of one of the MDA adducts, the lysyl-MDA crosslink, employing a [13C12] labeled internal standard. We demonstrated that levels of lysyl-MDA crosslink in human platelets are increased following its activation with arachidonic acid. This increase is inhibited by aspirin, the thromboxane synthase inhibitor, ozagrel and by γ-ketoaldehyde specific scavengers: 3-Methoxysalicylamine (3-MOSA) and Salicylamine (SA). To determine whether lysyl-MDA crosslinks reflect in vivo platelet activation, we analyzed samples from patients with medical conditions known to be associated with increased platelet activation. We employed traditional methods of measuring platelet activation: flow cytometry of p-selectin and reticulated platelets, and serum thromboxane, to measure platelet activation in patients with metabolic syndrome and sickle cell disease. These assays were compared with the levels of lysyl-MDA-crosslinks. In both populations, the levels of MDA-lysine-crosslink are increased by 2.5 fold compared to healthy volunteers and provide greater discrimination between groups than p-selectin expression and reticulated platelets. The inhibition of the lysyl-MDA crosslink adduct in patients taking NSAIDs further confirms the specificity for thromboxane synthase-dependent MDA modifications on platelet proteins. Discussion The results of this study provide compelling evidence that MDA-protein adducts in platelets may be a useful marker of in vivo platelet activation in humans and potentially helpful in predicting thrombotic risk and the benefit of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interrelationships between structure and function during the hemostatic response to injury

2019 ◽  
Vol 116 (6) ◽  
pp. 2243-2252 ◽  
Author(s):  
Maurizio Tomaiuolo ◽  
Chelsea N. Matzko ◽  
Izmarie Poventud-Fuentes ◽  
John W. Weisel ◽  
Lawrence F. Brass ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Antiplatelet Agents ◽  
Coagulation Factors ◽  
Jugular Veins ◽  
Spatial Regulation ◽  
Injury Model ◽  
And Function ◽  
Damaged Vessel

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y12signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.

Increased Thromboxane Biosynthesis in Essential Thrombocythemia

1995 ◽  
Vol 74 (05) ◽  
pp. 1225-1230 ◽  
Author(s):  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Raffaele Tartaglione ◽  
Sergio Cortelazzo ◽  
Tiziano Barbui ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Essential Thrombocythemia ◽  
Therapeutic Strategy ◽  
Low Dose ◽  
Thrombotic Risk ◽  
Dose Aspirin ◽  
Gender And Age ◽  
Low Dose Aspirin

SummaryIn order to investigate the in vivo thromboxane (TX) biosynthesis in essential thromboeythemia (ET), we measured the urinary exeretion of the major enzymatic metabolites of TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 40 ET patients as well as in 26 gender- and age-matched controls. Urinary 11-dehydro-TXB2 was significantly higher (p <0.001) in thrombocythemic patients (4,063 ± 3,408 pg/mg creatinine; mean ± SD) than in controls (504 ± 267 pg/mg creatinine), with 34 patients (85%) having 11-dehydro-TXB2 >2 SD above the control mean. Patients with platelet number <1,000 × 109/1 (n = 25) had significantly higher (p <0.05) 11 -dehydro-TXB2 excretion than patients with higher platelet count (4,765 ± 3,870 pg/mg creatinine, n = 25, versus 2,279 ± 1,874 pg/mg creatinine, n = 15). Average excretion values of patients aging >55 was significantly higher than in the younger group (4,784 ± 3,948 pg/mg creatinine, n = 24, versus 2,405 ± 1,885 pg/mg creatinine, n = 16, p <0.05). Low-dose aspirin (50 mg/d for 7 days) largely suppressed 11-dehydro-TXB2 excretion in 7 thrombocythemic patients, thus suggesting that platelets were the main source of enhanced TXA2 biosynthesis. The platelet count-corrected 11-dehydro-TXB2 excretion was positively correlated with age (r = 0.325, n = 40, p <0.05) and inversely correlated with platelet count (r = -0.381, n = 40, p <0.05). In addition 11 out of 13 (85%) patients having increased count-corrected 11-dehydro-TXB2 excretion, belonged to the subgroup with age >55 and platelet count <1,000 × 1099/1. We conclude that in essential thrombocythemia: 1) enhanced 11-dehydro-TXB2 excretion largely reflects platelet activation in vivo;2) age as well as platelet count appear to influence the determinants of platelet activation in this setting, and can help in assessing the thrombotic risk and therapeutic strategy in individual patients.

