Epinephrine-induced Platelet Aggregation Research Articles

Abstract 3097: Platelet COX-1 Dependent And Independent Heterogeneity In Response To Low-Dose Aspirin

Background: Platelets are key mediators of thrombosis and hemostasis. While aspirin decreases cardiovascular events in high-risk populations, its pharmacological and therapeutic effect is limited by heterogeneity in response and its effect on COX-1 dependent and independent pathways. We sought to investigate the heterogeneity of response of low-dose (81mg/day) aspirin. Methods: Following an overnight fast, 39 healthy controls had 4 weekly phlebotomies and received 81 mg aspirin daily between weeks 3 and 4. At each collection, platelet activity was measured using light transmission aggregation (LTA) in response to arachidonic acid (AA [1.5 mM]; COX-1 dependent ) and epinephrine (2.0 μM), ADP (2.0 μM), and collagen (1.0 μM; COX-1 independent pathways). Results: Median age was 27 (21, 59) years, 48.7% were female, 53.9% were white, and 7.7% were Hispanic. Median platelet aggregation in response to AA (1.5 mM) was 90.9%, 91.6%, and 91.4% in weeks 1, 2, and 3, demonstrating a robust reproducible response. Baseline AA-induced platelet aggregation was modestly correlated with ADP- (r=0.28, p=0.054), collagen- (r=0.42, p=0.0036), and epinephrine-induced (r=0.32, p=0.029) platelet aggregation. Following 1 week of aspirin, AA-induced platelet aggregation decreased to 11.8% (p<0.0001). The median (interquartile range) platelet inhibition in response to AA was 87.3% (78.5%, 92.5%) demonstrating a robust COX-1 dependent response. Platelet inhibition in response to epinephrine (52.1% [46.4, 62.6]), collagen (79.5% [70.3, 87.4]), and ADP (29.8% [22.0, 36.0]), albeit statistically significant, was less robust. While platelet inhibition in response to AA was correlated to platelet inhibition in response to collagen (r=0.64 p=1.12E-5) and epinephrine (r=0.44, p=0.0052), there was no correlation in response to ADP (r=-0.04, p=0.81) induced platelet inhibition. Conclusions: Our findings demonstrate a robust COX-1 dependent aggregation response and a more heterogeneous COX-1 independent aggregation response to low-dose aspirin. The mechanism and clinical significance of these findings require further study.

The Prediction of Milk Whey Extract (MWE) Bioactive Compounds Based on Proximate Analysis and its Effects on Blood Coagulation: A New Approach

The present work demonstrates the milk whey extraction, proximate analysis, and biochemical characterization of bioactive compounds in MWE. Effects of MWE on plasma coagulation and platelet aggregation. Proximate analysis of MWE was done according to AOAC. Whey protein banding was confirmed in 12%, 15%, and 18% SDS-PAGEs. A quantitative analysis of bioactive compounds was done. The anticoagulant effects of MWE were tested using plasma recalcification time in both PRP and PPP, further confirmed by PT and APTT assays and in tail bleeding assays at concentrations of 0-100 µg. The non-toxic property of MWE was screened by edema, hemorrhage, and direct hemolytic activities. MWE proximate analysis showed the presence of both micro- and macronutrients. Qualitative analysis confirmed the presence of proteins and carbohydrates. A similar protein-banding pattern was observed in 12%, 15%, and 18% SDS-PAGE’s in both reduced and non-reduced conditions. MWE prolonged the clotting time of human citrated plasma, both PRP and PPP, against the control 210 sec to 770 sec, suggesting its anticoagulant property. MWE delayed the clot progress of only the APTT ratio (8.99 ± 0.09), and PT was not altered, suggesting its role in an intrinsic pathway of blood coagulation. MWE exhibited antiplatelet activity in PRP against ADP and epinephrine-induced platelet aggregation. The percentage of inhibition was 74% and 100% for ADP and epinephrine, respectively. Interestingly, MWE exhibits nontoxic properties, it does not cause hemolysis, hemorrhage, or edema. Milk whey extraction and studies confirm bioactive compounds, and these exhibit antithrombotic, antiplatelet, and non-toxic properties. Hence, MWE not only acts as a good nutritive source but also may prove tobe a therapeutic bioactive compound in the management of cardiovascular disease.

