Growth Differentiation Factor 15 Is Associated with Platelet Reactivity in Patients with Acute Coronary Syndrome.

Bleeding events in patients with acute coronary syndrome (ACS) are a risk factor for adverse outcomes, including mortality. We investigated the association of growth differentiation factor (GDF)-15, an established predictor of bleeding complications, with on-treatment platelet reactivity in ACS patients undergoing coronary stenting receiving prasugrel or ticagrelor. Platelet aggregation was measured by multiple electrode aggregometry (MEA) in response to adenosine diphosphate (ADP), arachidonic acid (AA), thrombin receptor-activating peptide (TRAP, a protease-activated receptor-1 (PAR-1) agonist), AYPGKF (a PAR-4 agonist) and collagen (COL). GDF-15 levels were measured using a commercially available assay. GDF-15 correlated inversely with MEA ADP (r = -0.202, p = 0.004), MEA AA (r = -0.139, p = 0.048) and MEA TRAP (r = -0.190, p = 0.007). After adjustment, GDF-15 was significantly associated with MEA TRAP (ß = -0.150, p = 0.044), whereas no significant associations were detectable for the other agonists. Patients with low platelet reactivity in response to ADP had significantly higher GDF-15 levels (p = 0.005). In conclusion, GDF-15 is inversely associated with TRAP-inducible platelet aggregation in ACS patients treated with state-of-the-art antiplatelet therapy and significantly elevated in patients with low platelet reactivity in response to ADP.

Platelet-to-Lymphocyte Ratio as Marker of Platelet Activation in Patients on Potent P2Y12 Inhibitors.

A high platelet-to-lymphocyte ratio (PLR) has recently been associated with ischemic outcomes in cardiovascular disease. Increased platelet reactivity and leukocyte-platelet aggregate formation are directly involved in the progress of atherosclerosis and have been linked to ischemic events following percutaneous coronary intervention (PCI). In order to understand the relation of PLR with platelet reactivity, we assessed PLR as well as agonist-inducible platelet aggregation and neutrophil-platelet aggregate (NPA) formation in 182 acute coronary syndrome (ACS) patients on dual antiplatelet therapy with aspirin and prasugrel (n = 96) or ticagrelor (n = 86) 3 days after PCI. PLR was calculated from the blood count. Platelet aggregation was measured by multiple electrode aggregometry and NPA formation was determined by flow cytometry, both in response to ADP and SFLLRN. A PLR ≥91 was considered as high PLR based on previous data showing an association of this threshold with adverse ischemic outcomes. In the overall cohort and in prasugrel-treated patients, high PLR was associated with higher SFLLRN-inducible platelet aggregation (67 AU [50-85 AU] vs 59.5 AU [44.3-71.3 AU], P = .01, and 73 AU [50-85 AU] vs 61.5 AU [46-69 AU], P = .02, respectively). Further, prasugrel-treated patients with high PLR exhibited higher ADP- (15% [11%-23%] vs 10.9% [7.6%-15.9%], P = .007) and SFLLRN-inducible NPA formation (64.3% [55.4%-73.8%] vs 53.8% [44.1%-70.1%], P = .01) as compared to patients with low PLR. These differences were not seen in ticagrelor-treated patients. In conclusion, high PLR is associated with increased on-treatment platelet reactivity in prasugrel-treated patients, but not in patients on ticagrelor.

Microvascular rarefaction in patients with cerebrovascular events.