scholarly journals Impact of Diabetes Mellitus on Antithrombotic Management Patterns and Long‐Term Clinical Outcomes in Patients With Acute Coronary Syndrome: Insights From the EPICOR Asia Study

2020 ◽  
Vol 9 (22) ◽  
Author(s):  
Shaoyi Guan ◽  
Xiaoming Xu ◽  
Yi Li ◽  
Jing Li ◽  
Mingzi Guan ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Acute Coronary Syndrome ◽  
Antiplatelet Therapy ◽  
Dual Antiplatelet Therapy ◽  
Antiplatelet Agents ◽  
Coronary Syndrome ◽  
Increased Risk ◽  
Antithrombotic Management

Background Long‐term use of antiplatelet agents after acute coronary syndrome in diabetic patients is not well known. Here, we describe antiplatelet use and outcomes in such patients enrolled in the EPICOR Asia (Long‐Term Follow‐up of Antithrombotic Management Patterns in Acute Coronary Syndrome Patients in Asia) registry. Methods and Results EPICOR Asia is a prospective, observational study of 12 922 patients with acute coronary syndrome surviving to discharge, from 8 countries/regions in Asia. The present analysis included 3162 patients with diabetes mellitus (DM) and 9602 patients without DM. The impact of DM on use of antiplatelet agents and events (composite of death, myocardial infarction, and stroke, with or without any revascularization; individual components, and bleeding) was evaluated. Significant baseline differences were seen between patients with DM and patients without DM for age, sex, body mass index, cardiovascular history, angiographic findings, and use of percutaneous coronary intervention. At discharge, ≈90% of patients in each group received dual antiplatelet therapy. At 2‐year follow‐up, more patients with DM tended to still receive dual antiplatelet therapy (60% versus 56%). DM was associated with increased risk from ischemic but not major bleeding events. Independent predictors of the composite end point of death, myocardial infarction, and stroke in patients with DM were age ≥65 years and use of diuretics at discharge. Conclusions Antiplatelet agent use is broadly comparable in patients with DM and patients without DM, although patients with DM are more likely to be on dual antiplatelet therapy at 2 years. Patients with DM are at increased risk of ischemic events, suggesting an unmet need for improved antithrombotic treatment. Registration URL: https://www.clini​caltr​ials.gov ; Unique identifier: NCT01361386.

Understanding and Evaluating Platelet Function

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 387-396 ◽  
Author(s):  
Lawrence Brass
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Tumor Angiogenesis ◽  
Clinical Setting ◽  
Molecular Level ◽  
Current Knowledge ◽  
Antiplatelet Agents ◽  
Normal Hemostasis ◽  
The Impact

Abstract The contribution of platelets to normal hemostasis and vascular disease is well described. However, recent studies make it clear that much remains to be learned about platelet activation at the single cell and the molecular level, and about the contribution of platelets to inflammation, tumor angiogenesis, and embryonic development. This article is divided into two themes. The first is an overview of current knowledge of the mechanisms that drive platelet function in vivo and a brief summary of some of the emerging ideas that are modifying older views. The second theme is a consideration of the strengths and weaknesses of the tools we have as hematologists to assess platelet function in the clinical setting, identify mechanisms, and evaluate the impact of antiplatelet agents.

Developments in Oral Antiplatelet Agents for the Treatment of Acute Coronary Syndromes

2015 ◽  
Vol 29 (3) ◽  
pp. 239-249 ◽  
Author(s):  
David S. Roffman
Keyword(s):  
Clinical Trials ◽  
Major Bleeding ◽  
Antiplatelet Agent ◽  
Antiplatelet Agents ◽  
Clinical History ◽  
Bleeding Risk ◽  
Phase Iii ◽  
Coronary Syndrome ◽  
Phase Iii Clinical Trials ◽  
Increased Risk

