Spontaneous Platelet Aggregation Research Articles

PLATELET AGGREGATION FUNCTION IN PATIENTS WITH TYPE 1 DIABETES MELLITUS WITH KETOACIDOSIS OF VARYING DEGREES OF SEVERITY

The aim of the research. To study the aggregation function of platelets in the blood of patients with type 1 diabetes mellitus complicated by ketoacidosis, depending on the severity of ketoacidosis in the dynamics of the disease.Materials and methods. 135 patients with type 1 diabetes mellitus complicated by mild and moderate ketoacidosis were examined, respectively, on the 1st, 5th and 10th days of the disease. The average age of the patients was 29,2±8,9 years. Platelet aggregation was studied using a Biola two-channel laser platelet aggregation analyzer (model LA230, Russia).Results. It was found that in patients with type 1 diabetes mellitus complicated by moderate and severe ketoacidosis, spontaneous platelet aggregation was sharply increased compared with patients with type 1 diabetes mellitus complicated by mild ketoacidosis. The most pronounced changes were registered on the 1st and 5th days of the disease in patients with type 1 diabetes mellitus complicated by moderate and severe ketoacidosis, and on the 5th day in the same cohort of patients complicated by mild ketoacidosis.Conclusion. Clinically significant laboratory parameters in patients with type 1 diabetes mellitus complicated by ketoacidosis, indirectly indicating hypercoagulation, require drug correction.

The shrapnel-crushed wounds in dogs: status evaluation and therapy for wound healing

Determining the clinical and laboratory status of animals with crushed wounds as a result of the traumatic effect of ammunition fragments, as well as the wound healing effect of ecologically safe drugs based on natural beekeeping products is of great importance under the conditions of the animals' stay in the war zone and territories subject to rocket-bomb and artillery fire. The aim of the research is to study the clinical condition of dogs with shrapnel-crushed wounds, observing its dynamics during systemic therapy and evaluating the therapeutic effect of wound healing using the universal biological ointment Unibiol®. During the research, various physical, hematological, biochemical, hemorheological, hemodynamic, and statistical methods were employed. Six animals with shrapnel-crushed wounds on their limbs and groin were analyzed. It was established that due to the traumatic factor, injuries in dogs are characterized by extensive tissue destruction and deconstruction. The overall condition of the animals, based on clinical signs, is described as toxemic-septic. Hematological changes such as anemia, leukocytosis, thrombocytopenia, left shift in the nucleus, monocytosis, and lymphocytosis indicate a systemic inflammatory reaction and a septic state in the dogs' bodies. Biochemical studies revealed hypoalbuminemia leading to hypoproteinemia, creatinine elevation, uremia, elevated levels of indicator transaminases, suggesting multi-organ failure. A significant decrease in circulating blood volume, increased spontaneous platelet aggregation, characterizes a moderate traumatic circulatory shock, while a 5-fold increase in the circulation of D-dimer levels indicates a significant potential for disseminated intravascular coagulation syndrome. Following infusion and antibiotic therapy, wound sanitation over 5 days led to a significant restoration of the clinical-laboratory status of the injured dogs, setting the stage for Unibiol® ointment wound-healing therapy. As a result of the treatment conducted over 21 days, the wound defect healed with the formation of complete granulation tissue and an elastic scar. The clinical trials conducted allow recommending Unibiol® ointment for effective and environmentally safe healing of severe shrapnel-crushed wounds.

Spontaneous and stimulated platelet aggregation activity in patients with atrial fibrillation and thrombotic complications

