Paradoxical Rebound Platelet Activation After Painkillers Cessation: Missing Risk for Vascular Events?

Abstract Background Platelet activation plays a major role in the atherothrombotic complications of diabetes. Thromboxane (TX)A2 is a pro-thrombotic prostanoid, synthesized via cyclooxygenase-1 and released by activated platelets. The metabolism of TXA2 in vivo leads to a major stable end-product, 11-dehydro-TXB2 (TXM), measurable in urine and reflecting the whole-body rate of TXA2 biosynthesis. In two large trials of high-risk, aspirin-treated (mostly, without diabetes) patients, (CHARISMA and HOPE trials), the baseline rate of urinary TXM excretion was an independent predictor of future cardiovascular events. Purpose The aim of the ASCEND (A Study of Cardiovascular Events in Diabetes) TXM sub-study was to investigate the association between baseline urinary TXM and future serious vascular events or revascularization (SVE-R), major bleeds and incident cancer independent of other risk factors and treatment, in people with diabetes and no manifest cardiovascular disease at trial entry. Methods Urinary TXM was measured by a previously GC/MS-validated, immunoassay in 6,487 participants with eligible baseline samples. Analyses excluded 539 participants using NSAIDs. TXM appeared log-normally distributed, so analyses were by quintiles and per SD (=0.622) of continuous loge TXM. The association of loge TXM with outcome was adjusted by basic factors (age, sex, sample volume and randomized treatment allocation) and by the predictors of log TXM (smoking, type 2 diabetes treated with insulin or oral hypoglycaemics, HDL cholesterol, body mass index, urinary albumin/creatinine ratio, eGFR). The association of log TXM with non-vascular, non-cancer MedDRA outcomes was investigated to determine whether TXM had a general effect on outcome. During a mean of 6.6 years follow-up there were 618 SVE-Rs, 206 bleeds and 700 cancers among these patients. Results Log TXM correlated significantly with SVE-R, hazard ratio (HR) per 1 SD of log TXM: 1.13 (1.04–1.23), p=0.003 (Figure 1, panel a), non-significantly with major bleeds [HR 1.15 (1.00–1.32), p=0.055] (Figure 1, panel b), and marginally significantly with cancer [HR 1.09 (1.01–1.17), p=0.03] (Figure 1, panel c). There was no association of log TXM with non-vascular non-cancer MedDRA outcomes (HR per 1 SD, 0.99; 99% CI, 0.94–1.05). Conclusion The rate of urinary TXM excretion, a non-invasive biomarker of TXA2-mediated platelet activation in vivo, is log-linearly associated with serious vascular events independent of other risk factors in people with diabetes. Its potential association with cancer must be viewed as hypothesis-generating and needs confirmation. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): IMI1: Surrogate markers for micro- and macro-vascular hard endpoints for innovative diabetes tools (SUMMIT).

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The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029608316014 ◽

2008 ◽

Vol 14 (3) ◽

pp. 295-302 ◽

Cited By ~ 11

Author(s):

I. Anita Jagroop ◽

Dimitri P. Mikhailidis

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Shape Change ◽

Adenosine Diphosphate ◽

Vascular Events ◽

Spontaneous Platelet Aggregation ◽

Induced Aggregation ◽

Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

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Extracellular Matrix-Specific Platelet Activation Leads to a Differential Translational Response and Protein De Novo Synthesis in Human Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms21218155 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8155

Author(s):

Bjoern F. Kraemer ◽

Marc Geimer ◽

Mirita Franz-Wachtel ◽

Tobias Lamkemeyer ◽

Hanna Mannell ◽

...

Keyword(s):

Extracellular Matrix ◽

Platelet Activation ◽

De Novo ◽

Proteome Analysis ◽

Human Platelets ◽

Protein Sequencing ◽

Translation Initiation Factor ◽

Initiation Factor ◽

De Novo Synthesis ◽

Vascular Events

Platelets are exposed to extracellular matrix (ECM) proteins like collagen and laminin and to fibrinogen during acute vascular events. However, beyond hemostasis, platelets have the important capacity to migrate on ECM surfaces, but the translational response of platelets to different extracellular matrix stimuli is still not fully characterized. Using 2D-gel electrophoresis, confocal microscopy, polysome analysis and protein sequencing by mass spectrometry, we demonstrate that platelets show a differential expression profile of newly synthesized proteins on laminin, collagen or fibrinogen. In this context, we observed a characteristic, ECM-dependent translocation phenotype of translation initiation factor eIF4E to the ribosomal site. eIF4E accumulated in polysomes with increased binding of mRNA and co-localization with vinculin, leading to de novo synthesis of important cytoskeletal regulator proteins. As the first study, we included a proteome analysis of laminin-adherent platelets and interestingly identified upregulation of essentially important proteins that mediate cytoskeletal regulation and mobility in platelets, such as filamin A, talin, vinculin, gelsolin, coronin or kindlin-3. In summary, we demonstrate that platelet activation with extracellular matrix proteins results in a distinct stimulus-specific translational response of platelets that will help to improve our understanding of the regulation of platelet mobility and migration.

