Reduced Platelet Reactivity Research Articles

Variation of platelet function in clinical phenotypes of acute venous thromboembolism – Results from the GMP‐VTE project

BackgroundThe role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. ObjectiveTo characterize platelet function according to VTE phenotypes. Patients/MethodsIn total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)‐200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. ResultsPFA‐200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC‐1‐positive platelets after in‐vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet‐related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). ConclusionThis explorative study suggests an important distinction between PE‐related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet‐dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE‐dependent phenotypes compared to iDVT.

Platelet Activation and Reactivity in a Large Cohort of Patients with Gaucher Disease

Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data. Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface-activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels (r = 0.17, p-value = 0.042). Splenectomized GD patients had a higher level of unstimulated αIIbβ3 integrin and P-selectin expression. Reduced platelet reactivity (-2 standard deviation of reference range) was found in 79 (53%, 95% confidence interval [CI]: 44-61%) patients, of whom 10 (6.7%, 95% CI: 3.3-12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (odds ratio [OR]: 1.05, 95% CI: 1.01-1.1) and low P-selectin reactivity (OR: 2.03, 95% CI: 1.25-3.35) were associated with more than one bleeding manifestation. Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not αIIbβ3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity

Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice.Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16.Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents.Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice.Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamic evaluation of the platelet antiaggregant effect of swallowed Ticagrelor versus chewed Ticagrelor in patients with acute coronary syndrome

Abstract Introduction Dual antiplatelet therapy represents the standard of care for the treatment of patients with acute coronary syndrome (ACS). Ticagrelor is a direct-acting P2Y12 inhibitor and does not require metabolic activation. Objective To assess whether the chewed 180 mg of ticagrelor loading dose (LD), compared to conventional oral administration, increases platelet inhibition and reduces high on-treatment platelet reactivity (HTPR) in patients with ACS. Method Single-center randomized clinical trial in patients with ACS. We measured platelet reactivity and % platelet aggregation (%IPA) using VerifyNow P2Y12 (Accumetrics) at baseline, 30 minutes, 1, 4 and 24 hours after LD. HTPR was defined as ≥208 PRU and/or % platelet inhibition ≤15%. Results We studied 40 consecutive patients (Pts.), 21 Pts. in the swallowed ticagrelor group and 19 Pts. in the chewed ticagrelor group. At 30 minutes, platelet reactivity was 180.81±64.16 PRU vs. 117±58.62 PRU p&amp;lt;0.01 (95% CI 24.3 to 103.3); At one hour 154.29±84.55 PRU vs 54.84±59.6 PRU p&amp;lt;0.001 (95% CI 52.1 to 146.7) respectively. The baseline results, at 4 and 24 hours, had no differences (p=NS). %IPA was 11.19±22.42 vs 42.26±27.3 &amp;lt;0.0001, (95% CI 15.3, 47); 25.33±34.03 vs 75.47±26.27 p&amp;lt;0.0001, 95% CI (27.5, 66.7) Platelet reactivity in the chewed ticagrelor group was significantly reduced by 52.65% at 30 minutes after LD, compared to 16.79% in the swallowed group (95% CI −0.63 to −0.083; p&amp;lt;0.01). The frequency of HTPR at one hour was 57.14% in the swallowed group vs. 5.26% in the chewed group; p&amp;lt;0.001. After 4 hours, HTPR was not observed in any group. Conclusions Chewed ticagrelor allowed a significant reduction in platelet reactivity and HTPR from the first 30 minutes and the first hour. Funding Acknowledgement Type of funding sources: None.

PHARMACODYNAMIC EVALUATION OF THE PLATELET ANTIAGGREGANT EFFECT OF SWALLOWED TICAGRELOR AGAINST CHEWED TICAGRELOR IN PATIENTS WITH ACUTE CORONARY SYNDROME. TICA-MASTICA-SICA TRIAL

