The ABO Locus is Associated with Increased Fibrin Network Formation in Patients with Stable Coronary Artery Disease.

BACKGROUND: The ABO locus has been associated with increased risk of myocardial infarction (MI) in patients with coronary artery disease (CAD), but the underlying mechanisms are unknown. As altered fibrin clot structure has been demonstrated to predict MI in CAD patients, we examined the association between the ABO risk variant and fibrin clot properties, and investigated the effects of other CAD-associated risk variants. METHODS: We included 773 stable CAD patients. Patients were genotyped for 45 genome-wide CAD risk variants, including rs495828 at the ABO locus. We used a genetic risk score (GRS) for CAD calculated as the weighted sum of the number of risk alleles based on all 45 variants. Fibrin clot properties were evaluated using a turbidimetric assay. We studied clot maximum absorbance, a measure of clot density and fiber thickness, together with clot lysis time, an indicator of fibrinolysis potential. RESULTS: The rs495828 risk allele was present in 13.2% of patients and associated with higher clot maximum absorbance (adjusted effect size per risk allele: 1.05 [1.01 - 1.09], p = 0.01) but not with clot lysis time (p = 0.97). The rs12936587 (p = 0.04), rs4773144 (p = 0.02), and rs501120 (p = 0.04) were associated with clot lysis time; however, after Bonferroni correction, no significant associations were found between any of the remaining 44 CAD-associated variants and fibrin clot properties. The GRS was not associated with fibrin clot properties (p-values > 0.05). CONCLUSION: The ABO risk allele was associated with a more compact fibrin network in stable CAD patients, which may represent a mechanism for increased MI risk in ABO risk variant carriers.

Imbalance between Fibrin Clot Formation and Fibrinolysis Predicts Cardiovascular Events in Patients with Stable Coronary Artery Disease.

BACKGROUND: Despite long-term antiplatelet therapy with aspirin, recurrent cardiovascular events remain common in patients with coronary artery disease (CAD). OBJECTIVE: We aimed to determine whether fibrin network characteristics are predictive of vascular events in patients with stable CAD treated with aspirin monotherapy. METHODS: We included 786 patients with angiographically documented CAD and either prior myocardial infarction, type 2 diabetes mellitus, or both. Median follow-up time was 3 years. At inclusion, fibrin clot properties were evaluated using a turbidimetric assay and the following clot parameters were studied: (1) maximum absorbance, a measure of clot density and fiber thickness; (2) lysis time, assessing fibrinolysis potential; and (3) area under the curve (AUC), a measure of clot formation and lysis. The primary endpoint was the composite of nonfatal myocardial infarction, ischemic stroke, and cardiovascular death. Hazard ratios (HRs) were estimated using multivariable Cox proportional hazards regression. RESULTS: A total of 70 primary endpoints occurred. The primary endpoint occurred more frequently in CAD patients with increased clot AUC (crude HR for first vs. fourth quartile: 2.4 [95% confidence interval 1.2-4.6], p = 0.01). This finding remained significant after adjusting for potential confounders (adjusted HR: 2.4 [1.2-4.8], p = 0.01). Neither clot maximum absorbance nor lysis time showed significant association with future vascular events (adjusted HR for maximum absorbance 1.8 [0.9-3.7]; p = 0.09) and lysis time (1.6 [0.8-3.0]; p = 0.18). CONCLUSION: We demonstrate that increased clot AUC predicts future cardiovascular events in stable CAD patients receiving aspirin monotherapy.

The ABO locus is associated with increased platelet aggregation in patients with stable coronary artery disease.