Evidence based Scientific Narrative Review on the Anticoagulant Mechanisms of Allium sativum (Garlic)

Introduction: Garlic (Allium sativum, Family: Amaryllidaceae) is a seasoning plant cultivated all over the world. Garlic contains a lot of bioactive compounds including organosulfur compounds, saponins, phenolic compounds, and polysaccharides. Anticoagulant activity is one of the pharmacological properties of Garlic. The aim of this study was to review the mechanism of the anticoagulant activity of garlic and summarise the potential evidence about the anticoagulant activity of garlic from past studies. Method: Literature searches were conducted on PubMed and Google Scholar databases. 44 full articles were reviewed in detail in this study. The findings were reviewed and potential evidence for the anticoagulant mechanisms of garlic was summarised. Results: Organosulfur compounds in garlic play a major role in the anticoagulant property of garlic. Garlic exhibits anticoagulant activity mainly through three mechanisms. Those are inhibiting platelet aggregation, retarding thrombin formation, and promoting fibrinolysis. The anticoagulant activity of garlic is proven by several human, animal and in-vitro studies. Garlic significantly inhibits adenosine diphosphate and epinephrine induced platelet aggregation in healthy subjects. Garlic administration inhibit the thrombus formation in rats. Aqueous and methanolic extracts of garlic prolong prothrombin time. Extension in clotting time is reported with increasing concentrations of garlic extract. Conclusions: Garlic has been reported as a medicinal plant showing anticoagulant activity by resisting platelet aggregation, inhibiting thrombin formation and enhancing fibrinolysis and this study suggests that garlic reduces the risk of thrombosis therefore cardiovascular complications.
 
 Keywords: Anticoagulant Activity, Garlic, Thrombosis

Antioxidant and Antithrombotic Activities of Kenaf Seed (Hibiscus cannabinus) Coat Ethanol Extract in Sprague Dawley Rats.

Oxidative stress has been implicated in deadly lifestyle diseases, and antioxidants from plant sources are the primary option in the treatment regime. Kenaf seeds are the storehouse of potential natural antioxidant phytoconstituents. Perhaps, none of the studies documented the phytoconstituents and their antioxidant potential from Kenaf seed coat so far. Thus, the current study focuses on exploring the protective effect of Kenaf Seed Coat Ethanol Extract (KSCEE) against sodium nitrite and diclofenac-induced oxidative stress in vitro (red blood cell and platelets model) and in vivo (female Sprague Dawely rat's model) along with the antithrombotic activity. The infrared spectrophotometry data showed the heterogeneous functional groups (CH, OH, C = C, C = C-C) and aromatic rings. Reverse phase high-performance liquid chromatography and gas chromatography-mass spectrometry chromatogram of KSCEE also evidenced the presence of several phytochemicals. KSCEE displayed about 76% of DPPH scavenging activity with an IC50 value of 34.94µg/ml. KSCEE significantly (***p < 0.001) normalized the stress markers such as lipid peroxidation, protein carbonyl content, superoxide dismutase, and catalase in sodium nitrite and diclofenac-induced oxidative stress in RBC, platelets, liver, kidney, and small intestine, respectively. Furthermore, KSCEE was found to protect the diclofenac-induced tissue destruction of the liver, kidney, and small intestine obtained from seven groups of female Sprague Dawely rats. KSCEE delayed the clotting time of platelet-rich plasma and platelet-poor plasma and activated partial thromboplastin time, suggesting its anticoagulant property. In addition, KSCEE also exhibited antiplatelet activity by inhibiting both adenosine diphosphate and epinephrine-induced platelet aggregation. In conclusion, KSCEE ameliorates the sodium nitrite and diclofenac-induced oxidative stress in red blood cells, platelets, and experimental animals along with antithrombotic properties.