BACKGROUND Dual antiplatelet therapy represents the standard of care for the treatment of patients with acute coronary syndrome (ACS). Ticagrelor is a direct-acting P2Y12 inhibitor and does not require metabolic activation. The objective was to assess whether the chewed 180 mg of ticagrelor loading dose (LD), compared to conventional oral administration, increases platelet inhibition and reduces high on-treatment platelet reactivity (HTPR) in patients with ACS. METHODS AND RESULTS Single-center randomized clinical trial in patients with ACS. We measured platelet reactivity and % platelet aggregation (%IPA) using VerifyNow P2Y12 (Accumetrics) at baseline, 30 minutes, 1, 4 and 24 hours after LD. HTPR was defined as ≥208 PRU and/or % platelet inhibition ≤15%. We studied 40 consecutive patients (Pts.), 21 Pts. in the swallowed ticagrelor group and 19 Pts. in the chewed ticagrelor group. At 30 minutes, platelet reactivity was 180.81 ± 64.16 PRU vs. 117 ± 58.62 PRU p < 0.01 (95%CI 24.3 to 103.3); At one hour 154.29 ± 84.55 PRU vs 54.84 ± 59.6 PRU p < 0.001 (95%CI 52.1 to 146.7) respectively. The baseline results, at 4 and 24 hours, had no differences (p=NS). %IPA was 11.19 ±22.42 vs 42.26 ±27.3 < 0.0001, (95%CI 15.3, 47); 25.33 ±34.03 vs 75.47 ±26.27 p < 0.0001, 95%CI (27.5, 66.7) Platelet reactivity in the chewed ticagrelor group was significantly reduced by 52.65% at 30 minutes after LD, compared to 16.79% in the swallowed group (95%CI -0.63 to -0.083; p < 0.01). The frequency of HTPR at one hour was 57.14% in the swallowed group vs. 5.26% in the chewed group; p < 0.001. After 4 hours, HTPR was not observed in any group. CONCLUSION Chewed ticagrelor allowed a significant reduction in platelet reactivity and HTPR from the first 30 minutes and the first hour. Dual antiplatelet therapy represents the standard of care for the treatment of patients with acute coronary syndrome (ACS). Ticagrelor is a direct-acting P2Y12 inhibitor and does not require metabolic activation. The objective was to assess whether the chewed 180 mg of ticagrelor loading dose (LD), compared to conventional oral administration, increases platelet inhibition and reduces high on-treatment platelet reactivity (HTPR) in patients with ACS. Single-center randomized clinical trial in patients with ACS. We measured platelet reactivity and % platelet aggregation (%IPA) using VerifyNow P2Y12 (Accumetrics) at baseline, 30 minutes, 1, 4 and 24 hours after LD. HTPR was defined as ≥208 PRU and/or % platelet inhibition ≤15%. We studied 40 consecutive patients (Pts.), 21 Pts. in the swallowed ticagrelor group and 19 Pts. in the chewed ticagrelor group. At 30 minutes, platelet reactivity was 180.81 ± 64.16 PRU vs. 117 ± 58.62 PRU p < 0.01 (95%CI 24.3 to 103.3); At one hour 154.29 ± 84.55 PRU vs 54.84 ± 59.6 PRU p < 0.001 (95%CI 52.1 to 146.7) respectively. The baseline results, at 4 and 24 hours, had no differences (p=NS). %IPA was 11.19 ±22.42 vs 42.26 ±27.3 < 0.0001, (95%CI 15.3, 47); 25.33 ±34.03 vs 75.47 ±26.27 p < 0.0001, 95%CI (27.5, 66.7) Platelet reactivity in the chewed ticagrelor group was significantly reduced by 52.65% at 30 minutes after LD, compared to 16.79% in the swallowed group (95%CI -0.63 to -0.083; p < 0.01). The frequency of HTPR at one hour was 57.14% in the swallowed group vs. 5.26% in the chewed group; p < 0.001. After 4 hours, HTPR was not observed in any group. Chewed ticagrelor allowed a significant reduction in platelet reactivity and HTPR from the first 30 minutes and the first hour.

N-Terminal Pro-Brain Natriuretic Peptide and Right Ventricular Diameter Are Related to Aspirin Resistance in Coronary Artery Disease Patients

Background and Objectives: Resistance to ASA (ASAres) is a multifactorial phenomenon defined as insufficient reduction of platelet reactivity through incomplete inhibition of thromboxane A2 synthesis. The aim is to reassess the prevalence and predictors of ASAres in a contemporary cohort of coronary artery disease (CAD) patients (pts) on stable therapy with ASA, 75 mg o.d. Materials and Methods: We studied 205 patients with stable CAD treated with daily dose of 75 mg ASA for a minimum of one month. ASAres was defined as ARU (aspirin reaction units) ≥550 using the point-of-care VerifyNow Aspirin test. Results: ASAres was detected in 11.7% of patients. Modest but significant correlations were detected between ARU and concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) (r = 0.144; p = 0.04), body weight, body mass index, red blood cell distribution width, left ventricular mass, and septal end-systolic thickness, with trends for left ventricular mass index and prothrombin time. In multivariate regression analysis, log(NT-proBNP) was identified as the only independent predictor of ARU—partial r = 0.15, p = 0.03. Median concentrations of NT-proBNP were significantly higher in ASAres patients (median value 311.4 vs. 646.3 pg/mL; p = 0.046) and right ventricular diameter was larger, whereas mean corpuscular hemoglobin concentration was lower as compared to patients with adequate response to ASA. Conclusions: ASAres has significant prevalence in this contemporary CAD cohort and NT-proBNP has been identified as the independent correlate of on-treatment ARU, representing a predictor for ASAres, along with right ventricular enlargement and lower hemoglobin concentration in erythrocytes.