BACKGROUND: Genome-wide association studies of patients with coronary artery disease (CAD) suggest that several risk loci increase the risk of CAD and myocardial infarction (MI) equally. In contrast, the ABO locus is stronger associated with MI than with CAD, but the underlying mechanisms are unknown. PURPOSE: To investigate the association between the ABO risk variant and platelet activation and aggregation. Moreover, to explore the effects of other CAD-associated risk variants. METHODS: We included 879 stable CAD patients receiving low-dose aspirin. All patients were genotyped for 45 genome-wide significant CAD risk variants, including rs495828 at the ABO locus. A genetic risk score (GRS) was calculated to assess the combined risk of all genetic variants. Serum soluble P-selectin (sP-selectin) and thromboxane B2 were used as measures of platelet activation, and platelet aggregation was assessed by multiple electrode aggregometry (MEA) using arachidonic acid and collagen as agonists and VerifyNow. RESULTS: The rs495828 CAD risk allele was associated with higher MEA platelet aggregation; arachidonic acid: 14.9% (6.7-23.7%, p = 0.0002) higher AUC (Area Under aggregation Curve) per risk allele, and collagen: 13.1% (5.8%-20.9%, p = 0.0003). Conversely, sP-selectin levels were 7.5% (3.1%-11.7%, p = 0.001) lower per risk allele. Rs495828 genotypes were not associated with aggregation assessed by VerifyNow (p = 0.30) or S-thromboxane B2 levels (p = 0.98). None of the remaining variants or the GRS were associated with platelet activation or aggregation. CONCLUSIONS: The ABO risk allele was associated with increased platelet aggregation as assessed by MEA. This finding may contribute to explain the increased MI risk in ABO risk variant carriers.

Effect of remote ischaemic conditioning on platelet aggregation and platelet turnover.

Remote ischaemic conditioning (RIC) is a new beneficial treatment for patients with ST-elevation myocardial infarction. RIC may inhibit thrombus formation and, therefore, we investigated whether RIC affects platelet aggregation and turnover. 30 healthy male volunteers were subjected to intervention on day 1 (sham intervention, no aspirin), day 2 (RIC, no aspirin), and day 16 (RIC, treated 7 days with aspirin 75 mg/day). RIC was performed as four cycles of 5 min interchangeable inflation and deflation using an automated cuff. Blood samples were collected 5 min before, as well as 5 and 45 min after RIC. Platelet aggregation was measured by Multiplate® using collagen (COLtest), adenosine diphosphate (ADPtest), and arachidonic acid (ASPItest) as agonists. Platelet turnover was evaluated by flow cytometry. Serum thromboxane B2 was determined by ELISA to confirm aspirin compliance. We found no significant change in platelet aggregation at visit 1 (COLtest: p = 0.32; ADPtest: p = 0.24; ASPItest: p = 0.07), visit 2, except for ADP-induced platelet aggregation evaluated 5 min after RIC (COLtest: p = 0.39; ADPtest: p = 0.02; ASPItest: p = 0.39), or visit 3 (COLtest: p = 0.48; ADPtest: p = 0.61; ASPItest: p = 0.90). Platelet turnover was not influenced by RIC, neither on nor off aspirin (all p-values > 0.07). (1) RIC did not affect platelet aggregation in healthy young men. (2) RIC did not affect platelet turnover in healthy young men. (3) Aspirin did not influence the effect of RIC on platelet aggregation and turnover. (4) Future studies exploring the effect of RIC on platelet aggregation and turnover in patients with ischaemic heart disease are warranted.

Influence of Exercise on Platelet Function in Patients with Cardiovascular Disease.