Capillary density rarefaction and endothelial dysfunction contribute to chronic hypoperfusion and cerebral small vessel disease. Previous animal experiments revealed spatiotemporal microvascular remodeling directing post-stroke brain reorganization. We hypothesized that microcirculatory changes during acute cerebrovascular events could be reflected systemically and visualized sublingually. In a prospective observational trial in vivo sublingual sidestream darkfield videomicroscopy was performed in twenty-one patients with either acute stroke (n = 13 ischemic, n = 1 ischemic with hemorrhagic transformation and n = 2 hemorrhagic stroke) or transitory ischemic attacks (n = 5) within 24 h after hospital admission and compared to an age- and sex-matched control group. Repetitive measurements were performed on the third day and after one week. Functional and perfused total capillary density was rarefied in the overall patient group (3060 vs 3717 µm/mm2, p = 0.001 and 5263 vs 6550 µm/mm2, p = 0.002, respectively) and in patients with ischemic strokes (2897 vs. 3717 µm/mm2, p < 0.001 and 5263 vs. 6550 µm/mm2, p = 0.006, respectively) when compared to healthy controls. The perfused boundary region (PBR), which was measured as an inverse indicator of glycocalyx thickness, was markedly related to red blood cell (RBC) filling percentage (regarded as an estimate of microvessel perfusion) in the overall patient group (r = -0.843, p < 0.001), in patients with ischemic strokes (r = -0.82, p = 0.001) as well as in healthy volunteers (r = -0.845, p < 0.001). In addition, there were significant associations between platelet count or platelet aggregation values (as measured by whole blood impedance aggregometry) and microvascular parameters in the overall patient collective, as well as in patients with ischemic strokes. In conclusion, cerebrovascular events are associated with altered systemic microvascular perfusion.

Sex-specific platelet activation through protease-activated receptor-1 in patients undergoing cardiac catheterization.

BACKGROUND AND AIMS: Protease-activated receptor (PAR)-1-mediated platelet activation may vary according to sex and clinical situation. In order to investigate sex-specific platelet activation through PAR-1, we assessed platelet response to thrombin receptor-activating peptide (TRAP) in 562 patients undergoing cardiac catheterization without (Group 1A) and with (Group 1B) acute coronary syndrome (ACS). Subsequently, we sought to confirm our findings in 287 patients undergoing elective (Group 2A) or acute (Group 2B) percutaneous coronary intervention. METHODS: TRAP-stimulated platelet surface expression of P-selectin and activated glycoprotein IIb/IIIa (GPIIb/IIIa) were measured by flow cytometry in Group 1. Light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) in response to TRAP were assessed in Group 2. RESULTS: In Group 1A, platelet activation in response to TRAP was significantly higher in women compared to men (P-selectin: 511 MFI [443-597 MFI] vs. 471 MFI [393-552 MFI]; GPIIb/IIIa: 84 MFI [58-119 MFI] vs. 70 MFI [47-103 MFI]; both p ≤ 0.002). In contrast, in Group 1B, TRAP-stimulated P-selectin and activated GPIIb/IIIa were similar in men and women (both p ≥ 0.3). Likewise, TRAP-stimulated platelet aggregation was significantly higher in female patients in Group 2A (LTA: 66% [54-76%] vs. 51% [41-65%]; MEA: 78 AU [66-107 AU] vs. 62 AU [52-88 AU]; both p ≤ 0.02), whereas men and women in Group 2 B had similar platelet aggregation (p = 0.5). The occurrence of ischemic endpoints did not differ significantly between men and women in Group 1A and Group 1B. CONCLUSIONS: Platelet PAR-1 signaling is more pronounced in women than in men without ACS. In ACS, however, PAR-1-mediated platelet activation is similar in male and female patients.

Circulating MicroRNAs and Monocyte-Platelet Aggregate Formation in Acute Coronary Syndrome.

BACKGROUND: Monocyte-platelet aggregates (MPAs) are a sensitive marker of in vivo platelet activation in acute coronary syndrome (ACS) and associated with clinical outcomes. MicroRNAs (miRs) play an important role in the regulation of platelet activation, and may influence MPA formation. Both, miRs and MPA, could be influenced by the type of P2Y12 inhibitor. AIM: To study the association of platelet-related miRs with MPA formation in ACS patients on dual antiplatelet therapy (DAPT), and to compare miRs and MPA levels between prasugrel- and ticagrelor-treated patients. METHODS AND RESULTS: We analyzed 10 circulating platelet-related miRs in 160 consecutive ACS patients on DAPT with low-dose aspirin and either prasugrel (n = 80) or ticagrelor (n = 80). MPA formation was measured by flow cytometry without addition of platelet agonists and after simulation with the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, adenosine diphosphate (ADP), or arachidonic acid (AA). In multivariate regression analyses, we identified miR-21 (ß = 9.50, 95% confidence interval [CI]: 1.60-17.40, p = 0.019) and miR-126 (ß = 7.50, 95% CI: 0.55-14.44, p = 0.035) as independent predictors of increased MPA formation in vivo and after TLR-1/2 stimulation. In contrast, none of the investigated miRs was independently associated with MPA formation after stimulation with ADP or AA. Platelet-related miR expression and MPA formation did not differ significantly between prasugrel- and ticagrelor-treated patients. CONCLUSION: Platelet-related miR-21 and miR-126 are associated with MPA formation in ACS patients on DAPT. miRs and MPA levels were similar in prasugrel- and ticagrelor-treated patients.

Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers in Acute Coronary Syndrome: Implications for Platelet Reactivity?

BACKGROUND: In patients with acute coronary syndrome (ACS), angiotensin-converting enzyme (ACE) inhibitors are preferred over angiotensin receptor blockers (ARBs). However, in a recent pilot study, treatment with ACE inhibitors was associated with increased platelet reactivity compared to ARBs. Therefore, we sought to investigate the impact of renin-angiotensin-aldosterone system (RAAS) blockade with ACE inhibitors and ARBs on platelet aggregation in patients with ACS undergoing percutaneous coronary intervention. METHODS: On-treatment residual platelet reactivity in response to arachidonic acid (AA), adenosine diphosphate (ADP), SFLLRN, AYPGKF, and collagen was assessed by multiple electrode aggregometry (MEA) in 197 ACS patients on dual antiplatelet therapy (DAPT) with aspirin and either prasugrel or ticagrelor. RESULTS: One hundred sixty-five (83.7%) patients were treated with ACE inhibitors, 32 (16.3%) with ARBs. On-treatment residual AA- and ADP-inducible platelet reactivity was significantly higher in patients with ACE inhibitors (both p < 0.05). Likewise, SFLLRN was significantly higher in patients with ACE inhibitors (p = 0.036) and there was a trend for higher AYPGKF- and collagen-inducible platelet reactivity (p = 0.053 and p = 0.082). The incidence of high on-treatment residual platelet reactivity AA was significantly higher in patients with ACE inhibitors (52 [31.5%] vs. 3 [9.4%] patients; p = 0.019). CONCLUSION: ACE inhibitors are associated with increased on-treatment residual platelet reactivity in ACS patients with potent DAPT. Further clinical trials are needed to elucidate the role of RAAS blockade with ACE inhibitors and ARBs in ACS patients treated according to current standards.

Comparison of Light Transmission Aggregometry With Impedance Aggregometry in Patients on Potent P2Y12 Inhibitors.

Since data on the agreement between light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) in patients on the more potent P2Y12 inhibitors are missing so far, we investigated if the evaluation of the responsiveness to therapy by LTA can be replaced by MEA in 160 acute coronary syndrome (ACS) patients on dual antiplatelet therapy with aspirin and prasugrel or ticagrelor (n = 80 each). Cut-off values for high on-treatment residual platelet reactivity (HRPR) in response to adenosine diphosphate (ADP) or arachidonic acid (AA) were defined according to previous studies showing an association of HRPR with the occurrence of adverse ischemic outcomes. ADP- inducible platelet aggregation was 33% and 37% (P = 0.07) by LTA and 19 AU and 20 AU (P = 0.38) by MEA in prasugrel- and ticagrelor-treated patients, respectively. AA- inducible platelet aggregation was 2% and 3% by LTA and 15 AU and 16 AU by MEA, (all P ≥ 0.3) in patients on prasugrel and ticagrelor, respectively. By LTA, HRPR ADP and HRPR AA were seen in 5%/5% and in 4%/ 13% of patients receiving prasugrel- and ticagrelor, respectively. By MEA, HRPR ADP and HRPR AA were seen in 3%/ 25% and 0%/24% of prasugrel- and ticagrelor-treated patients, respectively. ADP-inducible platelet reactivity by MEA correlated significantly with LTA ADP in prasugrel-treated patients (r = 0.4, P < 0.001), but not in those receiving ticagrelor (r = 0.09, P = 0.45). AA-inducible platelet aggregation by LTA and MEA did not correlate in prasugrel- and ticagrelor-treated patients. Sensitivity/specificity of HRPR by MEA to detect HRPR by LTA were 25%/99% for MEA ADP and 100%/79% for MEA AA in prasugrel-treated patients, and 0%/100% for MEA ADP and 70%/83% for MEA AA in ticagrelor-treated patients. In conclusion, on-treatment residual ADP-inducible platelet reactivity by LTA and MEA shows a significant correlation in prasugrel- but not ticagrelor-treated patients. However, in both groups LTA and MEA revealed heterogeneous results regarding the classification of patients as responders or non-responders to P2Y12 inhibition.