Transferrin Saturation and Its Association with Platelet Reactivity

Introduction. While a plethora of risk factors have been well established as predictors for atherosclerosis and coronary heart disease, the possible association between body iron status and the risk of cardiovascular disease has yielded conflicting results. Epidemiological studies have found a significant negative association of transferrin saturation and coronary heart disease or myocardial infarction suggesting that higher body iron stores possibly confer a protective effect towards developing cardiovascular diseases. Despite the pivotal role of platelets on the pathogenesis of thrombotic complications, little information is available about the relationships between transferrin saturation and platelet reactivity. Aim of this study was to evaluate the influence of transferrin saturation on platelet function.Methods. The study included two groups: 32 healthy volunteers (17M/15F, age: 55±5 years) and 45 hereditary hemochromatosis (HH) patients (34M/11F, age: 62±2 years). HH patients were characterized by high prevalence of C282Y and/or H63D mutations of the hemochromatosis gene (HFE) and showed, just before the last therapeutic bloodletting, serum transferrin saturation and ferritin levels of 51±4% and 176±34 ng/ml, respectively.In platelet samples from healthy subjects and HH patients, we evaluated the effects of a 15-min preincubation with holotransferrin (holoTf) or apotransferrin (apoTf) (10-80 nmol/l) on: i) maximal aggregation to ADP (10 µmol/l), collagen (4 mg/l), epinephrine (5 µmol/l) and arachidonic acid (AA)(1 µmol/l) (Born's method); ii) expression of platelet activation marker CD62P in response to collagen (4mg/l) (flow-cytometry); iii) activation of signaling pathways PI-3K/Akt and MAPK/Erk-1/2 in response to collagen (4mg/l) and AA (100 µmol/l) (Western Blot); iv) reactive oxygen species (ROS) stimulated by AA (100 µmol/l) (DCF-DA fluorescence).Results. In healthy subjects we found that platelet exposure to holoTf, but not apoTf, significantly decreased platelet reactivity. In particular, 40 µmol/l HoloTf: i) reduced maximal aggregation by 39±1% to ADP, 40±4% to collagen, 49±3% to epinephrine, and 48±4% to AA (versus each agonist alone: p<0.05); ii) reduced platelet membrane expression of CD62P by 50±3% to collagen (p<0.0001); iii) reduced pAkt levels by 40±5% with collagen (p<0.001), and 38±2% with AA (p<0.0001); reduced pErk-2 levels by 66±5% with collagen (p<0.0001), and 38±4% with AA (p<0.005); iv) reduced the AA-induced ROS production by 35±3% (p<0.001). Also in platelets from HH patients holoTf significantly attenuated platelet reactivity. In fact, 40 µmol/l HoloTf: i) reduced platelet aggregation by 45±4% to ADP, 38±9% to collagen, 57±4% to epinephrine, and 49±5% to AA (versus each agonist alone: p<0.05); ii) reduced platelet membrane expression of CD62P by 54±6% to collagen (p<0.0001); iii) reduced pAkt levels by 37±6% with collagen (p<0.005), and 41±7% with AA (p<0.0001); reduced pErk-2 levels by 59±7% with collagen (p<0.0001), and 42±5% with AA (p<0.005); iv) reduced the AA-induced ROS production by 37±5% (p<0.001). The levels of transferrin saturation, but not of ferritin, were inversely correlated with the main platelet reactivity parameters; in particular, we found negative and significant correlations with the maximal aggregation to ADP (r:-0.332, p=0.05), collagen (r=-0.511, p=0.002), epinephrine (r=-0.404, p=0.02), AA (r=-0.460, p=0.009) and the CD62P expression at both basal conditions (r=-0.456, p=0.02) and after stimulation with collagen (r=-0.459, p=0.05) or AA (r=-0.629, p= 0.001).Conclusions. In healthy subjects, platelet exposure to holoTf, but not apoTf, reduced platelet reactivity as showed by a significant decrease of: i) aggregation, ii) membrane expression of CD62P, iii) activation of PI3K and MAPK pathways, and iv) oxidative stress. The effects of holoTf on platelet response were preserved in HH patients were transferrin saturation levels inversely correlated with platelet aggregation and activation. These results induce to hypothesize that in the association of high transferrin saturations and lower risk of cardiovascular diseases a role could be exerted by transferrin saturation effects on platelets. DisclosuresNo relevant conflicts of interest to declare.

Oral Squamous Cell Carcinoma Is Associated with a Low Thrombosis Risk Due to Storage Pool Deficiency in Platelets.