Aim: To study spontaneous and stimulated by adenosine diphosphate (ADP), adrenaline and collagen platelet aggregation activity, CD40-CD40 ligand system in patients with atrial fibrillation (AF) with previous thromboembolic complications (TEC) and newly diagnosed thrombosis.Material and Methods. The study included patients with «non-valvular» AF and anamnesis of TEC and newly identified thrombosis on anticoagulants in the period 01.2020-01.2023: 13 patients had anamnesis of TEC (group 1), in 18 patients were initially diagnosed thrombosis/spontaneous atrial echocontrast of high grade (group 2). The comparison group was the healthy individuals (n = 31) (group 3). The spontaneous aggregation ability of platelets and stimulated with solutions of ADP, adrenaline and collagen, the level of CD40 markers, soluble CD40 ligand (CD40L) were studied.Results. The spontaneous platelet aggregation activity was significantly higher in patients of group 2 compared with healthy individuals with a formation trend of larger aggregates in patients with TEC. When aggregation was induced by collagen, the rate of aggregate formation was significantly lower in the 1st and 2nd groups compared with healthy individuals. When using ADP, a comparable decrease in the degree and speed was noted, and when using adrenaline, only the rate of platelet aggregation was observed in groups 1 and 2. The concentration of soluble CD40L was significantly higher in patients with a history of TEC and newly thrombosis.Conclusion. Spontaneous platelet aggregation activity and the concentration of soluble CD40L are increased both in patients with past of TEC and in patients with newly diagnosed thrombosis due to AF and anticoagulant monotherapy.

Adhesion and Activation of Blood Platelets on Laser-Structured Surfaces of Biomedical Metal Alloys.

The laser surface modification of metallic implants presents a promising alternative to other surface modification techniques. A total of four alloyed metallic biomaterials were used for this study: medical steel (AISI 316L), cobalt-chromium-molybdenum alloy (CoCrMo) and titanium alloys (Ti6Al4V and Ti6Al7Nb). Samples of metallic biomaterials after machining were subjected to polishing or laser modification in two different versions. The results of surface modification were documented using SEM imaging and roughness measurement. After modification, the samples were sterilized with dry hot air, then exposed to citrate blood, washed with PBS buffer, fixed with glutaraldehyde, sputtered with a layer of gold and imaged using SEM to enable the quantification of adhered, activated and aggregated platelets on the surface of biomaterial samples. The average total number, counted in the field of view, of adhered platelets on the surfaces of the four tested biomaterials, regardless of the type of modification, did not differ statistically significantly (66 ± 81, 67 ± 75, 61 ± 70 and 57 ± 61 for AISI 316L, CoCrMo, Ti6Al4V and Ti6Al7Nb, respectively) and the average number of platelet aggregates was statistically significantly higher (p < 0.01) on the surfaces of AISI 316L medical steel (42 ± 53) and of the CoCrMo alloy (42 ± 52) compared to the surfaces of the titanium alloys Ti6Al4V (33 ± 39) and Ti6Al7Nb (32 ± 37). Remaining blood after contact was used to assess spontaneous platelet activation and aggregation in whole blood by flow cytometry. An in-depth analysis conducted on the obtained results as a function of the type of modification indicates small but statistically significant differences in the interaction of platelets with the tested surfaces of metallic biomaterials.

