Increased Platelet Aggregation Research Articles

Prognostic value of the psychoemotional status in patients with coronary artery disease: focus on platelet hemostatic system

Aim. To assess the effect of psychological distress on platelet morphology depending on the severity of mixed anxiety-depressive disorder (MADD) in patients with coronary artery disease (CAD). Material and methods . The study included 112 patients (mean age — 60,9±1,46 years), 84 of whom were diagnosed with CAD, class II-III angina of effort and stage I-III hypertension. The assessment of psychoemotional status and type D personality was carried out using the Hospital Anxiety and Depression Scale (HADS) and the Type D Scale-14 (DS14), respectively. The detection of platelet phenotype was performed on a high-tech XN-1000 hematology analyzer using flow cytometry. Results. Participants were divided into two groups: group I (n=40) — patients with MADD; group II (n=44) — patients without anxiety and depression. Assessment of the interconnected effect of MADD and psychological distress on the platelet profile in patients with CAD revealed differences between groups by the platelet morphology. The average platelet count (PLT) in patients of group I was 312,1±17,17x10 9 /L, and in patients with type D personality — 316,7±17,54x10 9 /L, which significantly prevailed the same parameter in comparison group — 220,4±1,48x109/L (p<0,001). The average platelet distribution width (PDW) was significantly higher in group I compared with group II (17,0±0,94 fL vs 13,6±0,76 fL (p<0,05)). The mean platelet volume (MPV) in patients of group I (11,3±0,42) was significantly higher than in the comparison and control groups: 9,7±0,23 and 8,7±0,28 fL, respectively (p<06001). According to logistic regression, the independent predictors of increased platelet aggregation in cardiac patients were depression Conclusion. Increased functional activity of platelets in patients with CAD and hypertension is due to the effect of MADD associated with type D personality.

Periodontal status, vascular reactivity, and platelet aggregation changes in rats submitted to hypercholesterolemic diet and periodontitis

Periodontitis can corroborate with development and progression of atherosclerosis and a possible bidirectional interaction between both pathologies has been hypothesized. The aim of this work was to study the interactions between diet-induced hypercholesterolemia and ligature-induced periodontitis in Wistar rats submitted to both conditions. Animals were divided into four experimental groups: C (control: standard diet without periodontitis), Perio (periodontitis plus standard diet), HC (high cholesterol diet without periodontitis), and HC+Perio (high cholesterol diet plus periodontitis). The diets were offered for 45days and a silk ligature was applied in the lower first molars of Perio and HC-Perio animals on day 34 and maintained for 11days until euthanasia. The mandibles were excised, and alveolar bone loss was determined by macroscopic and micro-tomographic (µ-CT) imaging. Blood samples were obtained, and platelet aggregation was induced in plasma rich in platelets by adenosine diphosphate (ADP) and collagen. Endothelium-dependent vascular reactivity and protein expression of endothelial (eNOS), phosphorylated endothelial (peNOS), and inducible (iNOS) nitric oxide synthases were evaluated in aorta samples. The HC diet combined with periodontitis (HC+Perio group) was associated with an increased alveolar bone loss, when compared to the other groups. Both in Perio and HC groups, platelet aggregation induced by ADP or collagen was increased, while maximum aortic relaxation induced by acetylcholine was decreased. Periodontitis or HC diet alone decreased the expression of peNOS and HC diet increased the expression of iNOS. In contrast, no additive or synergistic effects were found in vascular reactivity or in platelet aggregation when the two conditions were associated (HC+Perio group). Hypercholesterolemia accelerated the process of bone loss induced by periodontitis while a high cholesterol diet or periodontitis individually increased platelet aggregation and vascular reactivity in rats without additive or synergistic effects, when associated.

Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes

20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite which is known to increase platelet aggregation and cardiovascular risk. In this study, nine non-steroidal anti-inflammatory drugs (NSAIDs) selected by chemical structures were screened to determine their effects on the glucuronidation of 20-HETE using human liver microsomes (HLMs). Then, the combined effects of the selected NSAID and genetic polymorphisms in UDP-glucuronosyltransferase (UGT) were investigated. Among the tested NSAIDs, diclofenac was the strongest inhibitor of 20-HETE glucuronidation with an IC50 value of 3.5 μM. Celecoxib, naproxen, mefenamic acid, ibuprofen, and indomethacin showed modest inhibition with IC50 values of 77, 91, 190, 208, and 220 μM, respectively, while acetylsalicylic acid, rofecoxib, and meloxicam did not inhibit 20-HETE glucuronidation. Glucuronidation of 20-HETE by UGT2B7 and UGT1A9 recombinant enzymes was significantly inhibited by indomethacin, mefanemic acid, diclofenac, ibuprofen, naproxen, and celecoxib (P < 0.001). In addition, diclofenac exhibited a competitive inhibition mechanism with the Km value of 20-HETE glucuronidation increasing from 23.5 μM to 62 μM in the presence of 3.5 μM diclofenac. Diclofenac further decreased 20-HETE glucuronidation in HLMs carrying UGT2B7*2 alleles compared with the wild-type HLMs. The results from this study would be useful in understanding the alteration of 20-HETE levels in relation to NSAID and UGT genetic polymorphisms.