Venous thrombo-embolism (VTE) disease is the second most common cause of mortality in cancer patients, and evaluation and prevention of thrombosis risk is essential. VTE-associated risk varies according to the type of tumor disease. Oral cancer is the most frequent type of head and neck cancer, and it represents approximately 2.1% of all cancers worldwide. Most tumors are squamous cell carcinomas and are mainly due to tobacco and alcohol abuse. VTE risk associated with oral squamous cell carcinoma (OSCC) is low. However, many studies have shown that OSCC has the following biological features of cancers associated with a high thrombosis risk: modified thrombosis and fibrinolysis mechanisms; strong expression of procoagulant proteins; secretion of procoagulant microparticles; and production of procoagulant cytokines. Using an original mouse model of tongue squamous cell carcinoma, our study aimed to clarify this paradoxical situation. First, we showed that OSCC tumors have a pro-aggregatory phenotype and a high local thrombosis risk. Second, we found that tongue tumor mice do not have an elevated systemic thrombosis risk (the risk of an “at distance” thrombosis event such as lower extremity deep venous thrombosis or pulmonary embolism) and even show a reduction in risk. Third, we demonstrated that tongue tumor mice show a reduction in platelet reactivity, which explains the low systemic thrombosis risk. Finally, we found that tongue tumor mice present granule pool deficiency, thereby explaining the reduction in platelet reactivity and systemic thrombosis risk.

Ex vivo synergistic effects of apixaban with dual anti-platelet therapy (DAPT) in lowering platelet reactivity and thrombin generation (SEARCH)

Abstract Direct oral anticoagulants such as apixaban are increasingly being evaluated clinically for the secondary prevention of cardiovascular events; however, their effects on platelet function in combination with dual anti-platelet therapy (DAPT) have not been fully investigated. The purpose of this translational, in vitro study was to determine if apixaban via inhibition of thrombin generation exhibits synergistic activity with DAPT to reduce platelet reactivity. Consented subjects with a prior history (&amp;lt;12 mo) of ACS on DAPT regime with aspirin and clopidogrel (n=15; DAPT-C) or aspirin and ticagrelor (n=15; DAPT-T) were recruited, along with the age-matched healthy subjects as controls. Enrolled DAPT subjects had taken their prescribed regimen &amp;gt;7 days prior to blood collection. Platelet-rich plasma from TSC anticoagulated blood was prepared and treated in vitro with nothing, a carrier control or apixaban (40, 90 and 220 ng/mL). The range of 40 to 220 ng/mL brackets the expected apixaban exposure at steady state with all three approved regimens with the 40 ng/mL treatment corresponding to &amp;lt;5th percentile for the 2.5 mg bid dose, the 90 ng/mL corresponding to Cmax after the 2.5 mg bid or to Cmin after the 5 mg bid dose, and the 220 ng/mL corresponding to the Cmax after 10 mg bid dose. Platelet aggregation was measured by light transmission aggregometry (LTA) with tissue factor (TF) as agonist. Platelet p-selectin expression was measured by flow cytometry and thrombin generation was quantified. TF agonist was chosen to evaluate endogenous thrombin effects via Factor Xa activation (fXa). The CaCl2 concentration in the TF was titrated in the presence of peptide GPRP which minimized fibrin generation. The baseline maximal aggregation (MA) response was similar for both DAPT-T and DAPT-C (64%). Compared to DAPT alone, 90 and 220 ng/mL apixaban treatments decreased MA from 64% to 36% and 17% in the DAPT-T group and from 64% to 28% and 9% in the DAPT-C group (p&amp;lt;0.009), respectively. Platelet P-selectin expression decreased by 53% in the DAPT-T group with 220 ng/mL apixaban treatment (p&amp;lt;0.02) and in the DAPT-C group by 70% and 76% with 90 and 220 ng/mL apixaban treatment (p&amp;lt;0.004), respectively, compared to DAPT alone. Apixaban treatment (90 and 220 ng/mL) significantly increased thrombin generation lag time and time-to-peak results and significantly decreased peak thrombin in both DAPT groups (p&amp;lt;0.05). ACS patients on a DAPT regimen were susceptible to thrombin-mediated platelet activation via fXa. Apixaban treatment in vitro caused a larger reduction in thrombin-mediated platelet activation in the clopidogrel group compared to the ticagrelor group, consistent with ticagrelor having a more potent anti-platelet effect compared to clopidogrel. The in vitro addition of apixaban that corresponded to currently approved dosing regimens and at plasma drug levels routinely achieved demonstrated synergy with DAPT to reduce platelet reactivity and thrombin generation. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Bristol-Myers Squibb

Very preterm birth results in later lower platelet activation markers.