A review of the literature was conducted for clinical trials evaluating the antiplatelet P2Y12 receptor antagonists, clopidogrel, prasugrel, and ticagrelor, as well as the guidelines for the management of acute coronary syndrome (ACS) or myocardial infarction. Clinical guidelines recommend that patients with ACS be treated with dual oral antiplatelet therapy of aspirin plus clopidogrel, prasugrel, or ticagrelor. The selection of an appropriate antiplatelet agent depends on the treatment approach and a patient’s bleeding risk and clinical history. With respect to antiplatelet activity, prasugrel and ticagrelor demonstrate greater potency and less interpatient variability than clopidogrel. In phase III clinical trials, prasugrel and ticagrelor reduced the incidence of ischemic events in patients with ACS compared with clopidogrel. Ticagrelor and clopidogrel were associated with a similar risk of major bleeding, whereas patients receiving prasugrel had an increased risk of major bleeding versus those receiving clopidogrel. Pharmacists can provide guidance on the appropriate use of antiplatelet agents as well as the use of concomitant medications, while being vigilant for any potential drug interactions.

Platelet and Coagulation Activation in Myeloproliferative Diseases (MPD), and Relationship to JAK2(V617F) Mutation Status and Clonality.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4082-4082
Author(s):  
Beverley J. Robertson ◽  
Colin Urquhart ◽  
Isobel Ford ◽  
Henry G. Watson ◽  
Mark Vickers ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Significant Proportion ◽  
Jak2 V617f ◽  
Coagulation Activation ◽  
Jak2 Mutation ◽  
Activation Markers ◽  
Thrombotic Risk ◽  
Mutation Status ◽  
Increased Risk ◽  
Significant Difference

Abstract Patients with MPD have an increased risk of thrombosis. Previous reports suggest an association between clonality and thrombosis in ET. The contribution of the JAK2 mutation to thrombotic risk is unclear. In our cohort of patients with MPD (n=122) (PRV n = 54, ET n = 64, IMF n= 4), we compared coagulation and platelet activation markers (D-Dimers, thrombin antithrombin complexes (TAT), prothrombin fragments 1+2 (F1+2), soluble E-selectin(sE-selectin) and soluble P-selectin(sP-selectin)) between MPD patients and hypertensive controls. sP-selectin was significantly increased in patients with MPD (p=<0.001). The JAK2 mutation status was determined in our cohort by ARMS PCR. Of the total MPD cohort, 59% were JAK2 positive, (76% of PRV, 45% of ET, and 50% of IMF). Coagulation activation markers were compared in JAK2 positive and JAK2 negative patients. sP-selectin levels were highly significantly elevated in JAK2 positive patients compared to JAK2 negative (p= 0.002), or controls (p<0.001). There was no significant difference in platelet count between JAK2 positive and negative patients (p=0.19). The clonality of the MPD was determined in 54 female patients using an x-chromosome inactivation pattern (XCIP) assay (HUMARA). A significant proportion of “polyclonal” patients, as defined by XCIPs were positive for the JAK2 mutation. We therefore calculated the proportion clonality of samples and found no correlation between proportion clonality and any coagulation or platelet activation marker. Our results show an increase in platelet activation, as determined by sP-selectin levels, in patients with MPD compared to controls. Furthermore, platelet activation was a feature of patients positive for the JAK2 mutation when compared with wild type patients and controls. The role of the JAK2 mutation as a risk factor for thrombosis in MPD is still unclear but our study indicates that the presence of the mutation may be linked to platelet activation in MPD. Whether this translates into a higher clinical thrombosis risk requires further evaluation in a large prospective study.

scholarly journals In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4066-4072 ◽  
Author(s):  
Bethan Psaila ◽  
James B. Bussel ◽  
Matthew D. Linden ◽  
Bracken Babula ◽  
Youfu Li ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Immune Thrombocytopenia ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Adenosine Diphosphate ◽  
High Dose ◽  
Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

Abstract 57: Platelet Extracellular-signal-regulated Kinase 5 is a Redox Switch which Regulates Myocardial Infarct Expansion via Matrix Metalloproteinases

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Scott J Cameron ◽  
Sara K Ture ◽  
Deanne Mickelsen ◽  
Enakshi Chakrabarti ◽  
Kristina L Modjeski ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Ex Vivo ◽  
Myocardial Infarct ◽  
Left Ventricular ◽  
Fluorescent Imaging ◽  
Lv Function ◽  
Coronary Artery Ligation ◽  
Dependent Manner ◽  
Platelet Receptor