Decreased Platelet Inhibition by Thienopyridines in Hyperuricemia.

PURPOSE: Hyperuricemia carries an increased risk of atherothrombotic events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). This may at least in part be due to inadequate P2Y12 inhibition. The aim of this study was to prospectively investigate the potential association between hyperuricemia and decreased platelet inhibition by P2Y12 antagonists. METHODS: Levels of uric acid as well as on-treatment residual platelet reactivity in response to adenosine diphosphate (ADP) were assessed in 301 clopidogrel-treated patients undergoing elective angioplasty and stenting, and in 206 prasugrel- (n = 118) or ticagrelor-treated (n = 88) ACS patients following acute PCI. Cut-off values for high on-treatment residual ADP-inducible platelet reactivity (HRPR) were based on previous studies showing an association of test results with clinical outcomes. RESULTS: Hyperuricemia was significantly associated with increased on-treatment residual ADP-inducible platelet reactivity in clopidogrel- and prasugrel-treated patients in univariate analyses and after adjustment for differences in patient characteristics by multivariate regression analyses. In contrast, ticagrelor-treated patients without and with hyperuricemia showed similar levels of on-treatment residual platelet reactivity to ADP. HRPR occurred more frequently in clopidogrel- and prasugrel-treated patients with hyperuricemia than in those with normal uric acid levels. In contrast, hyperuricemic patients receiving ticagrelor did not have a higher risk of HRPR compared with those with normal uric acid levels. CONCLUSION: Hyperuricemia is associated with decreased platelet inhibition by thienopyridines but a normal response to ticagrelor. It remains to be established if lowering uric acid increases the antiplatelet effects of clopidogrel and prasugrel in hyperuricemic patients with HRPR.

Ticagrelor Inhibits Toll-Like and Protease-Activated Receptor Mediated Platelet Activation in Acute Coronary Syndromes.

PURPOSE: Since ticagrelor inhibits the cellular uptake of adenosine, thereby increasing extracellular adenosine concentration and biological activity, we hypothesized that ticagrelor has adenosine-dependent antiplatelet properties. In the current study, we compared the effects of ticagrelor and prasugrel on platelet activation in acute coronary syndrome (ACS). METHODS: Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa in response to adenosine diphosphate (ADP), the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, the TLR-4 agonist lipopolysaccharide (LPS), the protease-activated receptor (PAR)-1 agonist SFLLRN, and the PAR-4 agonist AYPGKF were measured by flow cytometry in blood from 80 ticagrelor- and 80 prasugrel-treated ACS patients on day 3 after percutaneous coronary intervention. Residual platelet aggregation to arachidonic acid (AA) and ADP were assessed by multiple electrode aggregometry and light transmission aggregometry. RESULTS: ADP-induced platelet activation and aggregation, and AA-induced platelet aggregation were similar in patients on ticagrelor and prasugrel, respectively (all p ≥ 0.3). Further, LPS-induced platelet surface expression of P-selectin and activated GPIIb/IIIa did not differ significantly between ticagrelor- and prasugrel-treated patients (both p > 0.4). In contrast, Pam3CSK4-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly lower in ticagrelor-treated patients (both p ≤ 0.005). Moreover, SFLLRN-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly less pronounced in patients on ticagrelor therapy compared to prasugrel-treated patients (both p < 0.03). Finally, PAR-4 mediated platelet activation as assessed by platelet surface expression of activated GPIIb/IIIa following stimulation with AYPGKF was significantly lower in patients receiving ticagrelor (p = 0.02). CONCLUSION: Ticagrelor inhibits TLR-1/2 and PAR mediated platelet activation in ACS patients more strongly than prasugrel.