Premature birth entails an adverse cardiovascular risk profile, but the underlying mechanisms are insufficiently understood. Here, we employed an unbiased cardiovascular proteomics approach to profile former very preterm-born preschoolers. This observational study investigated differences in plasma concentrations of 79 proteins, including putative cardiovascular biomarkers between very preterm- and term-born children on average 5.5 years old (53.1% male) using multiple-reaction monitoring mass spectrometry. Very preterm-born (n = 38; median gestational age 29.6 weeks) compared to term-born (n = 26; 40.2 weeks) children featured lower plasma concentrations of platelet factor 4 (PLF4; -61.6%, P < 0.0001), platelet basic protein (CXCL7; -57.8%, P < 0.0001), and hemoglobin subunit beta (-48.3%, P < 0.0001). Results remained virtually unchanged when adjusting for complete blood count parameters, including platelet count. Conversely, whole blood hemoglobin was higher (+7.62%, P < 0.0001) in preterm-born children. Very preterm birth was associated with decreased markers of platelet activation among preschoolers. These findings are consistent with reduced platelet reactivity persisting from very preterm birth to a preschool age. Former very preterm-born preschoolers featured reduced levels of platelet activation markers. While lower platelet reactivity in very preterm-born compared to term-born infants in the first days of life was established, it was unknown when, if at all, reactivity normalizes. The current study suggests that platelet hyporeactivity due to very preterm birth persists at least up to a preschool age. "Immaturity of the hemostatic system" may be a persistent sequel of preterm birth, but larger studies are needed to investigate its potential clinical implications.

Perioperative Bridging/Cessation of Antiplatelet Agents: 2020 Update

Perioperative management of patients receiving antiplatelet therapy is common and must carefully balance the risk of ischemia or thrombosis with bleeding. Here we describe pathways of platelet aggregation unique to the perioperative period and mechanisms of commonly encountered antiplatelet medications, and review current literature evaluating strategies for antiplatelet management surrounding elective noncardiac and cardiac surgery. Antiplatelet therapies demonstrate unique risk profiles for stent thrombosis and bleeding that may be dependent on individual genetic polymorphisms. Use of scoring systems or point-of-care platelet function assays may identify patients especially vulnerable to alterations in perioperative antiplatelet management, and guide timing of surgery. Prior guidelines, which recommend a minimum 6-month delay in elective surgery for patients receiving dual-antiplatelet therapy following percutaneous coronary intervention (PCI), may be amended to 3 months in certain cases in which newer generation antiplatelet therapies are administered. While use of intravenous bridging agents may reduce platelet reactivity during cardiac surgery, there is no single antiplatelet strategy which consistently reduces rates of major bleeding or cardiovascular events. There is insufficient evidence to support any specific perioperative antiplatelet strategy. Cases should be individualized to balance the risks of stent thrombosis and bleeding. Current recommendations may be modified if the risk of delaying surgery outweighs the risk of stent thrombosis. It is reasonable to guide management by utilizing scoring systems and point-of-care platelet function assays.

Association of CYP2C19*2 polymorphisms and high on-treatment platelet reactivity in acute myocardial infarction or coronary artery in-stent restenosis patients during dual antiplatelet therapy

ObjectiveCytochrome CYP2C19 mutant allele is associated with high on-treatment platelet reactivity (HTPR) after standard clopidogrel treatment, which is accompanied by an increased risk of ischemic events. This study assesses the association of CYP2C19 mutant allele with HTRR and its response to antiplatelet therapy in acute myocardial infarction (AMI) or coronary artery in-stent restenosis (ISR). MethodsCYP2C19 (*2, *3, *17) and ABCB1 genotyping were performed in a series of 98 AMI or ISR patients treated with standard-dose clopidogrel (75 mg, term daily). Platelet activity was measured by VerifyNow assay, with HTPR defined as P2Y12 reaction units (PRUs) >208. The patients with HTPR (n = 48) were randomly assigned to receive either high-dose clopidogrel (150 mg, term daily; n = 24) or ticagrelor (90 mg, bid; n = 24). ResultsNo association was observed between CYP2C19*3, ABCB1 genotype and platelet reactivity (PR) (P > .05 for all). A significantly higher frequency of HTPR were observed in the patients carrying CYP2C19*2 homozygotes (n = 9; 9/11, 81.8%) than the CYP2C19*2 allele carriers (n = 26; 26/48, 54.2%, P < .05) and the non-carriers (n = 13; 13/39, 33.3%, P < .001). No patient exhibited HTPR after treatment with ticagrelor irrespective of CYP2C19 genotype, whereas 5 (100%) patients with CYP2C19*2 homozygotes, 7 (50%) CYP2C19*2 allele carriers and 3 (60%) non-carriers after high-dose clopidogrel remained HTPR (P < .05 for all). ConclusionA higher rate of HTPR was observed in AMI or ISR patients with CYP2C19*2 homozygotes than those with one or none allele. In patients carrying CYP2C19*2 homozygotes, ticagrelor is significantly more effective in reducing platelet reactivity than high-dose clopidogrel.