Impact of active smoking on the immature platelet fraction and its relationship with the extent of coronary artery disease.

Smoking represents a major cardiovascular risk factor, due to the induction of oxidative stress and low-grade, continuous, inflammation that contribute to promote atherothrombosis. However, the mechanisms leading to increased platelet aggregability associated with smoking are only partially defined. A potential role has been hypothesized for immature platelets, a younger and potentially more reactive fraction, previously associated with the main determinants of coronary artery disease (CAD). Therefore, the aim of our study was to define the impact of smoking on the immature platelet fraction (IPF) and its relationship with prevalence and extent of coronary artery disease. We enrolled a cohort of consecutive patients undergoing coronary angiography in a single centre. Significant CAD was defined as at least 1 vessel stenosis >50%, while severe CAD was defined as left main and/or three-vessel disease. IPF was measured at admission by routine blood cell count (Sysmex XE-2100). We included in our study 2553 patients who were divided according to smoking status (active smokers: 512; nonactive smokers: 2041). Smokers were younger, more frequent males, with lower rate of diabetes mellitus, previous PCI and previous CABG (P<.001, respectively) and were in treatment less often with ARB, BB, nitrates, statins, ASA, clopidogrel, CCB and diuretics (P<.001, respectively) as compared to nonactive smokers. Higher percentage of smokers was observed in patients with higher IPF values, and at multivariate analysis, active smoking resulted as an independent predictor of higher IPF (adjusted OR [95% CI]=1.59[1.03-2.45], P=.035). Among smokers, higher IPF was associated with lower ejection fraction (P=.034), percentage of acute coronary syndrome (P=.002) and platelet count (P<.001) compared to ones with lower IPF. However, the IPF (according to quartiles values) was not associated with the prevalence and extent of CAD (82.5%, 80.4%, 86.1% and 80.9%, from 1st to 4th quartile, respectively, adjusted OR[95% CI]=0.98[0.79-1.23], P=.89) and severe CAD (31%, 31.1%, 39.1% and 35.2%, from 1st to 4th quartile, respectively, adjusted OR[95% CI]=1.03[0.86-1.23], P=.76). The present study shows an independent association between active smoking and the levels of immature platelet fraction in patients undergoing coronary angiography. However, among active smokers, IPF did not result as an independent predictor of CAD or severe CAD.

Physiological response of hemostasis of piglets to excessive thermal effects

Currently, the pig feels the need to further clarify aspects of the physiology of pigs. It is very important to clarify the changes of functional parameters of piglets at the impact of any environmental factors. Negative environmental impact can often particularly adversely affect hemostasis. However, changes its parameters clarified weakly. Objective: to assess changes in hemostasis in piglets undergoing overheating. The research involved 42 pigs at the age of 2.5 months breed: large white, which has experienced overheating during 3 hours as a result of breakdown of air conditioning system. The control group included 32 healthy animals kept in normal conditions of a pigsty. The experienced piglets determined significant increase of platelet aggregation that occur spontaneously and in response to stimulation. Experienced pigs blood coagulation was found high and significant weakening of fibrinolysis. Apparently, the overheating affects animals’ haemorheology and significantly inhibits anabolism. It is clear that in piglets, which suffered from overheating, activated hemostatic system, which affects the microcirculation and inhibits growth. For this reason, there is a great need in the search approaches for leveling in piglets undergoing an episode of overheating, excessive activity of blood coagulation and increased platelet aggregation.

Dosadašnja saznanja o etiopatogenezi i mogućnostima terapije Kovid-19

Currently, over 35 million people in the world are infected with the COVID-19 and over a million have died. The pandemic character of the disease has imposed dynamic research both in the field of clarification of the etiopathogenetic mechanisms of the disease and in the field of possible therapy. The amount of scientific knowledge has increased dozens of times in the last nine months, but we are still not even close to define an effective and safe therapy. The knowledge that COVID-19 is not only a respiratory but also a multisystem disease, which affects almost all organs, gave us opportunities for therapeutic adjustments. Strong oxidative stress, silent chronic hypoxia, increased coagulability, and increased platelet aggregation are just some of the mechanisms in the development of the disease. Cardiovascular consequences and vascular endothelial dysfunction are thought to be mainly responsible for death with a dangerous cytokine storm and acute respiratory distress syndrome. Having all this in mind in the therapeutic sense, it is proposed to use antibiotics, high doses of vitamin C, blood transfusion, immunoglobulin, interferon, anti-IL-6 antibodies, small doses of Dexamethasone or other corticosteroids, specific antiviral drugs such as favipiravir and remdesivir. The use of chloroquine is excluded from the therapeutic protocols of the World Health Organization and the Centers for Disease Control and Prevention (United States of America). For preventive and supportive purposes, high doses of vitamin D, vitamin C, zinc, probiotics, Alpha-lipoic acid, and other supplements are recommended. The use of anti-inflammatory, analgo-antipyretics drugs is considered justified, as well as the use of small doses of acetylsalicylic acid. Over 70 other drugs are currently being tested in over 400 clinical studies. In patients who already use corticosteroids by inhalation or intranasally due to asthma, chronic obstructive pulmonary disease, allergic rhinitis, or biological drugs due to immunomodulatory inflammatory diseases, this therapy should not be changed and it is not a prerequisite condition for more severe forms of the disease. The specifics of the infection in special populations such as children or pregnant women must also be taken into consideration.

