Immature platelets and platelet reactivity in patients with acute ST-segment Elevation Myocardial Infarction using whole blood flow cytometry with SYTO-13 staining.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets. OBJECTIVES: To investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients. METHODS: We conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B2 levels and standard immature platelet markers. RESULTS: Immature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05). CONCLUSIONS: Our study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy.

Platelet Function and Maturity and Related microRNA Expression in Whole Blood in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). MicroRNAs (miRs) may influence platelet function and maturity, and subsequently the effect of antiplatelet therapy. OBJECTIVES: We aimed to explore the association between miR expression and platelet function and maturity in patients with acute STEMI and healthy individuals. METHODS: We performed an observational study of STEMI patients admitted directly to primary percutaneous coronary intervention. Patients were treated with antiplatelet therapy according to guidelines. Within 24 hours after admission, blood samples were obtained to measure: the expression of 10 candidate miRs, platelet function markers using advanced flow cytometry, platelet aggregation, serum thromboxane B2, and platelet maturity markers. Furthermore, blood samples from healthy individuals were obtained to determine the normal variation. RESULTS: In total, 61 STEMI patients and 50 healthy individuals were included. STEMI patients had higher expression of miR-21-5p, miR-26b-5p, and miR-223-3p and lower expression of miR-150-5p, miR423-5p, and miR-1180-3p than healthy individuals. In STEMI patients, the expression of miR-26b-5p showed the most consistent association with platelet function (all p-values <0.05, Spearman's rho ranging from 0.27 to 0.41), while the expression of miR-150-5p and miR-223-3p showed negative associations with platelet function. No association between miR expression and platelet maturity markers was observed. CONCLUSION: In patients with STEMI, the expression of six miRs was significantly different from healthy individuals. The expression of miR-26b-5p may affect platelet function in acute STEMI patients and potentially influence the effect of antiplatelet therapy.

Immature platelets and cardiovascular events in patients with stable coronary artery disease.

Many patients with coronary artery disease (CAD) have reduced the effect of aspirin, which may partly be explained by immature platelets. We aimed to investigate whether immature platelet markers can predict cardiovascular events in a large cohort of stable CAD patients. A total of 900 stable CAD patients were included and followed for a median of 3 years. We measured markers of immature platelets (platelet count, immature platelet count, immature platelet fraction, mean platelet volume, platelet distribution width, platelet mass, and thrombopoietin) using automated flow cytometry and studied their relation to cardiovascular events. Our primary endpoint was a composite of acute myocardial infarction (MI), ischemic stroke, and cardiovascular death. A composite of MI, ischemic stroke, stent thrombosis and all-cause mortality was analyzed as a secondary endpoint. We found no difference in immature platelet markers between CAD patients with or without cardiovascular events. Regression analysis using hazards rates showed that markers of immature platelets did not have any predictive value for endpoints (p-values >.05). Markers of immature platelets did not predict future cardiovascular events during a 3-year follow-up period in CAD patients. This suggests that immature platelets measured in a stable phase does not have a major role in predicting future cardiovascular events.

What is the context? Many patients with coronary artery disease (CAD) have reduced antiplatelet effect of aspirinThe reduced antiplatelet effect of aspirin is most likely multifactorial and may partly be explained by immature plateletsWhat is new? In a cohort of 900 stable CAD patients, we measured markers of immature platelets and studied their relation to cardiovascular events during a 3-year follow-upOur study demonstrated that markers of immature platelets did not predict cardiovascular events in our cohortWhat is the impact? The findings from the present study suggest that immature platelets, measured in a stable phase, do not have a major role in predicting future cardiovascular events in CAD patients.

Effect of regular exercise training on platelet function in patients with cardiovascular disease and healthy individuals: A systematic review.