Effect of remote ischaemic conditioning on platelet reactivity and endogenous fibrinolysis in ST-elevation myocardial infarction: a substudy of the CONDI-2/ERIC-PPCI randomized controlled trial.

Remote ischaemic conditioning (RIC) has been shown to reduce myocardial infarct size in animal models of myocardial infarction. Platelet thrombus formation is a critical determinant of outcome in ST-segment elevation myocardial infarction (STEMI). Whether the beneficial effects of RIC are related to thrombotic parameters is unclear. In a substudy of the Effect of Remote Ischaemic Conditioning on clinical outcomes in STEMI patients undergoing Primary Percutaneous Coronary Intervention (ERIC-PPCI) trial, we assessed the effect of RIC on thrombotic status. Patients presenting with STEMI were randomized to immediate RIC consisting of an automated autoRIC™ cuff on the upper arm inflated to 200 mmHg for 5 min and deflated for 5 min for four cycles (n = 53) or sham (n = 47). Venous blood was tested at presentation, discharge (48 h) and 6-8 weeks, to assess platelet reactivity, coagulation, and endogenous fibrinolysis using the Global Thrombosis Test and thromboelastography. Baseline thrombotic status was similar in the two groups. At discharge, there was some evidence that the time to in vitro thrombotic occlusion under high shear stress was longer with RIC compared to sham (454 ± 105 s vs. 403 ± 105 s; mean difference 50.1 s; 95% confidence interval 93.7-6.4, P = 0.025), but this was no longer apparent at 6-8 weeks. There was no difference in clot formation or endogenous fibrinolysis between the study arms at any time point. RIC may reduce platelet reactivity in the first 48 h post-STEMI. Further research is needed to delineate mechanisms through which RIC may reduce platelet reactivity, and whether it may improve outcomes in patients with persistent high on-treatment platelet reactivity.

P4454N-terminal pro-brain natriuretic peptide and resistance to chronic therapy with 75 mg acetylsalicylic acid assessed by point-of-care test - prospective single center study

Abstract Background Acetylsalicylic acid (ASA) remains the principal medication for secondary prevention of atherosclerotic complications. Resistance to ASA (ASAres) is multifactorial and results in insufficient reduction of platelet reactivity through incomplete inhibition of thromboxane A2 (TXA2) synthesis. There is controversy regarding the optimal preventive ASA dose with common daily use of 75 mg in many European countries. Purpose The aim of our study is to reassess the prevalence and predictors of ASAres in contemporary cohort of coronary disease (CAD) patients (pts) on stable therapy with 75 mg ASA. Methods We studied 205 patients (36,6% females) with stable CAD and concomitant atherosclerotic disease history (ischemic stroke 10,2%, peripheral vascular disease 8,3%,) and type 2 diabetes in 39,5% on stable regimen 75 mg ASA for a minimum of 1 month (mean age 68,2±9,7 years, mean BMI 27,3±4,7 kg/m2). ASAres was defined as ARU (aspirin reaction unit) ≥550 using point-of-care VerifyNow Aspirin test. Exclusion criteria were: recent (up to 2 months) acute coronary syndrome, cancer, dermatological disease, epilepsy or other chronic neurological diseases, exacerbation of allergic disease, rheumatoid arthritis, periodontal disease, alcoholism, drug addiction, vegetarianism, veganism and other specific diets, and known thrombophilia. The population received standard concomitant preventive treatments including RAA blockade in 88,3%, beta-blockers in 85,9%, statins in 93,2%, and proton pump inhibitors (PPI) in 65,4%. History of infarction was present in 37% and mean left ventricular ejection fraction was 47% (18–75%). Results ASAres was detected in 11,7% of patients. Modest but significant correlations (Spearman's coefficient of rank correlation rho) were detected between ARU and C-reactive protein (CRP) (rs=0,15; p=0,030), N-terminal pro-brain natriuretic peptide (NT-proBNP) (rs=0,15; p=0,039), body weight (rs=0,22; p=0,0014), BMI (rs=0,207, p=0,0029). No significant differences in ASAres we found with regard to sex, other risk factors or concomitant medication, including PPI. However, in ASAres pts median concentrations of NT-proBNP were significantly higher (median 311 vs. 646pg/ml; p=0,046). In multivariate analysis NT-proBNP emerged as the only independent predictor of ASAres (AUC=0,626; p=0,027 with threshold value of 327,3 pg/ml resulting with negative predictive value of 16,98% and positive predictive value of 93,95% for ASAres). Conclusion ASAres has significant prevalence in this secondary prevention CAD cohort treated with 75 mg daily dose. NT-proBNP was identified as the only independent predictor in multivariate analysis. This finding may be important especially for pts with heart failure of ischemic etiology. The implications of switching into 100 mg or higher ASA doses remain to be investigated. Acknowledgement/Funding study was supported from unrestricted research grant from Aflofarm SA