Restoring the functional state of the hemostasis system in piglets experiencing an episode of transportation

Negative environmental impact can often adversely affect the functional properties of piglets and significantly weaken their viability. It is often possible to judge this by the dynamics of the activity of hemostasis indicators. The level of activity of platelets and coagulation systems very early change activity in the case of negative influences from the outside, causing the appearance of overt hemostasiopathy. This condition leads to the disruption of hemocirculation in the capillaries and a decrease in the severity of metabolism in the tissues. These changes lead to a slowdown in growth and a general weakening of animals, thereby leading to great economic damage. It was found that the appearance of transport stress in piglets leads to increased platelet aggregation and activation of homocoagulation mechanisms during inhibition of fibrinolysis. Due to the fact that this situation is very common, the possibility of eliminating these dysfunctions was studied. The study tested fumaric acid at 0.1 g/kg in the form of a feed additive for 20 days in piglets that underwent long-term transportation. As a result of using this supplement, it was possible to reduce platelet activity and blood coagulation and increase fibrinolysis activity, bringing them to control values. As a result of the study, it was found that the intake of fumaric acid in the body of piglets that have experienced the influence of a high-intensity medium optimizes the functional status of their hemostasis. This minimizes their risk of microthrombosis and weakened animal growth.

Oxygenation extremes after traumatic brain injury transiently affect coagulation

IntroductionTrauma patients may become hypoxic or iatrogenically hyperoxic in the early post-injury period. While both extremes of oxygenation may be harmful following injury, the mechanism has yet to be elucidated. We hypothesized that hypoxia or hyperoxia would induce changes in coagulation, creating a secondary insult exacerbating the primary injury. Materials and methodsMice underwent traumatic brain injury (TBI) or sham and were subsequently exposed to room air or brief hypoxia (15% FiO2) then sacrificed at intervals. Another cohort of uninjured mice underwent more prolonged hypoxia (10% FiO2) or hyperoxia (60% FiO2). Platelet function was evaluated by ADP- or ASPi-induced impedance aggregometry and viscoelastic coagulation parameters were assessed with rotational thromboelastometry. ResultsIn uninjured mice, ADP-induced platelet aggregation was acutely increased after prolonged hypoxia, but this difference did not persist at 6 h. Hypoxic and hyperoxic TBI mice had increased ADP induced platelet aggregation at 1 h compared to the normoxia group. TBI mice demonstrated an early increased platelet aggregation after hypoxia or hyperoxia. Viscoelastic coagulation parameters were similar between hypoxic, hyperoxic and room air groups at 1 h and 6 h. However, ROTEM clotting time and clot formation time were prolonged and maximal clot firmness were lower in sham TBI/hypoxia mice at 24 h. ConclusionPost-TBI hypoxia and hyperoxia resulted in transiently increased platelet aggregation in response to ADP, without lasting effecting on platelet function or coagulation. Hypoxia also induced delayed hypocoagulability after platelet aggregation normalized. Brief changes in oxygenation may temporally affect coagulation and platelet aggregation, which could contribute to physiologic secondary injury after trauma.

Effect of eugenolon white blood cells and corticosterone in sub-acute restraint stress-induced wistar albino rats