Protease-activated receptor-mediated platelet aggregation in acute coronary syndrome patients on potent P2Y12 inhibitors.

BACKGROUND: Despite the increasing use of potent P2Y12 inhibitors, further atherothrombotic events still impair the prognosis of many acute coronary syndrome (ACS) patients. This may in part be attributable to intact platelet aggregation via the human thrombin receptors protease-activated receptor (PAR)-1 and PAR-4. OBJECTIVE: We studied PAR mediated platelet aggregation in ACS patients following percutaneous coronary intervention (PCI) with stent implantation in a cross-sectional study. METHODS: Platelet aggregation to ADP as well as to the PAR-1 agonist SFLLRN and the PAR-4 agonist AYPGKF was assessed by multiple electrode aggregometry in 194 ACS patients on dual antiplatelet therapy with aspirin and either prasugrel (n = 114) or ticagrelor (n = 80) 3 days after PCI. RESULTS: Based on the consensus cutoff value, high on-treatment residual platelet reactivity to ADP (HRPR ADP) was observed in only 2 prasugrel-treated patients. Both patients with HRPR ADP had also a normal response to SFLLRN and AYPGKF. Among the 112 prasugrel-treated patients with adequate P2Y12 inhibition, 50 patients (45%) still had a normal response to SFLLRN, and 70 patients (63%) still had a normal response to AYPGKF. Among the 80 ticagrelor-treated patients with adequate P2Y12 inhibition, 25 patients (31%) still had a normal response to SFLLRN, and 50 (63%) still had a normal response to AYPGKF. CONCLUSION: Normal platelet aggregation via PAR-1 and PAR-4 is preserved in many patients with adequate P2Y12 inhibition by prasugrel and ticagrelor. The present findings may at least in part explain adverse ischemic events despite potent P2Y12 inhibition.

Functional capillary impairment in patients with ventricular assist devices.

The implantation of continuous - flow ventricular assist devices (VAD) is suggested to evoke angiodysplasia contributing to adverse events such as gastrointestinal bleeding. We evaluated in vivo capillary density and glycocalyx dimensions to investigate possible systemic microvascular changes in patients with chronic heart failure and VAD support vs. standard medical treatment. Forty-two patients with VAD support were compared to forty-one patients with ischemic and non-ischemic chronic heart failure (CHF) on standard pharmacotherapy and to a group of forty-two healthy subjects in a prospective cross-sectional study. Sublingual microcirculation was visualized using Sidestream Darkfield videomicroscopy and functional and perfused total capillary densities were quantified. Patients with VAD implantation were followed for one year and bleeding events were recorded. Median time after VAD implantation was 18 months. Patients were treated with centrifugal-flow devices (n = 31) or axial-flow devices (n = 11). Median functional capillary density was significantly lower in patients with VAD therapy as compared to CHF patients (196 vs. 255/mm2, p = 0.042, adjusted p-value). Functional and total capillary densities were 44% and 53% lower (both p < 0.001) in patients with VAD therapy when compared to healthy subjects. Cox regression analysis revealed loss of capillary density as a significant predictor of bleeding events during one -year follow-up of VAD patients (HR: 0.987, CI (95%): 0.977-0.998, p = 0.021 for functional and 0.992, CI (95%): 0.985-0.999, p = 0.03 for total capillary density). In conclusion, patients with VAD support exhibit capillary density rarefaction, which was associated with bleeding events. If confirmed independently, capillary impairment may be evaluated as novel marker of bleeding risk.

Oral antiplatelet agents in cardiovascular disease.