Regular exercise may reduce the risk of major cardiovascular thrombotic events. However, previous studies suggest that the risk of myocardial infarction or primary cardiac arrest is transiently increased during exercise. Thus, on the one hand, exercise seems to be able to protect against cardiovascular disease, but on the other hand, it seems to provoke sudden cardiac death. As platelets play a key role in arterial thromboembolic disease, the effect of exercise on platelet function is of special interest. This systematic review summarizes the evidence of the influence of exercise on platelet function in patients with coronary artery disease, angina pectoris, hypertension, or peripheral arterial disease. We specifically investigated, (1) if platelet function was increased in patients prior to exercise compared with healthy controls, (2) if exercise influenced platelet function differently in patients compared with healthy controls, and finally (3) if exercise reduced the effect of aspirin (acetylsalicylic acid). We performed a literature search in PubMed, Embase, Scopus, and Cochrane Library. In total, 18 articles were included and grouped into studies including patients with coronary artery disease (n = 7), angina pectoris (n = 5), hypertension (n = 5), and patients with peripheral arterial disease (n = 2). One study included both patients with coronary artery disease and patients with hypertension, and this study was therefore included in both groups. All studies performed short-term exercise either using treadmill (n = 12), primarily following the Bruce protocol, or bicycle ergometer test (n = 6). Overall, patients did not differ from healthy controls in platelet aggregation or activation prior to exercise. After exercise, conflicting results were reported with some studies reporting intensified platelet aggregation and/or platelet activation, some studies found no difference, whereas a few studies reported a reduction in platelet aggregation after exercise when compared with controls. Exercise seemed to impair the effect of aspirin during or shortly after exercise. In conclusion, the studies reported contradictive results. However, this review indicates that strenuous short-term exercise induces increased platelet activation also implying a reduced effect of aspirin during short-term exercise. Controlled studies on the effect of regular long-term low-intensity exercise are needed to further clarify the influence of long-term exercise on platelet function.

The SH2B3 and KCNK5 loci may be implicated in regulation of platelet count, volume, and maturity.

INTRODUCTION: In recent genome-wide association studies, coronary artery disease (CAD) and myocardial infarction (MI) have been linked to a number of genetic variants, but their role in thrombopoiesis is largely unknown. AIM: We investigated the association between CAD and MI-associated genetic variants and five thrombopoiesis-related indices: platelet count (PC), mean platelet volume (MPV), immature platelet count (IPC), immature platelet fraction (IPF), and serum thrombopoietin (TPO). METHODS: We genotyped 45 genome-wide significant CAD/MI-markers in 879 stable CAD patients. A genetic risk score was calculated to assess the combined risk associated with all the genetic variants. Platelet indices were analysed using the Sysmex XE-2100 haematology analyser. TPO was measured by ELISA. RESULTS: Two variants were nominally associated with several indices; for rs10947789 (KCNK5), the adjusted geometric mean was 2% higher for MPV (95% confidence interval: 1-2%, p=0.002), 6% for IPC (0-12%, p=0.033), and 9% for IPF (3-16%, p=0.004) per CAD risk allele. Moreover, an 11% lower TPO (3-19%, p=0.010) was observed. Rs3184504 (SH2B3) was associated with a higher adjusted geometric mean of 3% (1-6%, p=0.003) per CAD risk allele for PC, and an 11% (5-17%, p<0.001) lower TPO. Furthermore, the adjusted IPC was 5% (0-9%, p=0.037) lower per CAD risk allele for PC, whereas IPF levels did not vary across genotypes. CONCLUSION: As a novel finding, our study suggests a role for KCNK5 in the regulation of platelet size and maturity. Furthermore, our findings confirm an association between the SH2B3-locus and platelet count.

Reduced Antiplatelet Effect of Aspirin Does Not Predict Cardiovascular Events in Patients With Stable Coronary Artery Disease.

BACKGROUND: Increased platelet aggregation during antiplatelet therapy may predict cardiovascular events in patients with coronary artery disease. The majority of these patients receive aspirin monotherapy. We aimed to investigate whether high platelet-aggregation levels predict cardiovascular events in stable coronary artery disease patients treated with aspirin. METHODS AND RESULTS: We included 900 stable coronary artery disease patients with either previous myocardial infarction, type 2 diabetes mellitus, or both. All patients received single antithrombotic therapy with 75 mg aspirin daily. Platelet aggregation was evaluated 1 hour after aspirin intake using the VerifyNow Aspirin Assay (Accriva Diagnostics) and Multiplate Analyzer (Roche; agonists: arachidonic acid and collagen). Adherence to aspirin was confirmed by serum thromboxane B2. The primary end point was the composite of nonfatal myocardial infarction, ischemic stroke, and cardiovascular death. At 3-year follow-up, 78 primary end points were registered. The primary end point did not occur more frequently in patients with high platelet-aggregation levels (first versus fourth quartile) assessed by VerifyNow (hazard ratio: 0.5 [95% CI, 0.3-1.1], P=0.08) or Multiplate using arachidonic acid (hazard ratio: 1.0 [95% CI, 0.5-2.1], P=0.92) or collagen (hazard ratio: 1.4 [95% CI, 0.7-2.8], P=0.38). Similar results were found for the composite secondary end point (nonfatal myocardial infarction, ischemic stroke, stent thrombosis, and all-cause death) and the single end points. Thromboxane B2 levels did not predict any end points. Renal insufficiency was the only clinical risk factor predicting the primary and secondary end points. CONCLUSIONS: This study is the largest to investigate platelet aggregation in stable coronary artery disease patients receiving aspirin as single antithrombotic therapy. We found that high platelet-aggregation levels did not predict cardiovascular events.