Background: Dysregulated platelet activation in an ischemic microvascular environment may play a role in myocardial infarction (MI). Platelet receptor signaling is well-characterized, but mechanisms of receptor-independent activation, such as by reactive oxygen species (ROS) generated in ischemic conditions, are less well understood. We discovered that ERK5, a nuclear protein which is ROS-activated in others cells, is abundantly present in platelets. We investigated whether ERK5 could regulate platelet activation and thrombosis in healthy and diseased states. Methods: Human and mouse platelets were stimulated with agonists including ADP, U46619, TRAP, convulxin, or ROS (H 2 O 2 or 5% O 2 ). ERK5 activity was assessed by immunoblotting. Platelet activation was assessed via fluorescent-activated cell sorting (FACS) for P-selectin or activated GPIIb/IIIa. Intravascular thrombus (pulmonary embolus) or mesenteric thrombus (oxidative injury) formation was assessed by ex vivo fluorescent imaging and in vivo intravital microscopy, respectively. MI was performed in wild-type (WT) and in platelet specific ERK5 deficient (ERK5 -/- ) mice by LAD coronary artery ligation. Left ventricular (LV) function was determined by echocardiography. Matrix metalloproteinase (MMP) activity was determined by in-gel zymography. Results: Human and platelet ERK5 was activated by ROS and via the thrombin and thromboxane receptors, but not via the purinergic or collagen receptors. Murine in vivo thrombosis was regulated by platelet ERK5 only if the injury involved oxidative stress. MI in mice promoted sustained platelet activation over one week in an ERK5-dependent manner. Following MI, platelet ERK5 -/- mice had less reactive platelets, less platelet MMP activity and thromboxane production, attenuated MMP activity in the LV, less remodeling with smaller infarcts, and enhanced myocardial systolic performance. Conclusions: ERK5 is an ischemic sensor in platelets which regulates ongoing platelet activation after MI as well as remodeling via myocardial microvasculature. These observations may explain ischemic microvascular aberrations like the no-reflow phenomenon following percutaneous coronary intervention, suggesting a novel pharmacologic target.

Abstract 17216: Intracoronary Dual-modal OCT/NIRF Structural-Molecular Imaging with a Clinical Dose of Indocyanine Green (ICG) for Detection of High-risk Coronary Plaques in Diabetic Swine Model

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sunwon Kim ◽  
Min Woo Lee ◽  
Han Saem Cho ◽  
Joon Woo Song ◽  
Sunki Lee ◽  
...  
Keyword(s):  
High Risk ◽  
Near Infrared ◽  
Ex Vivo ◽  
Swine Model ◽  
Fully Integrated ◽  
Coronary Syndrome ◽  
Fibrous Cap ◽  
Nirf Imaging

Background: Acute coronary syndrome is frequently caused by rupture of macrophage abundant plaques with a large lipid-rich core. The present study aimed to investigate whether a fully integrated OCT/NIRF imaging combined with a clinically available near-infrared fluorescence (NIRF) enhancing ICG can detect the inflamed, lipid-rich plaques in swine coronary atheromata whose phenotype is similar to human vulnerable fibroatheroma. Methods and Results: Accelerated atherosclerosis was made by coronary balloon denudation in alloxan induced diabetic minipigs. A rapid coronary imaging (20 mm/sec pullback speed) using a fully integrated OCT/NIRF catheter was safely performed 30 minutes after I.V. injection of ICG (2.0 mg/kg) just under contrast purge. OCT clearly identified the lipid-rich plaques with fibrous cap. Simultaneously acquired, distance-calibrated NIRF imaging detected lipid-laden macrophage signals in OCT-proven plaques (figure). The in vivo plaque target-to-background ratio (pTBR) was significantly higher in ICG-injected swine compared to non-diabetic swines or saline-injected controls (p<0.05), which was validated on ex vivo fluorescence reflectance imaging (FRI) (figure). The in vivo and ex vivo peak pTBRs correlated significantly (p<0.05). In vitro experiments, and histopathology including fluorescence microscopic imaging and immunostaining of the plaque sections corroborated the findings in vivo . Conlusions: An OCT/NIRF imaging with a clinical use of ICG accurately identified macrophage abundant, lipid-rich coronary plaques in diabetic atheromatous minipigs. This highly translatable dual-modal molecular-structural imaging could be relevant for clinical intracoronary detection of high-risk plaques.

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