ДЕТЕРМИНАНТЫ ВЫСОКОЙ И НИЗКОЙ ОСТАТОЧНОЙ РЕАКТИВНОСТИ ТРОМБОЦИТОВ ПРИ ПРИЕМЕ КЛОПИДОГРЕЛА В 1-2 СУТКИ ИНФАРКТА МИОКАРДА

The aim of the study was to determine the clinical and laboratory factors that determine high and low platelet reactivity in patients taking clopidogrel on day 1-2 of myocardial infarction (MI). Material and Methods. A total of 405 patients (322 men and 83 women) with MI aged 31 to 74 years were examined. Group 1 included patients with low residual platelet reactivity (LRPR) – 89 people, group 2 consisted of patients with optimal platelet reactivity (OPR) – 223 people, group 3 was composed of patients with high residual platelet reactivity (HRPR) – 93 people. Platelet aggregation was assessed on a Multiplate impedance aggregometer (Germany) with several aggregation inducers. A blood test and a study of morphometric parameters of platelets were performed by the cytoflow method on an automated hematology analyzer Sysmex XS-500i (Japan). The study of the level of soluble CD40 ligand (sСD40L) and sP-selectin was performed in 140 patients, von Willebrand factor (VWF) and endothelin-1 were assessed in 150 people on the enzyme immunoassay analyzer Sunrise (TECAN, Austria) using Fine Biotech kits (China). All studies were performed upon admission to the hospital on day 1-2 of MI (at least 12 hours after the administration of a loading dose of clopidogrel). Results. A one-way linear regression analysis identified the following factors that independently affect the value of the ADP-test: age (β=-0.21, 95% CI: -0.42 – -0.0095; p=0.04); total cholesterol (β=1.4, 95% CI: 0.26–2.56; p=0.04); white blood cell count (β= 0.57, 95% CI: 0.003–1.14; p=0.049); hemoglobin (β=-0.16, 95% CI: -0.27 – -0.044; p=0.0067); platelet count (β=1.4, 95% CI: 0.26 – 2.56; p=0.04); PCT (β=76.8, 95% CI: 41.5 – 112.1; p=0.000024); TRAP-test (β=0.4, 95% CI: 0.33–0.46; p&lt;0.000001); CD40L (β=0.044, 95% CI: 0.013–0.074; p=0.005); sP-selectin (β=2.3, 95% CI: 0.63 – 3.97; p=0.007); VWF (β=0.06, 95% CI: 0.016 – 0.11; p=0.0085). To determine the probability of HRPR developing a binary logistic regression model was constructed based on the observational data of a sample of 398 patients, which included the following indicators: age, white blood cell count, mean platelet volume (MPV) and platelet distribution width (PDW) values, results of TRAP-test and the level of creatinine. At the probability cut-off p=0.4746, the sensitivity of the constructed model was 74.2%, specificity - 74.1%, classification accuracy – 74.1%, the area under the ROC-curve – 0.795 (CI: 0.745 – 0.844). To determine the probability of developing LRPR, a binary logistic regression model was constructed based on the observational data of 143 patients, which included the following indicators: the number of leukocytes, MPV and TRAP-test values, fibrinogen and VWF levels. With a probability cut-off p=0.5589, the sensitivity of this model was 84.6%, specificity – 77.8%, classification accuracy – 79.0%, the area under the ROC-curve – 0.826 (CI: 0.747 – 0.905). Conclusion. On days 1-2 of MI, 22% of patients with an excessive response to clopidogrel and 23% with an insufficient response to clopidogrel were identified. The determinants of HRPR are age, white blood cell count, MPV, PDW, TRAP-test and creatinine levels. The determinants of LRPR are white blood cell count, MPV values, spontaneous platelet aggregation, detected by the TRAP-test, the level of fibrinogen and VWF.

#4878 STUDYING THE EFFECTIVENESS OF PRASUGREL AND CLOPIDOGEL IN PATIENTS WITH CHRONIC LOWER LIMB ISCHEMIA WITH STAGE 2 CKD