Coronary artery disease-associated genetic variants and biomarkers of inflammation.

INTRODUCTION: Genetic constitution and inflammation both contribute to development of coronary artery disease (CAD). Several CAD-associated single-nucleotide polymorphisms (SNPs) have recently been identified, but their functions are largely unknown. We investigated the associations between CAD-associated SNPs and five CAD-related inflammatory biomarkers. METHODS: We genotyped 45 CAD-associated SNPs in 701 stable CAD patients in whom levels of high-sensitivity C-reactive protein (hsRCP), interleukin-6, calprotectin, fibrinogen and complement component 3 levels had previously been measured. A genetic risk score was calculated to assess the combined risk associated with all the genetic variants. A multiple linear regression model was used to assess associations between the genetic risk score, single SNPs, and the five inflammatory biomarkers. RESULTS: The minor allele (G) (CAD risk allele) of rs2075650 (TOMM40/APOE) was associated with lower levels of high-sensitivity C-reactive protein (effect per risk allele: -0.37 mg/l [95%CI -0.56 to -0.18 mg/l]). The inflammatory markers tested showed no association with the remaining 44 SNPs or with the genetic risk score. CONCLUSIONS: In stable CAD patients, the risk allele of a common CAD-associated marker at the TOMM40/APOE locus was associated with lower hsCRP levels. No other genetic variants or the combined effect of all variants were associated with the five inflammatory biomarkers.

A genetic risk score predicts cardiovascular events in patients with stable coronary artery disease.

BACKGROUND: Genetic risk scores (GRSs) may predict cardiovascular risk in community-based populations. However, studies investigating the association with recurrent cardiovascular events in patients with established coronary artery disease (CAD) are conflicting. METHODS: We genotyped 879 patients with high-risk stable CAD and created a GRS based on 45 single nucleotide polymorphisms previously reported to be associated with CAD in genome-wide association studies. Patients were categorised into high or low GRS according to the median GRS and followed for recurrent cardiovascular events using national Danish registries. The primary endpoint was a composite of myocardial infarction, coronary revascularisation, and cardiovascular death. RESULTS: Median (interquartile range) follow-up time was 2.8 (2.4-3.8)years. The cumulative incidence proportions of the primary endpoint at 1 and 3years were 6.4% and 11.5% in high-GRS patients vs. 2.5% and 7.3% in low-GRS patients. The corresponding relative risks were 2.56 (95% confidence interval (CI) 1.29-5.07), and 1.57 (95% CI 1.02-2.44). The adjusted hazard ratio (HR) of the primary endpoint was 1.50 (95% CI 1.00-2.25). The most pronounced effect of a high GRS was observed on coronary revascularisations (adjusted HR 2.10 [95% CI 1.08-4.07]). Risks of cardiovascular death (adjusted HR 1.07 [95% CI 0.46-2.48]) and all-cause death (adjusted HR 1.15 [95% CI 0.65-2.03]) were unaffected. CONCLUSIONS: A GRS predicts recurrent cardiovascular events in high-risk stable CAD patients. The observed effect was mainly driven by coronary revascularisations.

Thrombopoietin and platelet aggregation in patients with stable coronary artery disease.