MANAGEMENT OF MULTIPLE ORAL LESIONS IN A RARE SYNDROME WITH PLATELETS IMPAIRMENT

Hallerman-Streiff syndrome (HSS) is a rare congenital disease described in less than 200 cases worldwide. Skull changes, hypotrichosis, cataracts, a beaked nose, micrognathia, skin atrophy, dental anomalies, and short stature are characteristics described. A 28-year-old female patient was referred for dental care, complaining about recurrent spontaneous gingival bleeding. Intraoral examination revealed dental anomalies, periodontal disease, generalized gingival hyperplasia, and 3 nodules located on alveolar ridges. Due to extensive bleeding during periodontal treatment and medical history of bleeding episodes, a platelet aggregation test was requested, revealing impaired adrenaline-induced platelet aggregation. The following dental visits aimed to prevent hemorrhage during invasive procedures, using hemostatic agents. Nodules were removed with the aid of a high-power laser, with no intercurrences. Histologic analysis suggested the diagnosis of collagenous fibroma, a rare encounter. To date, this the first description of blood dyscrasia and oral lesions in a patients HSS.

Platelet aggregation under the conditions of vortex flow in vitro in patients with chronic heart failure

Aim. To compare the effect of increased concentrations of aggregation inducers (five-fold addition) under standard conditions and under the conditions of vortex flow in vitro on platelet aggregation in patients with chronic heart failure (CHF).Materials and methods. The study included 28 patients. The activity of platelet aggregation in platelet-rich plasma (PRP) was evaluated according to light transmission curves (%) and the average size of aggregates (relative units (rel. units)). The aggregation inducer was added once at 10 seconds of the study (standard procedure) and five times at 10 seconds, 1, 2, 3, and 4 minutes of the study with a constant stirring rate of 800 rpm. The same parameters were evaluated under the conditions of vortex flow, which was created by changing the stirring rate of the PRP from 800 rpm to 0 rpm and again to 800 rpm by pressing the centrifugation button on the analyzer.Results. In the course of the study, the size of the aggregates increased in patients with CHF only under the conditions of vortex flow. When a collagen aggregation inducer was added both at the concentration of 2 mmol / l and 10 mmol / l, platelet aggregation parameters increased under the conditions of vortex flow. During the study of epinephrine-induced platelet aggregation in patients with CHF, an increase in the aggregation parameters was revealed, both at five-fold addition of the inducer and under the conditions of vortex flow compared with the standard method.Conclusion. The proposed methodological approaches to creating the conditions for vortex flow in vitro and to five-fold addition of epinephrine showed an increase in the size of the aggregates and the degree of platelet aggregation. Collagen-induced aggregation under the conditions of vortex flow revealed 7 (25%) patients with high residual platelet reactivity (HRPR), and epinephrine-induced aggregation detected 15 (54%) patients with HRPR.

Monosubstituted Coumarins Inhibit Epinephrine-induced Platelet Aggregation.

AimThe aim of this study was to evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various pro-aggregatory agonists, particularly by epinephrine.BackgroundThe emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited.Objective The objective of this study was to establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Moreover, this study aimed to use in silico studies to suggest potential drug targets to which the molecules bind to produce antiplatelet effects.MethodsThe platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/μl) with epinephrine (10 µM), collagen (2 µg/ml) or ADP (10 µM). The aggregation of control platelets was considered 100% of the response for each pro-aggregatory agonist.ResultsEleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer.Conclusions In silico studies suggest that most active molecules might have antagonistic interactions in the α2 and β2 adrenoceptors. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

Pennisetum glaucum Protein Extract Protects RBC, Liver, Kidney, Small Intestine from Oxidative Damage and Exhibits Anticoagulant, Antiplatelet Activity