Introduction: The effect of eugenol on white blood cells (WBCs ) and corticosteroneis known. However, pharmacological properties and activities of eugenol such as anti-inflammatory, antioxidant, anesthetic, muscle relaxant, improved motor coordination, and increased red blood cells were reported in stress - induced rats. Objective: To evaluate the effect of eugenol on WBCs in sub -acute restraint stress-induced Wistar albino rats. Materials and Methods: Female Wistar albino rats (n = 30) weighing (150–220 g) were divided into five groups with six animals in each group, Group I: normal control; II : vehicle polyglycerol treated (PG); III: eugenol treated alone - TA (150 mg/kg/i.p for 15 days); IV : stress -induced (SA-stress alone), and V: stress followed by treated (T/S) with. Blood samples were collected at the end ofthestudyand total leucocyte count (TLC), differentiation leucocyte count (DLC), platelet count (PC) and concentration of corticosterone were estimated. Results: The TA group showed significant (p group. The platelet count was significantly less in the SA group as compared to T/ S group (p effect of eugenol on DLC did not show a significant difference with respect to neutrophil and lymphocytes count in T/S groups, when compared with other groups, whereas, no significant differences were observed between basophils, monocytes, and eosinophils of T/S when compared with SA group. No significant differences were observed in corticosterone levels in all groups. Conclusion: Acute restraint stress induced distribution of WBC was not significantly attenuated by eugenol and no changes were observed in corticosterone level. However, eugenol increases platelet aggregation. Keywords: Stress, Eugenol, Total leukocyte count, Differentiation leukocyte count, Platelets, Corticosterone.

AGGREGATION AND SIZE OF PLATELETS IN CHILDREN WITH BURN DISEASE

Introduction. One of the main factors involved in the pathogenesis of burn disease consists in the disturbance of microcirculation and haemostasis, caused by increased platelet aggregation. Mechanisms underlying the enhancement of platelet aggregation are poorly understood. Main results were obtained for adult patients at the onset of the burn disease, with no similar data on paediatric patients being available. There is evidence of a relationship between the size of platelets (MPV) and their functionality.Aim. To undertake a study of spontaneous and ADP-induced platelet aggregation and their size in children with burn disease.Materials and methods. We studied the aggregation and size of platelets in children aged 3–17 years, in whom burn areas covered 10–70 % of the body surface at the onset of the disease and before discharge. Spontaneous platelet aggregation was analysed under conditions of induced shear flow, whereas ADP-induced aggregation was studied employing a turbidimetric method. The study of platelet sizes was carried out using a conductometric method.Results. Spontaneous platelet aggregation increases significantly in children with burn disease. The integrated optical density of the formed aggregates, their area and perimeter were estimated for the first time. These indicators remained elevated even after burn wounds had been completely closed. In burn disease, changes in ADP-induced platelet aggregation were multidirectional in nature. Mean platelet volume (MPV) was increased during the acute period of burn disease, decreased during the period of toxaemia, and normalised by the time of patients’ discharge. Changes in MPV did not affect the aggregation properties of platelets. There was no correlation between the degree of aggregation and the severity of hyperfibrinogenemia. The blood of burn patients contained a large number of activated platelets, which was the reason for the increase in their spontaneous aggregation, not requiring the participation of exogenous inducers.Conclusion. An increase in spontaneous platelet aggregation was observed in children after a thermal injury, which remained elevated until the burn wounds were completely closed. A significant increase in the number of activated platelets constituted the reason for the increase in spontaneous platelet aggregation.Conflict of interest: the authors declare no confl ict of interest.Financial disclosure: the study had no sponsorship.

Platelet reactivity expressed as a novel platelet reactivity score is associated with higher inflammatory state after coronary artery bypass grafting.

Despite therapy, patients operated using a cardiopulmonary bypass demonstrate increased platelet aggregation, which rebounds to above preoperative levels. The aim of the study was to test the interaction between platelet reactivity/activation and selected inflammatory markers in the post-operative period. In total, 103 patients with non-ST elevation acute coronary syndrome (NSTE-ACS) who were not eligible for percutaneous coronary interventions (PCI), and required urgent revascularization, were included. Platelet reactivity was measured using the PFA-100 platelet analyser, multiple electrode aggregometry, and was expressed as a novel platelet reactivity score (PRS). Patients were divided using their PRS scores into high platelet relativity or low platelet reactivity subgroups (HPR or LPR). Platelet basal activation was measured using immunoassays for soluble P-selectin and soluble CD40L. We measured high-sensitivity C-reactive protein (CRP), and used immunoassays for tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) as inflammation markers. Significant differences between HPR and LPR groups were found for CRP (mg/l): 81.5 vs. 44.6, p < 0.02; and TNF-α (pg/l): 3.51 vs. 2.37, p < 0.02. A significant association was found between CRP, TNF-α, IL-6 and platelet reactivity (platelet reactivity score). Cohen's k showed: CRP = 0.49, p < 0.0001, TNF-α = 0.37, p < 0.002. Perioperative myocardial infarction and rhythm disturbances occurred more frequently in the high platelet reactivity group: 7 (16.3%) vs. 2 (3.3%), p < 0.04, and 9 (20.9%) vs. 4 (6.7%), p < 0.04, respectively. Inflammatory parameters CRP and TNF-α are strongly associated with platelet reactivity (expressed as PRS) in cardiopulmonary bypass graft patients. Platelet hyperreactivity in the early post-operative period combined with a systemic inflammatory state correlates with a higher risk of post-operative rhythm disturbances and myocardial infarction.