Platelet function and activation markers in primary hypercholesterolemia treated with anti-PCSK9 monoclonal antibody: A 12-month follow-up

Background and aimsIn the association between hypercholesterolemia (HC) and thrombotic risk platelet hyper-reactivity plays an important role. The inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) to reduce plasma LDL-cholesterol merges as effective therapeutic strategy to prevent cardiovascular (CV) events. Aim of this study was to verify whether a treatment up to 12 months with the monoclonal antibodies (mAbs) anti-PCSK9 influences platelet function in primary HC. Methods and resultsIn patients affected by primary HC (n = 24), all on background of statin and 17 on acetyl salicylic acid (ASA), platelet function parameters were evaluated at baseline up to 12 months of treatment with the mAb anti-PCSK9 alirocumab or evolocumab.From baseline, the treatment with anti-PCSK9 mAbs: i) in ASA HC patients, significantly decreased platelet aggregation detected in platelet-rich plasma by light transmission aggregometry and in whole blood Platelet Function Analyzer-100 assay; ii) in all HC patients, significantly decreased platelet membrane expression of CD62P and plasma levels of the in vivo platelet activation markers soluble CD40 Ligand, Platelet Factor-4, and soluble P-Selectin. Furthermore, CD62P expression, and sP-Selectin, PF-4, sCD40L levels significantly correlated with serum PCSK9. ConclusionBesides markedly lowering LDL-c levels, our results suggest that HC patients benefit from anti-PCSK9 mAb treatment also for reducing platelet reactivity and increasing platelet sensitivity to the inhibitory effects of aspirin. These effects on platelets could play a role in the reduction of CV event incidence in patients treated with PCSK9 inhibitors.

Physical proximity and functional cooperation of glycoprotein 130 and glycoprotein VI in platelet membrane lipid rafts

ObjectiveClinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)‐6 forms a complex with soluble IL‐6 receptor α (sIL‐6Rα) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase Cγ2 (PLCγ2) in collagen‐induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3. Methods and resultsWe conducted complementary in vitro experiments to show that the IL‐6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL‐6‐sIL‐6Rα complex to activate the kinase Syk and the substrate PLCγ2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130‐Janus tyrosine kinase (JAK)‐STAT3 signaling abolished the cross‐activation and reduced platelet reactivity to collagen. ConclusionThese results demonstrate cross‐talk between collagen and IL‐6 signal pathways. This cross‐talk could potentially provide a novel mechanism for inflammation‐induced platelet hyperactivity, so the IL‐6‐GP130‐JAK‐STAT3 pathway has been identified as a potential target to block this hyperactivity.

A polyphenol-rich diet is associated with decreased platelet aggregation in breast cancer patients

It is well documented that plant polyphenols have both anti-cancer and anti-platelet effects. Hence, the aim of this work was to investigate a relationship between dietary intake of polyphenols and platelet aggregation in newly-diagnosed breast cancer patients. The nutritional value of a diet, including dietary intake of plant polyphenols was estimated. Platelet aggregation was induced with arachidonic acid (0.5 mmol/l), collagen (3.2 μg/ml) or ADP (6.4 μmol/l) and measured using multiple electrode aggregometry (Multiplate&lt;sup&gt;®&lt;/sup&gt;) in whole blood. It was found that platelet aggregation was significantly higher in the low polyphenol intake group than the high intake group: the respective values (area under the aggregation curve recorded in units; U) were arachidonic acid: 84.8 vs. 65.3, P=0.003; ADP: 76.5 vs. 67.8, P=0.006; collagen 79.5 vs. 64.3, p=0.024 respectively. The study indicates, for the first time, an association between diet rich in polyphenols and reduced platelet reactivity in breast cancer patients.

Impact of lipoprotein apheresis on thrombotic parameters in patients with refractory angina and raised lipoprotein(a): Findings from a randomized controlled cross-over trial