Objective High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis. Method In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation. Results: PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation. Conclusion Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. Abbreviations PGE Pennisetum Glaucum protein Extract APPT Activated Partial Thromboplastin Time PT Prothrombin Time ROS Reactive Oxygen Species PRP Platelet Rich Plasma PPP Platelet Poor Plasma SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis PAS Periodic Acid-schiff Staining OD Optical Density INR International Normalized Ratio PBS Phosphate Buffered Saline SOD Superoxide Dismutase TCA Trichloro Acetatic Acid DTNB Di-Thio-bis-NitroBenzoic acid SGOT Serum Glutamate Oxaloacetate Transaminase SGPT Serum Glutamate Pyruvate Transaminase ALP Alkaline Phosphatase DFC Diclofenac Syl Silymarin MED Minimum Edema Dose MHD Minimum Hemorrhagic Dose

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Monitoring of Aspirin Therapy in Patients with Cerebrovascular Pathology

Aim. Evaluation of various methods for determining the effectiveness of aspirin therapy and aspirin resistance in patients with cerebrovascular disease (CVD) depending on the presence of type 2 diabetes mellitus (T2DM).Materials and methods. The prospective study included 78 patients with various manifestations of CVD. All patients received acetylsalicylic acid (ASA) 75 mg daily. Along with a comprehensive clinical examination, a laboratory assessment of platelet function with the usage of Born method with aggregometer, a detailed biochemical blood test (including the determination of the small subunit of low density lipoprotein [s-LDL]), as well as therapeutic drug monitoring of salicylates were performed on a gas chromatograph-mass spectrometer.Results. In 53% of cases, the absence or insufficiency of the effect of ASA on platelet aggregation was noted. Two subgroups were separately identified: with T2DM; (n=40) and without T2DM (n=38). Insufficient effect of the drug on platelet aggregation characteristics were observed in 65% of patients with T2DM and in 38% without T2DM. The differences were revealed between the groups in triglycerides, s-LDL, glycemic and glycated hemoglobin (p &lt;0.05). In the group of patients with T2DM, the median ASA concentration was 0.01 [0; 0.32] μg/ml, and salicylic acid (SA)– 0.20 [0; 0.39] μg/ml, while in patients without T2DM higher values were noted: ASA 0.29 [0.15; 0.55] μg/ml, SA – 0.33 [0.05; 0.73] μg/ml. A decrease in the concentration of ASA was associated with an increase in the level of ADP-induced platelet aggregation. There was an increase in the levels of ADPand adrenaline-induced platelet aggregation with a decrease in the concentration of ASA (p=0.004) and SA (p=0.006). Therapeutic drug monitoring revealed a more significant decrease in ASA level in T2DM than in the comparison group (p=0.025).Conclusion. It is advisable to monitor aspirin therapy both with the assessment of platelet aggregation and with therapeutic drug monitoring. The relationship between the level of ASA and SA in the blood plasma and the functional activity of platelets was confirmed. The presence of T2DM is associated with a decrease in the concentration of ASA and aspirin resistance detected in therapeutic drug monitoring.