Thrombopoietin (TPO) may facilitate platelet activation and aggregation. However, data on the impact of TPO on platelet aggregation in patients with stable coronary artery disease (CAD) are scarce. We aimed to investigate associations between TPO and platelet aggregation and activation in patients with stable coronary artery disease (CAD). We studied 900 stable CAD patients. Serum TPO was assessed by ELISA. Platelet aggregation was evaluated using the Multiplate Analyzer (agonists: arachidonic acid [AA] and collagen) and the VerifyNow Aspirin Assay. Platelet activation was evaluated by soluble (s)P-selectin. Cyclooxygenase-1 inhibition was evaluated by serum thromboxane B2 (TXB2). We found that TPO correlated weakly with platelet aggregation evaluated by Multiplate using AA (r = -0.09, p = 0.01) and collagen as agonists (r = -0.03, p = 0.43) and by VerifyNow (r = 0.07, p = 0.03). We found no correlation between TPO and sP-selectin (r = -0.01, p = 0.70). Independent predictors of AA-induced platelet aggregation by Multiplate included high levels of sP-selectin and serum TXB2, high platelet count, increasing age and body mass index, female sex, and active smoking. Independent predictors of TPO included low AA-induced platelet aggregation by Multiplate, high levels of hs-CRP, active smoking, and high platelet aggregation evaluated by VerifyNow. In conclusion, TPO levels did not correlate with platelet activation and only weak associations were found between TPO and platelet aggregation, suggesting that TPO did not substantially facilitate platelet aggregation in stable CAD patients.

Effect of long-term remote ischaemic conditioning on platelet function and fibrinolysis in patients with chronic ischaemic heart failure.

INTRODUCTION: Remote ischaemic conditioning (RIC) protects against ischaemia-reperfusion injury through cellular protective pathways, but may also modulate haemostasis. We aimed to investigate the effect of long-term RIC on platelet function and fibrinolysis in patients with chronic ischaemic heart failure (CIHF). MATERIAL AND METHODS: In a prospective, outcome-assessor blinded, paired study, 16 patients with CIHF and 21 age- and gender-matched controls without ischaemic heart disease (IHD) were treated with RIC once daily for 28±4days. RIC was performed as four cycles of 5min upper arm ischaemia and reperfusion. We evaluated collagen and arachidonic acid induced platelet aggregation (Multiplate® Analyzer), platelet turnover (Sysmex® XE-5000), platelet activation (plasma soluble-platelet-selectin) and fibrinolysis (clot lysis time, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1)). We compared blood samples assessed at baseline and following long-term RIC. RESULTS: Long-term RIC did not affect platelet aggregation, turnover or activation or PAI-1 in any study groups. Long-term RIC did not affect fibrin clot lysis time in patients with CIHF but reduced fibrin clot lysis time in matched controls without IHD (median: 773s (interquartile range: 689-936) vs. 658s (618-823), p=0.03). t-PA was increased following long-term RIC in CIHF patients (2.5 (1.7-3.4) vs. 2.9 (1.8-4.0), p=0.03) and in matched controls without IHD (1.5 (1.3-1.9) vs. 1.6 (1.4-2.3), p=0.03). CONCLUSIONS: While long-term RIC did not affect collagen or arachidonic acid induced platelet aggregation, platelet turnover or sP-selectin, fibrinolysis was increased although most consistently in matched controls without IHD. This finding suggests that RIC may stimulate fibrinolysis potentially reducing the risk of thrombosis.

Effect of remote ischaemic conditioning on coagulation and fibrinolysis.