Identifying miRNA Biomarkers and Predicted Targets Associated with Venous Thromboembolism in Colorectal Cancer Patients

Introduction Venous thromboembolism (VTE) is the second leading cause of mortality among cancer patients and is potentially preventable through the use of anticoagulation therapy. Risk models exist to guide use of prophylactic therapy but have low positive predictive value. New biomarkers are needed, particularly in intermediate-risk cancer types. Extracellular small RNAs, such as miRNAs, are promising biomarkers that have been implicated in tumor-dependent modification of platelets, a key component of thrombus formation. In this pilot study, we explore miRNAs as potential biomarkers for VTE risk in patients with colorectal cancer. Methods We conducted a case-control study utilizing specimens from a population enrolled in a prospective colorectal cancer biorepository at the Cleveland Clinic. Cases were defined as patients who developed VTE, including deep vein thrombosis and pulmonary embolism, within 6 months after cancer diagnosis and had their blood drawn prior to VTE. Cases were matched to controls (who did not have VTE and had a minimum of 6 months survival after cancer diagnosis) on a 1:2 ratio based on age, sex, cancer stage at diagnosis, and cancer treatment received prior to blood collection for a total of 21 patients. Total RNA from plasma specimens were sequenced on a Ion Proton platform (Thermo Fisher). Sequencing data were analyzed using the limma-voom R package. As this study was meant to be exploratory, miRNA were determined to be differentially expressed at a corrected Benjamini-Hochberg false detection rate (FDR) &amp;lt; 0.2. Target genes of differentially expressed miRNAs were predicted using mirDB and target gene pathways constructed with PANTHER. Results The study population had a median age of 65 (IQR 51-72). Of these patients, 85.7% were male; 42.9% had stage I/II cancer, 42.9% stage III cancer, 14.3% stage IV cancer; 71.4% had received no treatment prior to blood collection, 14.3% received chemo/chemoradiation therapy, and 14.3% received surgery. A total of 2426 unique miRNAs (median 1524, IQR 1371-1665) were expressed in the study group. Of these, 9 miRNAs were significantly differentially expressed (FDR &amp;lt; 0.2) and downregulated in cases compared to controls: hsa-miR-4451, 942-3p, 8063, 3132, 3118, 105-5p, 891a-5p, 200a-5p, and 6832-3p. From these miRNAs, 609 target genes were predicted and classified into 75 pathways, including angiogenesis, G-protein coupled receptors (GPCRs), inflammation mediated by chemo/cytokines, and integrin signaling. Target genes within these notable pathways included EPHA3, PDGFA, PTK2/FAK1, and IL15. Conclusions We identified 9 significantly downregulated miRNAs in the blood of colorectal cancer patients who developed VTE compared to controls in this pilot study. These data suggest that colorectal cancer patients may express unique miRNA profiles prior to VTE development which may be useful as biomarkers in future predictive models. In addition, this study identified potential new mechanistic targets for understanding cancer-associated thrombosis. While the role of GPCRs, integrins, and inflammation in platelet activation and function is widely known, this study also identified factors within the angiogenesis pathway that have been linked to increased platelet aggregation and tissue factor activation. Thus, downregulation of inhibitory miRNAs may cause disinhibition of pathways important for platelet and vascular function and other prothrombotic factors. Disclosures Khorana: Janssen: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy. McCrae:Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Pitfall of Light Transmission Aggregometry-Based Assessment of Platelet Function in Acute Ischemic Stroke Patients

Background: The utility of light transmission aggregometry (LTA)–based assessment of platelet function in acute ischemic stroke patients remains controversial. This study aimed to clarify why LTA failed to estimate platelet function in acute ischemic stroke patients. Methods: Using LTA, we evaluated the platelet aggregation abilities of citrated blood samples from 22 acute noncardiogenic ischemic stroke patients prior to treatment and compared them with those of 65 heathy volunteer controls. Platelet counts and mean platelet volumes (MPV) of citrated blood and platelet-rich plasma (PRP) prepared for LTA were evaluated simultaneously. Using a hematology analyzer, we also measured and compared the aggregation-prone properties of platelets in the hematology analysis process between patient and control samples. Results: Although platelets aggregated more easily and frequently in patient samples (P < .01), the maximum aggregation rate (MA%) of LTA was paradoxically lower in patients than in controls (P < .05). The PRP/citrated blood ratio of platelet counts and MPV were significantly lower in patients than in controls (P < .05). Conclusions: Our results suggest that MA% of LTA is erroneously displayed as lower values than the actual status in patients with increased platelet aggregation ability such as acute ischemic stroke because activated large platelets are preaggregated and thus decreased in the PRP on LTA.