Platelet aggregation in patients with chronic heart failure in a "vortex" flow

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Исследование выполнено в рамках фундаментальной темы НИИ кардиологии АААА-А15-115123110026-3. Platelet aggregation mechanisms are studied using standard methods without taking turbulence into account. However, in cardiovascular diseases, the blood rheology changes, and the parameters of the turbulent flow acquire strong prothrombotic effects. The adhesion of several platelets creates a "snowball" effect with platelet hyperaggregation, leading to rapid vessel occlusion. Thus, the study of platelet aggregation in patients with cardiovascular diseases in conditions of creating a "vortex" flow in platelet-rich plasma is very relevant. Objective to study the effect of "vortex" flow in platelet-rich plasma on spontaneous and epinephrine-induced platelet aggregation in patients with CHF. Material and method. We studied 15 patients (75% of them men) with CHD, having CHF I-III FC. Platelet aggregation activity was studied using a turbidimetric method using a laser analyzer (220 LA "NPF Biola", Russia). Platelet aggregation activity in platelet-rich plasma (BTP) was estimated by light transmission curves in % and average aggregate size in relative units (Rel. units), with the inducer epinephrine in concentrations of 2 and 10 mg/ml, with constant stirring at 800 rpm. The same parameters were evaluated byour ownproposed approach with a creation ofa "vortex" plasma flow, which were achieved by changing the mixing rate of BTP from 0 to 800 rpm. Aggregation data is presented as a median with an interquartile range (Me (Q1; Q3)). Statistical data processing was performed using SPSS packages (version 19). The differences were considered significant at a significance level of p &amp;lt; 0.05. Results. In patients with CHF, the indicators of spontaneous aggregation measured by the standard method were 3.1 (1.5; 4.0) % and 1.7 (1.1; 2.0) Rel. units. Under the conditions of a"vortex" flow, the aggregate size increased to 5.4 (3.2; 6.1) Rel. units(p = 0.04). The indicators of standard epinephrine-induced aggregation at a concentration of 2 mg/ml were 46.7 (35.8; 66.2) % and 15.0(11.4; 18.9) Rel. units, and when the mixing speed was changed from 0 to 800 rpm, the indicators increased to 52.7 (41.3; 76.5) % (p = 0.003) and 19.4 (17.3; 20.6) Rel. units(p = 0.04). In conditions of increased epinephrine concentration of 10 mg/ml, the indicators were 52.5 (41.9; 74.5) % (p = 0.03) and 15.8 (12.2; 18.4) Rel. units. Under the conditions of"vortex" flow, aggregation indicators were 75.4 (62.0; 80.5)% (p = 0.04), and the size of aggregates increased to 356.0 (230.5; 462.5) Rel. units. Conclusion. Standard methods for studying of platelet aggregation are not always sufficient to detect an increased pro-aggregative potential of platelets. The proposed method for creationof "vortex" flow conditions showed an increase in the size of platelet aggregates and the degree of aggregation against the background of increased epinephrine concentration in patients with chronic heart failure, which proves its effectiveness in detecting platelet hyperaggregation.

Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Antiplatelet Effect of Mirtazapine via Co-blocking of the 5-HT2A and α2-Adrenergic Receptors on Platelets.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT2A and α2-adrenergic receptors on platelets and suppresses platelet aggregation mediated by ADP, increased by either 5-HT or adrenaline. Thus, a detailed monitoring of bleeding is recommended for patients taking MTZ.

Гемостаз у лиц с сахарным диабетом 2‑го типа и ожирением при острой и хронической цереброваскулярной патологии