INTRODUCTION: Remote ischaemic conditioning (RIC) reduces infarct size and may improve prognosis in patients with acute myocardial infarction. To explore the potential mechanisms, we investigated the effects of RIC on coagulation and fibrinolysis. METHODS: Interventional crossover study including 30 healthy drug-naïve males. Participants were exposed to a sham intervention (visit 1) and to RIC (visit 2 and 3) induced by intermittent arm ischaemia through four cycles of 5-min inflation of a blood pressure cuff followed by 5-min deflation. Prior to visit 3, all participants received aspirin 75mg daily for seven days. Blood samples were obtained at baseline as well as 5 and 45min after intervention. Whole blood coagulation was assessed by thromboelastometry (ROTEM®) and thrombin generation. Fibrinolysis was evaluated by clot turbidity-lysis, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1). RESULTS: No differences were found in clot initiation, clot propagation or clot strength evaluated by ROTEM® (all p-values≥0.98). During aspirin treatment, clot initiation and clot propagation decreased after RIC evaluated by ROTEM® EXTEM® clotting time (p=0.04) and ROTEM® EXTEM® maximum velocity (p=0.03). After sham and RIC, thrombin generation declined as evaluated by reduced endogenous thrombin potential (RIC: p=0.001) and peak (RIC: p=0.01). After RIC during aspirin treatment, changes in thrombin generation were inconsistent; increased peak (p=0.04) and time to peak (p<0.001) and a decrease in lag-time (p<0.001). Clot lysis time was prolonged both after sham and after RIC (p<0.001). After RIC, PAI-1 levels declined (p=0.03), but t-PA levels also declined after all interventions (p-values≤0.04). CONCLUSION: RIC did not have substantial effects on coagulation or fibrinolysis compared to sham. Overall, aspirin did not influence the results.

Platelets and Antiplatelet Therapy in Patients with Coronary Artery Disease and Diabetes.

Patients with diabetes mellitus are at increased risk of cardiovascular events. Despite advances in medical and interventional therapy, cardiovascular morbidity and mortality remains high in patients with diabetes. Although accelerated atherosclerosis has long been recognized as an underlying cause, recent studies suggest that changes in platelets and coagulation also play important roles. Patients with diabetes exhibit a prothrombotic milieu with hyperreactive platelets and coagulation abnormalities. Thus, prevention of cardiovascular events in patients with coronary artery disease (CAD) and diabetes involves a multifactorial approach including treatment of risk factors such as dyslipidemia, obesity, hypertension, hyperglycemia, and hypercoagulation. An impaired response to antiplatelet therapy has been consistently reported and optimization of this therapy seems appropriate to reduce the risk of cardiovascular events in these patients. In this review, platelet abnormalities are summarized together with an update of benefits and limitations of antiplatelet therapy in patients with CAD and diabetes.

The Influence of Haemoglobin A1c Levels on Platelet Aggregation and Platelet Turnover in Patients with Coronary Artery Disease Treated with Aspirin.

BACKGROUND: Hyperglycaemia may attenuate the antiplatelet effect of aspirin and thereby increase the risk of cardiovascular events. We investigated the influence of increased haemoglobin A1c (HbA1c) levels on platelet aggregation and turnover in a large cohort of patients with coronary artery disease (CAD) with type 2 diabetes, prediabetes or no diabetes. METHODS: In this observational study, we included 865 stable CAD patients on 75 mg aspirin as mono-therapy of whom 242 patients had type 2 diabetes and were receiving antidiabetic drugs. Among 623 patients without diabetes, we classified 303 patients with prediabetes (HbA1c ≥5.7-6.4% [39-47 mmol/mol]) naive to antidiabetic drugs. Platelet aggregation was evaluated by the Multiplate Analyzer using arachidonic acid and collagen and by the VerifyNow Aspirin. Platelet turnover was evaluated by immature platelets using flow cytometry and platelet activation by soluble P-selectin. RESULTS: CAD patients with type 2 diabetes had higher platelet aggregation (all p-values <0.01), platelet turnover (immature platelet count, p<0.01) and platelet activation (p<0.001) than patients without diabetes. CAD patients with prediabetes had increased platelet aggregation (p = 0.02) and platelet count (p = 0.02) compared with patients without diabetes. Increased levels of HbA1c correlated positively with increased platelet aggregation using arachidonic acid (r = 0.19, p<0.0001), collagen (r = 0.10, p<0.01) and VerifyNow (r = 0.15, p<0.0001), and with platelet count (r = 0.08, p = 0.01), immature platelet count (r = 0.11, p<0.001) and soluble P-selectin (r = 0.15, p<0.0001). These associations were mainly evident in non-diabetic and prediabetic CAD patients. CONCLUSIONS: CAD patients with prediabetes and diabetes may have attenuated antiplatelet effect of aspirin compared with CAD patients without diabetes. This may be related to increased platelet count in patients with prediabetes. Increased levels of HbA1c correlated positively, though weakly, with increased platelet aggregation, platelet turnover and platelet activation.