Raised lipoprotein(a) [Lp(a)] is a cardiovascular risk factor common in patients with refractory angina. The apolipoprotein(a) component of Lp(a) exhibits structural homology with plasminogen and can enhance thrombosis and impair fibrinolysis. The objective of the study was to assess the effect of lipoprotein apheresis on markers of thrombosis and fibrinolysis in patients with high Lp(a). In a prospective, single-blind, crossover trial, 20 patients with refractory angina and raised Lp(a)>50mg/dL were randomized to three months of weekly lipoprotein apheresis or sham. Blood taken before and after apheresis/sham was assessed using the Global Thrombosis Test, to assess time taken for invitro thrombus formation (occlusion time) and endogenous fibrinolysis (lysis time), as well as von Willebrand Factor, fibrinogen, D-dimer, thrombin/anti-thrombin III complex, prothrombin fragments 1+2, and thrombin generation assays. Lp(a) was significantly reduced by apheresis (100.2 [interquartile range {IQR}, 69.6143.0] vs 24.8 [17.2,34.0] mg/dL, P=.0001) but not by sham (P=.0001 between treatment arms). Apheresis prolonged occlusion time (576±116 s vs 723±142 s, P<.0001) reflecting reduced platelet reactivity and reduced lysis time (1340 [1128, 1682] s vs 847 [685,1302] s, P=.0006) reflecting enhanced fibrinolysis, without corresponding changes with sham. Apheresis, but not sham, reduced von Willebrand Factor (149 [89.0, 164] vs 64.2 [48.5, 89.8] IU/dL, P=.0001), and fibrinogen (3.12±0.68 vs 2.20±0.53g/L, P<.0001), and increased prothrombin fragments 1+2 (158.16 [128.77, 232.09] vs 795.12 [272.55, 1201.00] pmol/L, P=.0006). There was no change in D-dimer, thrombin/anti-thrombin III complex, or thrombin generation assay with apheresis or sham. Lipoprotein apheresis reduces Lp(a) and improves some thrombotic and fibrinolytic parameters in patients with refractory angina.

Half-dose ticagrelor versus high-dose clopidogrel in reducing platelet reactivity in acute coronary syndrome patients with high on-clopidogrel platelet reactivity (divide study).

High on-treatment platelet reactivity (HTPR) after clopidogrel administration in patients with acute coronary syndrome (ACS) has been associated with an increased risk of adverse events. Our previous studies reported that half-dose ticagrelor provides a similar inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation as standard-dose ticagrelor, but half-dose of ticagrelor has not been studied in Chinese ACS patients with HTPR. This study aimed to compare the antiplatelet action of half-dose ticagrelor with high-dose clopidogrel in ACS patients with HTPR. In this single-center randomized controlled trial, 80 (of 418 screened, 19.13%) ACS patients with HTPR while on clopidogrel were randomized to either half-dose ticagrelor (90mg LD, then 45mg twice daily) or high-dose clopidogrel (150mg once daily). Platelet function was assessed by thromboelastography (TEG) and light transmission aggregometry (LTA), and adverse events were monitored throughout the study for 30days. The ADP-induced platelet inhibition rate (IR) as measured by TEG was significantly higher for half-dose ticagrelor compared with high-dose clopidogrel (70.40% [61.10%-91.70%] vs. 44.25% [34.67%-79.07%], p = 0.001). The repeated HTPR rate was dramatically higher for high-dose clopidogrel compared with half-dose ticagrelor (6 of 32, 18.75% vs. 1 of 35, 2.85%; p = 0.04). No patients in either treatment group exhibited a major bleeding event or other adverse events. In ACS patients with HTPR, half-dose ticagrelor is more effective than high-dose clopidogrel in reducing platelet reactivity (NCT03062462).

A switch to a raltegravir containing regimen does not lower platelet reactivity in HIV-infected individuals.

Platelet hyperreactivity and increased platelet-monocyte aggregation (PMA) are associated with increased cardiovascular risk and inflammation. In a previous cross-sectional study, individuals using a raltegravir (RAL)-based regimen were found to have reduced platelet reactivity and PMA compared with other antiretroviral regimens. Our aim was to investigate whether switching from a nonintegrase inhibitor regimen to a RAL-based regimen reduces platelet reactivity or PMA. An investigator initiated, single-centre, prospective randomized, open-label, blinded endpoint trial. Forty HIV-infected adults using a nonintegrase inhibitor containing regimen with undetectable viral load were randomized to either continue their regimen or switch to a RAL-based regimen for 10 weeks, continuing the same backbone. The primary outcome was the change in platelet reactivity at week 10, which was determined as the expression of the platelet activation marker P-selectin and binding of fibrinogen before and after ex-vivo stimulation with different platelet agonists. Secondary outcomes included PMA, plasma markers of platelet activation and markers of inflammation and immune cell activation. Twenty-one participants were enrolled in the continuation group and 19 in the RAL group. Baseline characteristics were comparable between groups. There were no differences in the change in platelet reactivity to either platelet agonist at week 10, nor in plasma markers of platelet activation. PMA, C-reactive protein, T-cell activation (CD38HLA-DR) and monocyte (CD14CD16) subsets. Switching a nonintegrase inhibitor containing regimen to a RAL-based regimen does not reduce platelet reactivity, platelet-leukocyte aggregation, inflammation and immune activation in virologically suppressed HIV-infected individuals. NCT02383355.