Введение. В настоящее время цереброваскулярные заболевания (ЦВЗ) в России являются второй по частоте причиной смерти среди больных сахарным диабетом (СД). С гипергликемией и избыточной массой тела связывают протромботические и провоспалительные изменения, приводящие к неблагоприятным исходам инсульта и прогрессированию ЦВЗ. Цель исследования: оценить роль избыточной массы тела как дополнительного фактора риска развития нарушений гемостаза при острых и хронических формах цереброваскулярной патологии у пациентов с СД 2‑го типа (СД‑2). Материалы и методы. В исследование включено 305 пациентов с ишемическими ЦВЗ: группа 1 — 87 больных с ишемическим инсультом (ИИ) и СД‑2; группа 2 — 58 пациентов с ИИ без СД; группа 3 — 80 больных с хроническими ЦВЗ (ХЦВЗ) и СД‑2; группа 4 — 80 пациентов с ХЦВЗ без СД. Выполнены общесоматический и неврологический осмотр, магнитно-резонансная томография головного мозга, инструментальные исследования с расчетом индекса массы тела (ИМТ) и лабораторные исследования с определением гликемии и гликированного гемоглобина (HbA1c), АДФ-индуцированной (АДФ-АТ) и адреналин-индуцированной (Адр-АТ) агрегации тромбоцитов, коагулограммы, включающей содержание фибриногена и Д-димеров, международное нормализованное отношение, активированное частичное тромбопластиновое время, фибринолитическую активность (ФА), индекс фибринолиза, активность плазминогена и уровень тканевого активатора плазминогена (t-РА). Результаты. Наибольших значений глюкоза и HbA1c достигали у пациентов с ИИ и СД‑2. Во всех группах больных с цереброваскулярной патологией СД‑2 сопровождался ожирением и повышенным ИМТ. ЦВЗ (как ИИ, так и хроническая форма) в сочетании с СД‑2 протекали на фоне протромботического состояния крови, связанного с нарушением всех звеньев гемостаза, и проявлялись увеличением АДФ-АТ и Адр-АТ, повышением содержания фибриногена и Д-димеров при одновременном снижении ФА, индекса фибринолиза, активности плазминогена и уровня t-PA. Самые выраженные нарушения наблюдали у пациентов с ИИ и СД‑2, особенно со стороны фибринолиза. Выявлены значимые корреляции между ИМТ и агрегацией тромбоцитов, уровнями фибриногена и Д-димеров, ФА и t-PA. Заключение. У лиц с СД‑2 острые и хронические ЦВЗ сопровождаются ожирением и увеличенным ИМТ, который взаимосвязан с активацией тромбоцитарного и плазменного гемостаза и снижением фибринолиза. Наибольшие изменения отмечены у пациентов с острым инсультом и СД‑2, для которых характерно сочетание гиперфибриногенемии и значительное снижение фибринолиза. Повышение ИМТ у больных ЦВЗ ассоциировано с выраженностью протромботических изменений. Introduction. At present cerebrovascular diseases (CVD) in Russia is the second most common cause of death among patients with diabetes mellitus (DM). Prothrombotic and pro-inflammatory changes leading to adverse outcomes of stroke and progression of CVD are associated with hyperglycemia and obesity. Aim: to assess the role of obesity as an additional risk factor for the development of hemostasis disorders in acute and chronic forms of cerebrovascular pathology in patients with type 2 diabetes mellitus (DM‑2). Materials and methods. The study included 305 patients with ischemic CVD: group 1 — 87 patients with ischemic stroke (IS) and DM‑2; group 2 — 58 patients with IS without DM; group 3 — 80 patients with chronic CVD (CCVD) and DM‑2; group 4 — 80 patients with CCVD without DM. The following studies were completed: somatic and neurological examinations, magnetic resonance imaging of the brain, calculation of body mass index (BMI) and laboratory tests with the determination of glycemia and glycated hemoglobin (HbA1c), ADP-induced (ADP-AT) and adrenaline-induced (Adr-AT) platelet aggregation, coagulogram with fibrinogen and D-dimers content, international normalized ratio, activated partial thromboplastin time, fibrinolytic activity (FA), fibrinolysis index (FI), plasminogen activity and level of tissue plasminogen activator (t-PA). Results. The highest values of glucose and HbA1c were found in patients with IS and DM‑2. In all groups of patients with cerebrovascular pathology DM‑2 was accompanied with obesity and increased BMI. CVD (both IS and chronic form) in combination with DM‑2 were accompanied by prothrombotic blood state (associated with hemostasis disorders), and manifested by enhancement of ADP-AT and Adr-AT, increasing of fibrinogen and D-dimers content and decreasing of FA, FI, plasminogen activity and t-PA. Most expressed disturbances (especially in fibrinolysis) were observed in patients with IS and DM‑2. Significant correlations were found between BMI and platelet aggregation, levels of fibrinogen and D-dimers, FA and t-PA. Conclusion. In patients with DM‑2 acute and chronic CVD are accompanied by obesity and increased BMI and associated with activation of platelet and plasma hemostasis and decreased fibrinolysis. The largest changes were noted in patients with acute stroke and DM‑2 and characterized by a combination of hyperfibrinogenemia and a significantly decreased fibrinolysis. Increasing of BMI in patients with CVD was associated with the severity of prothrombotic changes.

Changes in the indicators of thromboelastography and platelet function in pregnant women with various forms of hypertensive disorders in the third trimester of pregnancy.