Association of T715P (RS6136), M62I (RS2228315), S290N (RS6131), V640L (RS6133) polymorphisms in the P-selectin gene and its ligand with acetylsalicylic acid resistance in patients with coronary artery disease after coronary artery bypass grafting

Aim.To study the association of T715P (rs6136), M62I (rs2228315), S290N (rs6131), V640L (rs6133) polymorphisms in the P-selectin gene with resistance to acetylsalicylic acid (ASA) in patients with coronary artery disease after coronary artery bypass grafting (CABG).Material and methods. The study included 90 patients aged 61,5±6,9 years (70 men and 20 women) with II-IV functional class (FC) angina pectoris, according to the Canadian Cardiovascular Society grading. The atherosclerotic nature of coronary artery disease is confirmed by coronary angiography. Patients stopped taking antiplatelet agents before CABG for at least 5 days. The aggregation study was carried out with an optical aggregometer using ADP inducers (5 pM) and arachidonic acid (1 µМ) before CABG, on 1-3 and 8-10 days after surgical treatment.DNA samples were examined for the presence of T715P (rs6136), M62I (rs2228315), S290N (rs6131), V640L (rs6133) polymorphisms in the P-selectin gene using realtime PCR with allele-specific primers.Results.When comparing aPTT, fibrinogen level, platelet aggregation activity with ADP inducers (5 µМ) and arachidonic acid (1 µМ), no differences were found among groups of patients with homozygous and heterozygous variants of the studied polymorphisms genotypes, both before and on 1-3, 8 -10 days after CABG. Regarding presence of ASA resistance, patient groups with homozygous variants of genotypes (T715P (rs6136), M62I (rs2228315), S290N (rs6131), V640L (rs6133)) did not statistically differ in prevailing or rare alleles from the corresponding groups with heterozygous genotypes. In the first 10 days of the postoperative period, thrombotic events (4,4%) were observed in 4 patients in the study group: acute myocardial infarction, acute cerebrovascular accident. Regarding frequency of adverse events in the first 10 days after CABG, between groups of patients with homozygous variants of the studied genotypes (T715P (rs6136), M62I (rs2228315), S290N (rs6131), V640L (rs6133) in prevailing allele and groups with heterozygous variants of the corresponding genotypes there were also no statistically significant differences.Conclusion.Rs6133, rs6163, rs2228315, rs6131 polymorphisms in the platelet P-selectin gene are not associated with ASA resistance and are not associated with increased platelet aggregation activity in patients with coronary artery disease. The rare T, C, G, A alleles of the studied polymorphisms do not lead to an increase in the risks of adverse events in the first 10 days after CABG.

Prothrombotic states in women with infertility and psychosomatic disorders

Failures of in vitro fertilization (IVF) may be associated with prothrombotic states, the circulation of antiphospholipid antibodies (APA). 93 women with infertility were screened: the first group 32 women without severe psychosomatic disorders; the second group 61 women with psychosomatic disorders. The control group consisted of 30 fertile women. We carried out measurements in the blood serum of the level of AFA to membrane phospholipids (phosphatidylethanolamine, phosphatidylserine, cardiolipinum), antibodies to β2-glycoprotein (Iβ2-GPI), hemostasis (platelet count, ADP-induced platelet aggregation index, fibrinogen concentration, prothrombin index, activated partial thromboplastin time APTT, test for soluble fibrin-monomeric complexes, D-dimer), homocysteine, molecular genetic study of polymorphous variants for β-fibrinogen (C148T, 455GA) genes. The frequency of significant AFA titres in the group of women without psychosomatic disorders was 18.9%, and in women with psychosomatic disorders 44.3%, the rate of β2-GPI 9.6% in the first group versus 24.5% in the second group. Only in 11.5% of women in the second group elevated levels of APA were associated with β2-GPI and/or one or more clinical criteria for antiphospholipid syndrome (APS). In patients with infertility and psychosomatic disorders, we found increased platelet aggregation in the context of relative thrombocytopenia, higher fibrinogen levels, soluble fibrin-monomeric complexes, and prolonged APTT with elevated D-dimer levels. Some patients had hyperhomocysteinemia. In patients with psychosomatic disorders, the frequency of the minor alleles of the locus C148T and 455GA of the β-fibrinogen gene was greater than 40% (25–30% in first group). We distinguished factors that adversely affect the efficiency of IVF in the patients with psychosomatic disorders: elevation of APA; reduction in the number of thrombocites; growth of the ADP-induced aggregation index; extension of APTT; increase of fibrinogen, D-dimer; homocysteine; the presence of the minor allele of the T polymorphic locus C148T of the β-fibrinogen gene. The presence of prothrombotic states associated with APS should be taken into account when preparing for IVF and the appropriate correction should be made for them.

Impedance Aggregometry Reveals Increased Platelet Aggregation during Liver Transplantation.