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Platelet bound complement split product (PC4d) is a marker of platelet activation and arterial vascular events in systemic lupus erythematosus

Clinical Immunology ◽

10.1016/j.clim.2021.108755 ◽

2021 ◽

pp. 108755

Author(s):

Yevgeniya Gartshteyn ◽

Adam Mor ◽

Daichi Shimbo ◽

Leila Khalili ◽

Teja Kapoor ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Platelet Activation ◽

Lupus Erythematosus ◽

Systemic Lupus ◽

Vascular Events ◽

Complement Split Product

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Platelets as Predictors of Vascular Risk: Is There a Practical Index of Platelet Activity?

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/107602960300900301 ◽

2003 ◽

Vol 9 (3) ◽

pp. 177-190 ◽

Cited By ~ 169

Author(s):

Stavroula Tsiara ◽

Moses Elisaf ◽

I. Anita Jagroop ◽

Dimitri P. Mikhailidis

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Vascular Risk ◽

Platelet Activity ◽

Vascular Events ◽

Bioactive Substances ◽

Platelet Surface ◽

Soluble P

Activated platelets play a role in the pathogenesis of coronary heart disease (CHD). Following activation, platelets change shape, aggregate, and release several bioactive substances. The aim of this review is to identify if there is a simple and cost-effective method that indicates platelet activation and predicts the risk of CHD and vascular events. The rationale for identifying high-risk patients is to reduce their risk of vascular events by administering appropriate and effective antiplatelet treatment, like aspirin, clopidogrel, or combination regimens. Many laboratory tests estimating platelet activity have been described. Some are relatively simple, such as spontaneous or agonist-induced platelet aggregation. Other tests include measuring the mean platelet volume (MPV) or plasma soluble P-selectin levels. Some more complex tests include flow cytometry to determine platelet GP Ilb/Illa receptors, platelet surface P-selectin, plateletmonocyte aggregates, and microparticles. Only few prospective studies assessed the predictive value of platelet activation in healthy individuals. Although the MPV seems an 'easy method, there are insufficient data supporting its ability to predict the risk of a vascular event in healthy adults. Platelet aggregation, in whole blood or in platelet-rich plasma was not consistently predictive of vascular risk. Soluble P-selectin measurement is a promising method but it needs further evaluation. Flow cytometry methods are costly, time-consuming, and need specialized equipment. Thus, they are unlikely to be useful in estimating the risk in large numbers of patients. There is as yet no ideal test for the detection of platelet activation. Each currently available test has merits and disadvantages. Simple methods such as the MPV and the determination of platelet release products need further evaluation.

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Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

Blood ◽

10.1182/blood-2018-99-118321 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1794-1794

Author(s):

Alessandro Lucchesi ◽

Silvia Carloni ◽

Martina Ghetti ◽

Serena De Matteis ◽

Gerardo Musuraca ◽

...

Keyword(s):

Blood Flow ◽

Platelet Count ◽

Platelet Activation ◽

Binding Capacity ◽

Myeloproliferative Neoplasms ◽

Positive Control ◽

Vascular Events ◽

Chronic Myeloproliferative Neoplasms ◽

Fibrinogen Receptor ◽

Microcirculatory Disorders

Abstract INTRODUCTION Patients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed. Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified. A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances. METHODS Blood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer. RESULTS Surprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04). CONCLUSION In untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. Figure. Figure. Disclosures Martinelli: Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

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Abstract 17186: In Vivo Platelet Activation in Impaired Glucose Tolerance and During Progression of Type 2 Diabetes: a Cross-sectional and Longitudinal Study

Circulation ◽

10.1161/circ.130.suppl_2.17186 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Flavia Ciaffardini ◽

Giovanni Davì ◽

Patrizia Di Fulvio ◽

Gloria Formoso ◽

Rossella Liani ◽

...