The aim of this research is the study of haemostasis of pregnant women suffering from various forms of hypertensive disorders in their III trimester of pregnancy. 165 women at 26-41 weeks of pregnancy were examined: 22 women had moderate preeclampsia, 31 had severe preeclampsia, 45 women suffered from chronic hypertension, 20 women have developed preeclampsia on the background of chronic hypertension and 47 women had no hypertensive disorders (control group). The hemostasis system has been assessed using the results of the following investigations: thromboelastography, induced platelet aggregation with ADP and adrenaline at a dosage of 1.25 and 2.5 μg/ml respectively and collagen at a dosage of 20 mg/ml, platelet ATP secretion and the average concentration of platelet components. Thromboelastography has been performed using TEG® 5000 thromboelastograph (Haemoscope Corporation, USA). The study of platelet aggregation and platelet ATP secretion has been performed at automatic aggregometer CHRONO-LOG® Model 700 (USA). The mean platelet component concentration has been measured using SIEMENS ADVIA 2120i automated hematology analyzer (Siemens Healthcare Diagnostics Inc., USA). Thromboelastogram analysis showed a decrease in the plasma hemostasis activity in all groups of women with hypertensive disorders. The functional activity of platelets of women with moderate preeclampsia and chronic arterial hypertension did not change in comparison with to the control group. The disorder of dense platelet granules degranulation and decrease in their aggregation ability have been detected in a cohort with severe preeclampsia. The decrease in adrenaline induced platelet aggregation has been noted in the group of women suffering from preeclampsia on the background of chronic arterial hypertension. Thromboelastography analysis (R, K, angle α, TMA, Cl, LY30) may be useful for the differential diagnosis of severe preeclampsia and chronic arterial hypertension. The results of the study led to the conclusion that it is advisable to use low doses of ADP and adrenaline as inducers of platelet aggregation, considering their granulocyticity and the ability to secrete ATP.

Epinephrine restores platelet functions inhibited by ticagrelor: A mechanistic approach

Ticagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y12 receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y1 and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca2+ mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y1 receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca2+ mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the cAMP pathway and activation of the PI3K pathway, thus enabling the P2Y1 receptor signalling and subsequent Ca2+ mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients.

High Prevalence of Sticky Platelet Syndrome in Patients with Infertility and Pregnancy Loss.

Platelet hyperaggregability, known as sticky platelet syndrome (SPS), is a prothrombotic disorder that has been increasingly associated with pregnancy loss. In this retrospective study, we aimed to investigate the clinical and diagnostic relevance of SPS in 208 patients with infertility and unexplained pregnancy loss history. We studied 208 patients that had been referred to undergo a dose-dependent platelet aggregation response to adenosine diphosphate and epinephrine using light transmission aggregometry modified by Mammen during an 11-year period. Patients’ platelet aggregation response was compared with platelet function in 29 female healthy controls of fertile age with no previous history of pregnancy loss. We found a prevalence of SPS type II (33.2%) in 208 female patients with infertility and pregnancy loss. ∆-epinephrine-induced platelet aggregation in patients with SPS was significantly decreased (median 7% and range −21 to 43%) compared to patients without SPS (median 59%, range 7–88% and p < 0.0001) and healthy controls (median 57%, range 8–106% and p < 0.0001). The optimum SPS-diagnostic cutoff value for ∆-epinephrine aggregation was ≤32% (sensitivity 95.7%, specificity 95.2%). SPS patients with low-dose acetylsalicylic acid (ASA) therapy (n = 56) showed improved pregnancy outcome (32 pregnancies; live births n = 18 (56%)) compared to SPS patients without low-dose ASA (n = 13) (3 pregnancies; live births n = 1 (33%)). Our study demonstrates the clinical and diagnostic relevance of platelet hyperaggregation in women with infertility and pregnancy loss history. Further studies should investigate the potential of SPS as a novel decisional tool with both diagnostic and clinical implications in infertility and pregnancy loss.