INTRODUCTION: Regular exercise training is essential in prevention and treatment of cardiovascular disease (CVD), yet the beneficial effects of exercise remain only partly explained. Platelets play a key role in CVD and may be affected by regular exercise training. We aimed to systematically summarise studies investigating the effect of regular exercise training on platelet function in patients with CVD and in healthy individuals. METHODS: Studies were identified by PubMed, Embase and Web of Science May 16, 2022. We selected studies investigating markers of platelet function in relation to regular exercise training in patients with CVD and in healthy individuals. Regular exercise was defined as exercise training for four weeks or more. RESULTS: Of the included studies, 11 investigated patients with CVD and 29 were on healthy individuals. Studies were heterogeneous regarding design, study population and methodology, and the results were ambiguous. In total, 52 different markers of platelet function were investigated with platelet aggregation, soluble P-selectin, and thromboxane B2 (TXB2) as the most frequently examined. When evaluating between-group changes after regular exercise, two studies found a reduced platelet aggregation in the exercise group whilst three studies did not find a difference between groups. With respect to TXB2, three studies reported a reduction and two studies an increase in the exercise group. There were no between-group differences in the seven studies examining soluble P-selectin. CONCLUSION: Regular exercise training has no clear impact on platelet function in patients with CVD or healthy individuals. PROSPERO REGISTRATION: CRD42022350539.

Expression of microRNA Predicts Cardiovascular Events in Patients with Stable Coronary Artery Disease.

BACKGROUND: New biomarkers are warranted to identify patients with coronary artery disease (CAD) at high risk of recurrent cardiovascular events. It has been reported that the expression of microRNAs (miRs) may influence the development of CAD. OBJECTIVES: We aimed to investigate whether the expression of selected candidate miRs is a predictor of cardiovascular events in a cohort of stable CAD patients. METHODS: We performed a single-center prospective study of 749 stable CAD patients with a median follow-up of 2.8 years. We investigated the expression of nine candidate miRs and their relation to cardiovascular events in this cohort. The primary endpoint was the composite of nonfatal myocardial infarction (MI), stent thrombosis (ST), ischemic stroke, and cardiovascular death. The composite of nonfatal MI and ST was analyzed as a secondary endpoint. Furthermore, nonfatal MI, ST, ischemic stroke, and all-cause mortality were analyzed as individual endpoints. RESULTS: Employing receiver operating characteristic curves, it was shown that compared with traditional cardiovascular risk factors alone, combining the expression of miR-223-3p with existing traditional cardiovascular risk factors increased the predictive value of ST (area under the curve: 0.88 vs. 0.77, p = 0.04), the primary composite endpoint (0.65 vs. 0.61, p = 0.049), and the secondary endpoint of the composite of nonfatal MI and ST (0.68 vs. 0.62, p = 0.04). CONCLUSION: Among patients with CAD, adding miR-223-3p expression to traditional cardiovascular risk factors may improve prediction of cardiovascular events, particularly ST. Clinical trials confirming these findings are warranted.

Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood.

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.

Cytoreductive treatment and association with platelet function and maturity in patients with essential thrombocythaemia.

Patients with essential thrombocythaemia (ET) have an increased risk of thromboembolic events, which may differ according to different cytoreductive drugs. We investigated the effect of cytoreductive treatment on platelet function and turnover in ET patients. Blood samples were obtained at 1 and 24 h after aspirin intake. Platelet function was evaluated by platelet aggregation and flow cytometry. Platelet turnover was assessed by immature platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV). A total of 47 ET patients were included and grouped into 21 patients not receiving cytoreductive treatment, 15 patients receiving hydroxycarbamide and 11 patients receiving pegylated interferon alpha (peg-IFN). Patients receiving peg-IFN had significantly higher IPF and MPV than the other ET groups. Patients not receiving cytoreductive treatment had significantly higher platelet aggregation 24 h after aspirin intake than the other ET groups (p-values from 0.03 to 0.0002). Patients receiving hydroxycarbamide had significantly higher expression of platelet granule makers, P-selectin and CD63, than patients receiving peg-IFN (p-values ≤0.003). Cytoreduction provides more consistent platelet inhibition compared with no cytoreductive treatment. Moreover, peg-IFN provides superior inhibition of platelet activation markers than hydroxycarbamide, which in part may explain differences in risk of thromboembolic events in ET patients.