In patients presenting for liver transplantation, increased platelet aggregation as well as thrombocytopenia have been demonstrated, but bedside assays have not been investigated. We compared platelet aggregation in liver transplantation patients and control surgical patients using impedance aggregometry. We hypothesized that platelet activity is not altered during liver transplantation. After the allowance of the ethics committee, platelet aggregation was determined using impedance aggregometry with the activators ristocetin, adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin receptor-activating peptide (TRAP) in liver transplantation patients at four time points (start of surgery, anhepatic phase, reperfusion, end of surgery) and in control surgical patients. Moreover, platelet count was determined using a Coulter counter. To compensate for the thrombocytopenia often present in patients presenting for liver transplantation, the ratio between impedance aggregometry finding and platelet count was used. For statistical evaluation, the t-test or the Mann–Whitney U-test were used, as appropriate. Platelet aggregation ratio showed a 3.1-fold increase in liver transplantation patients (n = 37) in comparison to control surgical patients (n = 10) when ristocetin was used as the activator (p = 0.001). Moreover, an approximately twofold increase of ADP-, arachidonic acid-, collagen-, and TRAP-induced platelet aggregation ratio was determined. Platelet aggregation normalized at the end of the transplantation procedure. Impedance aggregometry revealed a markedly increased platelet aggregation in some liver transplantation patients and might be suitable to guide platelet transfusion and antiplatelet therapy.

In vitro analysis of the effect of contrast agents on the antiaggregant effects of P2Y12 inhibitors

ObjectivesThe contrast agents have different molarities and ionic structures. The high osmolar contrast agents could increase platelet aggregation but the ionic contrast agents decrease platelet aggregation. However there is insufficient data on whether the antiaggregan effect of P2Y12 inhibitors used during coronary interventions are affected by the contrast agents. This study aimed to evaluate the in vitro effects of different contrast agents on the antiaggregant activity of P2Y12 inhibitors (clopidogrel, ticagrelor and prasugrel). Materials and methodsThirty patients (who underwent percutaneous coronary interventions and were treated with a P2Y12 inhibitor for a minimum of 10 days) and five healthy volunteers were divided into four groups: the clopidogrel (10 patients), ticagrelor (10 patients), prasugrel (10 patients) and control (five volunteers) groups. Antiaggregant activity was measured by using the Verify-Now method and was represented as P2Y12 reaction unit (PRU) values. Three tubes of blood were collected from the participants in the three patient groups and in the control group; as the contrast material, 10% iohexol was added to a second tube, and 10% iodixanol was added to a third tube. PRU values of the control and the contrast tubes were measured at 5 min and at 30 min after the contrast material was added. ResultsIohexol and iodixanol led to a significant decrease in the PRU values in the control group (iohexol: 188.4 ± 39.2 vs 142.4 ± 17.0, p = .04; iodixanol: 188.4 ± 39.2 vs 159.2 ± 33.7, p = .04) and in the clopidogrel group (iohexol: 140.8 ± 50.8 vs 106.3 ± 44.4, p = .04; iodixanol: 140.8 ± 50.8 vs 109.4 ± 47.6, p = .009) but not in the ticagrelor and prasugrel groups. The PRU values were significantly lower in the ticagrelor (23.1 ± 26.2) and prasugrel (23.4 ± 27.5) groups than in the clopidogrel (140.8 ± 50.8) and control (188.4 ± 39.2) groups (p < .01), and the PRU values for the ticagrelor and prasugrel groups were similar for both the 5-min and 30-min time periods (p > .05). The antiaggregant activities of iohexol and iodixanol were observed to be similar at the 5- and 30-minute time points for all of the groups (p > .05). ConclusionIohexol and iodixanol had in vitro antiaggregant effects, and their antiaggregant effects were similar. Iohexol and iodixanol increased the clopidogrel antiaggregant activity in vitro, but they did not significantly alter the antiaggregant activities of prasugrel and ticagrelor.

Relation of Functional Activity of Platelets to Prognosis of Unfavorable Cardiovascular Events in Patients with Acute Coronary Syndrome. Results of a Registry Study

to assess relation ofhigh functional activity ofplatelets to prognosis ofunfavorable cardiovascular events in patients with Acute Coronary Syndrome (ACS). The study was based on the data of a single center ACS registry conducted in the Central Clinical Hospital of the Presidential Affairs Department of Russian Federation. Of 529 included patients in 425 without contraindications to double antiplatelet therapy we carried out analysis of dependence of 30 days level of unfavorable events on parameters of functional activity of platelets. High on-treatment platelet reactivity (HTPR) was found to be associated with 3.5 increase of mortality in the group of patients with high cardiovascular risk. Logistic model of prognosis of unfavorable events based on multifactorial analysis of data from patients with measured platelet aggregation included chronic kidney disease, type of myocardial infarction, and degree ofplatelet aggregation &gt;45%. C -statistic was equal to 0.77. We also present in this paper discussion of problems related to studying approaches to individualization of anti-aggregation therapy in real clinical practice and problems of organization ofsimilar studies. The study showed that patients with ACS increased platelet aggregation, as well as chronic kidney disease and type 2 MI are associated with a 30 day prognosis of adverse events.