The influence of low-grade inflammation on platelets in patients with stable coronary artery disease.

Inflammation is likely to be involved in all stages of atherosclerosis. Numerous inflammatory biomarkers are currently being studied, and even subtle increases in inflammatory biomarkers have been associated with increased risk of cardiovascular events in patients with coronary artery disease (CAD). Low-grade inflammation may influence both platelet production and platelet activation potentially leading to enhanced platelet aggregation. Thrombopoietin is considered the primary regulator of platelet production, but it likely acts in conjunction with numerous cytokines, of which many have altered levels in CAD. Previous studies have shown that high-sensitive C-reactive protein (CRP) independently predicts increased platelet aggregation in stable CAD patients. Increased levels of CRP, fibrinogen, interleukin-6, stromal cell-derived factor-1, CXC motif ligand 16, macrophage migration inhibitory factor, RANTES, calprotectin, and copeptin have been associated with increased risk of cardiovascular events in CAD patients. Additionally, some of these inflammatory markers have been associated with enhanced platelet activation and aggregation. However, CRP and other inflammatory markers provide only limited additional predictive value to classical risk factors such as smoking, blood pressure, and cholesterol levels. Existing data do not clarify whether inflammation simply accompanies CAD and increased production and aggregation of platelets, or whether a causal relationship exists. In this review, we provide a comprehensive overview of inflammatory markers in stable CAD with particular emphasis on platelet production, activation, and aggregation in CAD patients.

Determinants of reduced antiplatelet effect of aspirin in patients with stable coronary artery disease.

BACKGROUND: Aspirin is a cornerstone in management of coronary artery disease (CAD). However, considerable variability in the antiplatelet effect of aspirin has been reported. AIM: To investigate independent determinants of reduced antiplatelet effect of aspirin in stable CAD patients. METHODS: We performed a cross-sectional study including 900 stable, high-risk CAD patients. Among these, 795 (88%) had prior myocardial infarction, 250 (28%) had type 2 diabetes, and 170 (19%) had both. All patients received 75 mg aspirin daily as mono antiplatelet therapy. The antiplatelet effect of aspirin was assessed by measurement of platelet aggregation employing 1) multiple electrode aggregometry (MEA, Multiplate Analyzer) in whole blood anticoagulated with citrate or hirudin using arachidonic acid (AA) or collagen as agonists, and 2) VerifyNow Aspirin Assay. Compliance was assessed by measurement of serum thromboxane B2. RESULTS: Platelet count, prior myocardial infarction, type 2 diabetes and body mass index were independent determinants of increased AA-induced MEA platelet aggregation in citrate and hirudin anticoagulated blood (p-values ≤ 0.045). Similar results were found with VerifyNow. Prior coronary artery bypass grafting, age, smoking (MEA, AA/citrate) and female gender (MEA, AA/hirudin) were also independent determinants of increased platelet aggregation (p-values ≤ 0.038). Compliance was confirmed by low serum thromboxane B2 levels in all patients (median [25%;75%]: 0.97 [0.52;1.97], range 0.02-26.44 ng/ml). CONCLUSION: Platelet count, prior myocardial infarction, type 2 diabetes and body mass index were independent determinants of increased platelet aggregation, indicating that these characteristics may be key factors in reduced antiplatelet effect of aspirin in stable CAD patients.

Fibrin clot structure and platelet aggregation in patients with aspirin treatment failure.

BACKGROUND: Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF. METHODS: We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics. RESULTS: Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31-0.35, p-values<0.001) and CRP levels (r = 0.60, p<0.001). CONCLUSIONS: Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.

The activation peptide cleft exposed by thrombin cleavage of FXIII-A(2) contains a recognition site for the fibrinogen α chain.

Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the ß-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.