Keyword(s):

Type 2 Diabetes ◽

Glucose Tolerance ◽

Impaired Glucose Tolerance ◽

Platelet Activation ◽

Circadian Variation ◽

Study Entry ◽

Vascular Events ◽

Over Time

Introduction: Impaired glucose tolerance (IGT) reflects the progression from normoglycemia to type 2 diabetes mellitus (DM). IGT and DM are associated with high cardiovascular disease (CVD) morbidity and mortality. Hypothesis: Enhanced platelet activation has been previously reported in DM, but whether this pertains to IGT is unknown. Methods: We investigated in vivo platelet activation, as reflected by the urinary excretion of 11-dehydro-TXB2 (TXM), in IGT and DM patients with a diagnosis documented either <12 months (new-DM) or ≥12 months (established-DM) from study entry. Patients were studied repeatedly up to 46 months to assess changes in platelet activation over time. Some patients were given standard 500 kcal meals at 8am, 1 and 6pm and TXM was measured repeatedly during a 12-hr period. Results: We enrolled 49 IGT patients (19M, age 56±9 yrs), 59 new-DM (36M, 61±10 yrs) and 61 established-DM (34M, 62±7 yrs, DM duration 9.7±6.1 yrs) patients diagnosed according to the ADA criteria, not on antiplatelet drugs. Median TXM values at study entry were comparably enhanced in the 3 groups (1,189 [897-1,503], 1,277 [897-1,870], and 1,217 [885-1,494] pg/mg creatinine in IGT, new- and established-DM, respectively; p=0.39). Urinary TXM excretion showed limited intra-subject variability over time, as shown in the Figure. There was no convincing evidence of a circadian variation in TXM excretion. Two-hour, post-meal glucose variations (8-10am or 1-3pm) did not affect subsequent TXM values. Conclusions: We conclude that the potential benefits and risks of antiplatelet therapy should be considered in IGT management, because persistent platelet activation might contribute to CVD progression and its thrombotic complications. Given the stability of high TXM excretion in DM, this non-invasive biomarker of platelet activation should be tested as a predictor of vascular events during long-term follow-up.

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Increased Thromboxane in Patients with Polycythemia Vera and Thrombosis: Evidence for Aspirin-Unsuppressible Platelet Activation In Vivo

Blood ◽

10.1182/blood-2019-122828 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5372-5372

Author(s):

Rossella Rosari Cacciola ◽

Elio Gentilini Cacciola ◽

Veronica Vecchio ◽

Emma Cacciola

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Vascular Events ◽

Platelet Factor ◽

Thrombotic Risk ◽

Closure Time ◽

The Mean

Polycyhemia vera (PV) is a myeloproliferative neoplasm characterized by increased thromboxane (TX) production and thrombotic risk. It is reported that serum TXB2 concentrations in PV patients are twofold higher than healthy controls and that low-dose aspirin (ASA) therapy reduces the risk of major vascular events by 50 to 60%. To evaluate this unusual size of the effect of ASA we have studied platelet count, hematocrit (HCT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4), as markers of platelet activation, TXB2, as primary indicator of platelet activation, the platelet function activity (PFA), as indicator of ASA platelet sensitivity, and the clotting time (CT), as parameter of thrombin formation. We studied 60 patients (38 men, 22 women; mean age 51 years, range 32-70) with PV according to WHO criteria. The mean duration of disease was 12 years. All patients were on ASA 100 mg once daily. All patients were on phlebotomy. None had inherited or acquired thrombotic risk factors. Of 60 patients, 30 had thrombosis (20 men, 10 women) and 30 had no thrombosis. Of 30 with thrombosis, 15 developed nonfatal myocardial infarction (10 men, 5 women) defined by chest pain of typical intensity and duration and ST-segment elevation in any limb lead on electrocardiography, 10 had nonfatal stroke (8 men, 2 women) confirmed with the use of magnetic resonance imaging, and 5 (2 men , 3 women) had deep venous thrombosis confirmed by ultrasonography. Platelet count and HCT were measured by automated analyzer. β-TG and PF4 were determined by ELISA. TXB2 was measured by radioimmunoassay technique. ASA platelet sensitivity was measured by Platelet Function Analyzer (PFA-100). CT was measured by thromboelastometry. The mean platelet count was 430±170x109/L. The mean HCT value was 42±3%. The patients with thrombosis had high β-TG, PF4 and TXB2 (110±45 IU/ml, 45±21 IU/ml, and 1.700±1.990 nmol/L, respectively), shortened C/EPI closure time (T, unit: s, n.v. 84-160 s) (55±10 s) and shortened CT (CT, unit: s. n.v. 100-240 s) (45±20 s) whereas the patients without thrombosis had normal β-TG, PF4 and TXB2 (20±11 IU/ml, 6±2 IU/ml, and 800±280 nmpl/L, respectively), prolonged C/EPI closure time (249±40 s) and normal CT (110±20 s). These findings might suggest that in PV patients and thrombotic complications might need a platelet-selective dosage of ASA. Disclosures No relevant conflicts of interest to declare.

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