Antiplatelet agents significantly reduce mortality and morbidity in ischemic heart disease, cerebrovascular disease and peripheral artery disease (PAD), and are therefore part of guideline-driven daily medical treatment in these patients. Due to its beneficial effects in the secondary prevention of atherothrombotic events, aspirin remains the most frequently prescribed antiplatelet agent in cardiovascular disease. In patients with acute coronary syndromes (ACS) and in those undergoing angioplasty with stent implantation dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) receptor antagonist is indicated. The development of the newer ADP P2Y12 inhibitors prasugrel and ticagrelor has further improved prognosis in ACS patients compared to clopidogrel. Moreover, vorapaxar allows the inhibition of platelet activation by thrombin via protease-activated receptor-1 and has been approved for the use in patients with PAD and in those with a history of myocardial infarction. This review article summarizes the current evidence on oral antiplatelet agents in cardiovascular disease. Keywords: Aspirin, clopidogrel, prasugrel, ticagrelor, vorapaxar, cardiovascular disease.

Decreased platelet inhibition by P2Y12 receptor blockers in anaemia.

BACKGROUND: Anaemic patients undergoing angioplasty and stenting are at an increased risk of ischaemic events, which may be caused by an inadequate response to antiplatelet therapy with adenosine diphosphate (ADP) P2Y12 inhibitors. In the current study, we investigated the associations between anaemia and on-treatment platelet reactivity in clopidogrel-treated (group 1, n = 306) and prasugrel-/ticagrelor-treated (group 2, n = 109) patients undergoing elective and acute angioplasty with stent implantation, respectively. MATERIALS AND METHODS: Monocyte-platelet aggregate (MPA) formation was determined by flow cytometry in both groups. On-treatment residual platelet reactivity in response to ADP was assessed by light transmission aggregometry (LTA) in both groups, and by the VerifyNow P2Y12 assay and the Impact-R in group 1. P-selectin expression was measured by flow cytometry in group 2. RESULTS: In both groups, anaemia was associated with significantly higher MPA formation in response to ADP (both P ≤ .02). Moreover, by LTA maximal aggregation in response to ADP was significantly higher in patients with anaemia in both groups (both P < .05), and anaemic patients in group 1 had a significantly higher on-treatment platelet reactivity by the VerifyNow P2Y12 assay and the Impact-R than those without anaemia (both P < .001). In group 2, significantly higher platelet surface expression of P-selectin was seen in anaemia after stimulation with ADP (P = .02). CONCLUSION: Anaemia is associated with decreased platelet inhibition by ADP P2Y12 receptor antagonists after elective and acute percutaneous interventions with stent implantation. However, due to inconsistencies between different platelet function tests additional data are needed to clarify the role of anaemia for platelet inhibition.

Disaggregation Following Agonist-Induced Platelet Activation in Patients on Dual Antiplatelet Therapy.

Disaggregation as the difference between maximal and final platelet aggregation by light transmission aggregometry indicates the stability of platelet aggregates. We evaluated the extent of disaggregation after platelet stimulation with adenosine diphosphate (ADP), arachidonic acid (AA), collagen, epinephrine, and thrombin receptor-activating peptide (TRAP)-6 in 323 patients on dual antiplatelet therapy with daily aspirin and clopidogrel (group 1), prasugrel (group 2), or ticagrelor (group 3) therapy. All patients in group 1 underwent elective angioplasty and stenting, whereas all patients included in groups 2 and 3 suffered from acute coronary syndromes (STEMI or NSTEMI) and underwent urgent PCI. Significant differences between maximal and final platelet aggregation were observed with all agonists throughout the groups (all p<0.001). Disaggregation was highest using AA (clopidogrel 36.5%; prasugrel/ticagrelor 100%) and ADP (clopidogrel 21.7%; prasugrel/ticagrelor 100%). In contrast, low disaggregation was observed after platelet stimulation with collagen and TRAP-6 in clopidogrel-treated patients, and after platelet stimulation with collagen and epinephrine in prasugrel- and ticagrelor-treated patients. In conclusion, pathways of platelet activation that are not inhibited by standard antiplatelet therapy allow persisting platelet aggregation and may at least in part be responsible for adverse ischemic events.