Flow Cytometric Assessment of Changes in Platelet Reactivity after Acute Coronary Syndrome: A Systematic Review.

Increased platelet activity is an important predictor for recurrent cardiovascular events in patients with acute coronary syndromes (ACS). Flow cytometry is an advanced method for evaluation of platelet activity. We aimed to summarize the current literature on dynamic changes in platelet activity analyzed by flow cytometry in patients with ACS. Employing the guidelines of Preferred Report Items for Systematic Reviews and Meta-Analyses (PRISMA), we searched PubMed and Embase on October 26, 2021, and identified studies measuring platelet activity with flow cytometry in ACS patients in the acute phase (baseline) and at follow-up in a more stable phase. In the 12 included studies, fibrinogen receptor, α-granule secretion, platelet reactivity index, monocyte-platelet aggregates, neutrophil-platelet aggregates, and reticulated platelets were measured. The fibrinogen receptor and α-granule secretion were either unchanged or lower during follow-up measurements than in the acute phase. Platelet reactivity index showed inconsistent results. Values of monocyte-platelet aggregates and neutrophil-platelet aggregates were lower at follow-up than at baseline (p-values <0.05). Reticulated platelets were either unchanged (p-value >0.64) or lower at 1 to 2 months follow-up (p-value 0.04), and also lower at 5 months to 1-year follow-up (p-value >0.005) compared with baseline. Overall, flow cytometric analyses of platelet function in ACS patients showed that platelet activity was lower at follow-up than at baseline. However, in some patients, platelet activity remained unchanged from baseline to follow-up, possibly indicating a sustained high platelet activity that may increase the risk of recurrent cardiovascular events.

Association of whole blood microRNA expression with platelet function and turnover in patients with coronary artery disease.

INTRODUCTION: The risk of recurrent cardiovascular events in patients with coronary artery disease (CAD) is determined by multiple factors including platelet function and turnover. MicroRNAs (miRs) may regulate both platelet function and turnover. We aimed to identify candidate miRs associating with platelet function and turnover in a cohort of stable CAD patients. Furthermore, we retrieved information on binding targets of the candidate miRs to obtain a more comprehensive biological insight into miR regulation of platelet function and turnover. METHODS: Based on existing literature and a pilot study, we identified nine candidate miRs. Subsequently, we investigated the expression of the candidate miRs in whole blood and their relation to platelet function and turnover in 749 CAD patients. Platelet function was analysed using impedance aggregometry, optical aggregometry and serum thromboxane B2 measurements. Platelet turnover markers (immature platelet count, immature platelet fraction and mean platelet volume) were measured using monochromatic automated flow cytometry. RESULTS: Expression of miR-93-5p, miR-126-3p, miR-150-5p, miR-423-3p and miR-1180-3p showed negative correlations with platelet function (p-values from <0.0001 to 0.0006, rho from -0.13 to -0.36). In addition, expression of miR-423-3p showed negative correlation with platelet turnover markers (p-values from 0.001 to 0.004, rho from -0.11 to -0.12). CONCLUSIONS: We identified several novel miRs that may regulate platelet function and turnover, thereby contributing to the increased risk of recurrent cardiovascular events in CAD patients.

MicroRNA as Biomarkers for Platelet Function and Maturity in Patients with Cardiovascular Disease.