Megakaryocytes and platelets from a novel human adipose tissue–derived mesenchymal stem cell line

AbstractThe clinical need for platelet transfusions is increasing; however, donor-dependent platelet transfusions are associated with practical problems, such as the limited supply and the risk of infection. Thus, we developed a manufacturing system for platelets from a donor-independent cell source: a human adipose-derived mesenchymal stromal/stem cell line (ASCL). The ASCL was obtained using an upside-down culture flask method and satisfied the minimal criteria for defining mesenchymal stem cells (MSCs) by The International Society for Cellular Therapy. The ASCL showed its proliferation capacity for ≥2 months without any abnormal karyotypes. The ASCL was cultured in megakaryocyte induction media. ASCL-derived megakaryocytes were obtained, with a peak at day 8 of culture, and ASCL-derived platelets (ASCL-PLTs) were obtained, with a peak at day 12 of culture. We observed that CD42b+ cells expressed an MSC marker (CD90) which is related to cell adhesion. Compared with peripheral platelets, ASCL-PLTs exhibit higher levels of PAC1 binding, P-selectin surface exposure, ristocetin-induced platelet aggregation, and ADP-induced platelet aggregation, as well as similar levels of fibrinogen binding and collagen-induced platelet aggregation. ASCL-PLTs have lower epinephrine-induced platelet aggregation. The pattern of in vivo kinetics after infusion into irradiated immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice was similar to that of platelet concentrates. ASCL-PLTs have similar characteristics to those of peripheral platelets and might have an additional function as MSCs. The establishment of the ASCL and its differentiation into ASCL-PLTs do not require gene transfer, and endogenous thrombopoietin is used for differentiation. The present protocol is a simple method that does not require feeder cells, further enhancing the clinical application of our approach.

Preoperative screening for the hemostatic system in ENT surgery in children

The analysis (1998-2018) of the causes of early postoperative bleeding in 68 children with chronic lymphoproliferative syndrome and late postoperative bleeding (on the 7-10th day) in 3 children aged 3 to 17 years was performed. It was found the disorder of the platelet functional activity (adrenaline-induced platelet aggregation, adrenaline concentration - 2.5 µmol/l) in 83.8% cases (57 children). Late postoperative bleedings were developed due to the uncontrolled intake of nonsteroidal anti-inflammatory painkillers, which led to the platelet dysfunction. It has been established the decrease in the prothrombin percent by Quick in 73.5% children, and the elongation of the activated partial thromboplastin time (APTT) in the quarter of children amid the bleeding. Half of the children showed the increase of D-dimer concentration to 1.5-2 fold amid the bleeding. We offer preoperative examination: platelet count, prothrombin percent by Quick (prothrombin time), APTT and fibrinogen level. We recommend an extended hemostasis study with the detection of adhesive, aggregative and secretory functions of platelets in the children with chronic lymphoproliferative syndrome if the results of the integral screening tests differ from normal range.

Potent procoagulant and platelet aggregation inducing serine protease from Tridax procumbens extract

Background: Tridax procumbens extract (TPE) has been widely used in tribal/folk medicine to stop bleeding and to enhance wound healing process. Based on its traditional medicinal importance, the TPE is evaluated for its possible role in blood coagulation cascade. Objective: This study has been conducted to evaluate the TPE for the presence of protease associated with fibrino (geno) lytic, collagenolytic properties, and its action on blood coagulation cascade and platelet aggregation to substantiate its procoagulant nature. Materials and Methods: The TPE was analyzed in vitro for proteolytic, fibrinogenolytic, and collagenolytic activity. It was also analyzed for its effect on blood coagulation and platelet aggregation. In vivo studies have been conducted for hemorrhagic and edema inducing activity. Results: A non-toxic serine protease having procoagulant property associated with fibrino (geno) lytic, collagenolytic (both type I and type IV), and platelet aggregation inducing was identified and evaluated from TPE. The TPE decreased the clotting time of human plasma as evaluated by recalcification time and partial thromboplastin time by 19.8 and 1.53 folds, respectively. This suggests its procoagulant nature. TPE also enhanced the adenosine diphosphate-/epinephrine-induced platelet aggregation by 1.35 and 1.38 folds, respectively. Conclusion: The TPE serine protease is a non-toxic procoagulant with fibrino (geno) lytic and collagenolytic activities and induces platelet aggregation. Further, isolation and characterization of active molecule in TPE will allow us to exploit the pharmacological potential of TPE on coagulation cascade.