256Heart transplant recipients with cardiac allograft vasculopathy have increased platelet aggregation before and after low-dose aspirin therapy

Abstract Background Following heart transplantation (HTx), long-term survival is reduced mainly due to development of a specific form of coronary artery disease, coronary allograft vasculopathy (CAV). Using optical coherence tomography to visualize the coronary artery wall, we have recently shown that layered fibrotic plaques resembling organized clots are the dominant plaque component in CAV. Thus, thrombosis is suggested as a possible mechanism contributing to development and progression of CAV. Aspirin is widely used after HTx despite limited evidence and lack of specific guidelines. The antiplatelet effect of aspirin has not been thoroughly examined in HTx-patients. Purpose To investigate baseline platelet aggregation and the antiplatelet effect of aspirin in HTx-patients with and without CAV. Methods We included 68 HTx-patients (median 8.6 years from HTx). In 66 patients taking 75 mg aspirin for a minimum of 7 days, platelet aggregation was measured in whole blood using impedance aggregometry with the following agonists: Adenosine diphosphate (ADP) stimulating ADP-receptors and arachidonic acid (AA) for monitoring of aspirin treatment. Aspirin compliance was confirmed by measuring serum-thromboxane B2. Platelet aggregation prior to aspirin therapy was measured in 59 patients as it was not considered clinically safe to interrupt ongoing aspirin treatment for one week prior to blood sampling in 9 patients mainly due to previous coronary stenting. CAV burden was determined by coronary angiography and echocardiography based on international classification. Patients were divided into two groups; no CAV (n=37) and CAV (n=29). Results In HTx-patients not treated with aspirin, we found significantly increased ADP-induced platelet aggregation in patients with CAV vs. patients without CAV (904 (95% CI 813–995) vs. 786 (95% CI 728–843) AU*min, P=0.02). Baseline AA-induced aggregation was also higher in patients with CAV vs. patients without CAV, though non-significant (994 (95% CI 907–1081) vs. 905 (95% CI 839–972) AU*min, P=0.10). Even though aspirin reduced AA-induced platelet aggregation in both groups, patients with CAV had significantly increased AA-induced platelet aggregation compared with patients without CAV on aspirin treatment (380 (95% CI 295–465) vs. 286 (95% CI 239–334) AU*min, P=0.04) (Fig. 1). Conclusions HTx-patients with CAV have increased platelet aggregation before and after aspirin treatment compared with HTx-patients without CAV. Aspirin monotherapy may not provide sufficient platelet inhibition in HTx-patients with CAV. Acknowledgement/Funding Aarhus University (PhD-salary)

P5454PCSK9 as a predictor of cardiovascular events in atrial fibrillation: role of platelet activation

Abstract Background High circulating levels of proprotein convertase subtilisin/kexin 9 (PCSK9) were shown to be predictive of cardiovascular events (CVEs) in patients with atrial fibrillation (AF). Because high PCSK9 plasma levels were significantly correlated with 11-dehydro-thromboxane B2 (11-dh-TxB2), a marker of platelet activation, it is conceivable to hypothesize a direct effect of PCSK9 on platelet activation but the mechanism is still unclear. Purpose We evaluated the association between PCSK9 and platelet activation in FA patients and investigate the possible molecular mechanism involved. Methods According to our previous prospective study, we conducted a post-hoc analysis including 50 patients with baseline PCSK9 below and 50 above the median value of 1200pg/ml. The two groups were balanced for age and sex. In vivo platelet activation was assessed by aggregation (PA), recruitment, plasma thromboxane B2 (TxB2) formation and sPselectin levels. As markers of oxidative stress we used sNox2-dp, H2O2 production, urinary isoprostanes and oxLDL. To asses the role of PCSK9 in platelet activation, we performed an in vitro study with platelets from healthy subjects (n=5) added with PCSK9 concentrations achievable in human circulation (1000pg/ml and 2000pg/ml) measuring PA, TxB2, isoprostanes production, Nox2 activation, H2O2 production, oxLDL, p38, p47 and PLA2 phosphorylation. Results We observed an increased of platelet activation and oxidative stress in patients with PCSK9 levels above median (1200pg/ml) compared to those below (p&lt;0.05). A significant correlation between plasma levels of PCSK9 and markers of platelet activation and markers of oxidative stress were found. In vitro study demonstrated that PCSK9, at the concentration similar to that of patients with CVEs, was able to increase platelet activation act by binding oxLDL receptor. PCSK9 dependent platelet activation is mediated by p47 phosphorylation, a key step in Nox2 activation and is mediated by the PLA2 phosporylation. Conclusions PCSK9, at concentration achievable in patients with CVEs, increased platelet aggregation via oxLDL receptor with a pathway involving Nox2 activation.