Patients with cardiovascular disease (CVD) are at increased risk of suffering myocardial infarction. Platelets are key players in thrombus formation and, therefore, antiplatelet therapy is crucial in the treatment and prevention of CVD. MicroRNAs (miRs) may hold the potential as biomarkers for platelet function and maturity. This systematic review was conducted using the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). To identify studies investigating the association between miRs and platelet function and maturity in patients with CVD, PubMed and Embase were searched on October 13 and December 13, 2020 without time boundaries. Risk of bias was evaluated using a standardized quality assessment tool. Of the 16 included studies, 6 studies were rated "good" and 10 studies were rated "fair." In total, 45 miRs correlated significantly with platelet function or maturity (rho ranging from -0.68 to 0.38, all p < 0.05) or differed significantly between patients with high platelet reactivity and patients with low platelet reactivity (p-values ranging from 0.0001 to 0.05). Only four miRs were investigated in more than two studies, namely miR-223, miR-126, miR-21 and miR-150. Only one study reported on the association between miRs and platelet maturity. In conclusion, a total of 45 miRs were associated with platelet function or maturity in patients with CVD, with miR-223 and miR-126 being the most frequently investigated. However, the majority of the miRs were only investigated in one study. More data are needed on the potential use of miRs as biomarkers for platelet function and maturity in CVD patients.

A 19-Year-Old Man with a History of Recreational Inhalation of Nitrous Oxide with Severe Peripheral Neuropathy and Central Pulmonary Embolism.

BACKGROUND Recreational use of nitrous oxide (laughing gas) is a growing phenomenon among young people due to easy accessibility and a presumed innocent effect. However, complications have been reported, especially following high and long-term use, including nerve damage, spontaneous pneumo-mediastinum, myocardial infarction, and macrocytic anemia. CASE REPORT We report a case of a 19-year-old previously healthy man with occasional recreational use of nitrous oxide of up to 10 times within recent months, who presented with severe peripheral neuropathy. Laboratory examination revealed severely elevated homocysteine values of 92 µmol/L (reference range, <10 µmol/L), strongly elevated methylmalonic acid level of >10 µmol/L (range, 0.1-0.4 µmol/L), vitamin B12 level of 234 pmol/L (range, 200-600 pmol/L), hemoglobin level of 9.3 mmol/L (range, 8.3-10.5 mmol/L), platelets of 384×109/L (range, 145-350×109/L), and leucocytes of 6.2×109/L (range, 3.5-10.0×109/L). Nitrous oxide can result in vitamin B12 inactivation and nerve damage due to lack of myelination. During hospitalization, the patient had a bilateral central pulmonary embolism, probably caused by a combination of nitrous oxide abuse and some extent of immobilization. After 6 months of nitrous oxide cessation and treatment with B vitamins, the patient experienced almost no residual symptoms, and homocysteine and methylmalonic acid levels normalized. CONCLUSIONS Our case shows that even moderate recreational use of nitrous oxide can lead to severe peripheral neuropathy as well as increase the risk of thromboembolic complications. Especially young and previously healthy individuals presenting with unexplained neuropathy or thromboembolic events should therefore be asked about possible use of nitrous oxide.

Lower Antiplatelet Effect of Aspirin in Essential Thrombocythemia than in Coronary Artery Disease.

Background Patients with essential thrombocythemia (ET) and coronary artery disease (CAD) have increased risk of thromboembolic complications. In addition, a reduced antiplatelet effect of aspirin has been demonstrated in both patient groups. As ET is a platelet disorder, platelets may be more important for the thromboembolic risk in ET than in CAD. We aimed to investigate the antiplatelet effect of aspirin and platelet turnover in ET versus CAD patients. Methods We included 48 ET patients and an age-matched group of 48 CAD patients. The effect of aspirin was evaluated by thromboxane B 2 (TXB 2 ) levels and platelet aggregation. Platelet turnover was assessed by immature platelet count (IPC) and immature platelet fraction (IPF). Results ET patients had reduced effect of aspirin compared with CAD patients, demonstrated by significantly higher TXB 2 levels (median of differences = 22.3 ng/mL, p < 0.0001) and platelet aggregation (median of differences = 131.0 AU*min, p = 0.0003). Furthermore, ET patients had significantly higher IPC ( p < 0.0001) and IPF ( p = 0.0004) than CAD patients. Conclusion ET patients have lower 24-hour antiplatelet effect of aspirin than CAD patients. This may be explained by an increased platelet production and turnover counteracting the antiplatelet effect of aspirin. These findings strengthen the rationale for exploring novel antiplatelet regimens in ET patients to reduce the risk of cardiovascular events.