Abstract Background and Aims To compare the clinical and rheological efficacy of prasugrel and clopidogrel in patients with chronic lower limb ischemia with stage 2 chronic kidney disease. According to statistics, in Europe, peripheral arterial disease is common in more than 40 million Europeans. According to the developed tactics, not only stroke should be recognized as a serious, life-threatening condition, but other peripheral vascular diseases can lead to disability and death. Methods The study included 101 patients with critical lower limb ischemia of atherosclerotic origin. The mean age of the patients was 51.51±8.33 years. As a control group, 20 healthy volunteers (mean age 53.90 ± 9.05 years) were examined without signs of pathology of the cardiovascular system, including those of a vegetative nature. All patients included in the study were hospitalized in the Department of Interventional Cardiology of the Republican Specialized Scientific and Practical Center for Therapy and Medical Rehabilitation, Tashkent city. Results In 94 patients (93.07%), CLCI was accompanied by hemodynamically significant multifocal atherosclerotic lesions. The first place in frequency of combined lesions was occupied by the coronary vessels, the second place - by the arteries of the brachiocephalic basin. 82 patients (81.19%) had previously suffered arterial occlusion, also most often in the form of acute myocardial infarction, somewhat less often - acute cerebrovascular accident. 47 patients (46.53%) underwent revascularization earlier, mainly coronary artery stenting, coronary artery bypass grafting, carotid endarterectomy. 8 patients (7.92%) underwent revascularization of the contralateral leg. The study studied spontaneous (SAT) and ADP-induced platelet aggregation. In the CG, SAT was noted only in 50% of cases and in all patients of the CLCI group. After comparing the results in the groups of patients with CLCI and representatives of the CG, in whom SAT was noted, it was found that both the rate and degree of SAT in the group of patients were significantly higher than in healthy individuals (2.95±0.59% versus 0.55± 0.21% and 6.05±1.54% versus 1.24±0.21%, respectively, p&amp;lt;0.001 significance of the intergroup difference of both parameters). All patients due to CINC underwent endovascular revascularization of the affected leg. After the procedure, patients were prescribed an antiplatelet agent at a maintenance dose (prasugrel 10 mg/day, clopidogrel 75 mg/day). After 3 months, a control examination was carried out, during which the dynamics of platelet aggregation activity was studied. It was found that the indicators of CAT and ADP induced aggregation decreased by 22-37%. During the test, patients with resistance to antiplatelet agents were identified - in whom the decrease in the degree of ADP5 induced aggregation was less than 20%. Such resistance was found in 19 patients: 1 patient in the prasugrel group (1.69%) and 18 patients in the clopidogrel group (42.86%, chi square = 27.00, p&amp;lt;0.001). Isolation of patients with normal and reduced response to the loading dose of clopidogrel revealed some differences (Table 3.3). So, already initially in this group of patients, the degree of ADP5-induced aggregation was lower than in the group of patients with an antiplatelet response of more than 20%. After the application of a loading dose of clopidogrel, differences in relative dynamics were noted only in terms of the degree of ADP%-induced aggregation (p&amp;lt;0.001). Conclusions Thus, the present study revealed that in patients with CLCI, there is a significant activation of CAT and an increase in the aggregation response to the introduction of high doses of the inducer of ADP5 aggregation. The loading dose of antiplatelet agents contributed to a significant decrease in platelet aggregation activity after 2 hours with a large effect of prasugrel. Chronic antiplatelet therapy led to a decrease in the rate and degree of aggregation, both spontaneous and ADP-induced, a decrease in the endogenous ADP release response, also with a great effect in patients taking prasugrel.

Evaluation Of Platelet Aggregation Activity Depending on The Duration of Antiplatelet Administration in Patients with Coronary Heart Disease

To study the aggregation activity of platelets in patients with ischemic heart disease depending on the long-term intake of acetylsalicylic acid (ASA). Material and methods. The study included: 116 patients with coronary artery disease with stable angina pectoris, taking a standard dose of aspirin 75 mg/day. during various terms. Patients with SSN were divided into 4 groups according to the duration of taking ASA. Platelet aggregation was measured using a laser platelet aggregation analyzer Alat-2 Biol, with computer processing according to the AGGR program. Adenosine diphosphate (ADP) in concentrations of 0.1, 1.0, 5.0 mmol/l was used as an inductor. The results of the study of patients taking ASA for more than 5 years showed that spontaneous aggregation and ADP-induced platelet aggregation were higher than in patients taking the drug for less than 5 years. Statistically significant differences were observed when compared with a group of patients taking ASA for less than a year (spontaneous aggregation of 1.7±0.09 (p&lt;0.05), 0.1 μm ADP-induced aggregation of 6.58±0.86 (p&lt;0.05), 1.0 μm ADP-induced aggregation of 16.51±1.63 (p&lt;0.01), 5.0 μm ADP-induced aggregation 58.44±3.72 (p&lt;0.01). Conclusion. Thus, the results showed that with an increase in the duration of aspirin intake, there was a change in both spontaneous and ADP-induced platelet aggregation with a statistically significant increase.

Reference range for ADAMTS13 antigen, activity and anti-ADAMTS13 antibody in the healthy adult Singapore population

ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin Type 1 motif, member 13) plays a fundamental role in the regulation of haemostasis and thrombosis. Its deficiency leads to an accumulation of ultra-large von Willebrand multimers, inducing spontaneous platelet aggregation, thrombosis in the microvasculature, and thrombotic thrombocytopenic purpura (TTP), a condition with 90% mortality when left untreated. Prompt quantification of ADAMTS13 antigen, activity and autoantibody plays a crucial role in the diagnosis and management of TTP and can help differentiate it from other thrombotic microangiopathies (TMAs). Reference ranges for ADAMTS13 are generally derived from Caucasian patients. Given that polymorphism in the ADAMTS13 gene can be associated with variable ADAMTS13 levels, we aimed to establish the first reference range in Singapore and provide a crucial laboratory test for institutions here and elsewhere. 150 healthy voluntary donors (75 men, 75 women) aged 21-60 years, with an ethnic mix mirroring Singapore's population profile, were recruited. ADAMTS13 antigen, activity and autoantibody levels were measured using the fluorescence resonance energy transfer-vWF73 and enzyme-linked immunosorbent assay methodologies. Levels (activity 0.65-1.79 IU/mL, antigen 0.36-1.17 IU/mL, autoantibody 1.4-12.5 U/mL) were not statistically different between the genders and various age groups. TTP and TMAs are encountered in a wide range of specialties. The availability of new assays in Singapore will aid clinicians in the timely management of these conditions. Standardising reference ranges established for Singapore against World Health Organization standards allows harmonisation of measurements between laboratories and for future research collaborations.

Comparative analysis of hemostasis system state indicators in severe COVID-19

Introduction. Clinical experience in managing patients with a new coronavirus infection caused by the SARS-CoV-2 allowed to identify specific hemostasis disorders, and enables to introduce the concept of COVID-associated coagulopathy. The aim of the study was to assess the direction of coagulogram parameter changes, whole blood clotting parameters and characteristics of platelet and plasma hemostasis in patients with severe COVID-19. Materials and methods. The parameters of the hemostasis system were assessed using venous blood of 12 patients with severe COVID-19 and 16 healthy volunteers. The whole blood clotting process was investigated by low-frequency piezothromboelastography. The platelet count and indicators of spontaneous and ADP-induced platelet aggregation were estimated with the help of a laser platelet aggregation analyzer. Fibrinolytic activity of plasma, plasminogen activity, content of fibrinogen, D-dimer, PTT, APTT, PTI and INR were assessed. Results. An increased level of fibrinogen, a 6-fold increased D-dimer level, and increased PTT were found in patients with severe COVID-19. The patient platelets count was reduced by 51 % (p &lt;0.05), spontaneous platelet aggregation remained at nearly normal level. Almost complete inhibition of ADP-induced platelet reactivity and inhibition of XIIa-dependent fibrinolysis was revealed, despite an increased by 19.3 % (p &lt;0.05) plasminogen activity. Parameters of the whole blood coagulation process pointed a pronounced activation of platelet hemostasis, a significant intensification of the polymerization stage of clot formation and an increased intensity of clot lysis and retraction. Conclusion. The significant increase of D-dimer level and paradoxical inhibition of plasma fibrinolytic activity revealed by test of XIIa-dependent fibrinolysis (in contrast to the increased intensity of clot lysis when assessing the coagulation of whole blood) indicate the complex pathogenic mechanisms of coagulopathy caused by SARS-CoV-2 infection, and the involvement of blood cells and the vascular wall in the process of pathological thrombus formation.

Spontaneous Platelet Aggregation in Blood Is Mediated by FcγRIIA Stimulation of Bruton's Tyrosine Kinase.

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2–3 min. In our cohort of healthy blood donors (n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

COVID-19 adenovirus vaccine triggers antibodies against PF4 complexes to activate complement and platelets

BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. ObjectiveTo assess antibodies in interaction with the activation of platelets and complement triggered by VITT. MethodsAntibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. ResultsNone of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. ConclusionsOur findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.

Comparative study of specific activity of rectal suppositories with clopidogrel

One of the most effective platelet antiaggregants is clopidogrel, inhibiting platelet activation by selectively binding adenosine diphosphate (ADP) with specific receptors. However, in a number of clinical situations it is necessary to have a pronounced antithrombotic effect in the shortest possible time which gives rise to interest in the transmucose use of the drug, in particular, rectal route. The theoretical preconditions for the development of a rectal administration of clopidogrel are based on the data that the anti-aggregation effect of a substance is carried out by its main metabolite formed after “first hepatic passage”, while clopidogrel itself in this aspect is inactive. On the base of complex physico-chemical, pharmaceutical, biopharmaceutical, rheological and microbiological investigations, the rational composition of clopidogrel rectal dosage form – suppository on a hydrophilic base to prevent atherothrombotic events in patients with myocardial infarction, acute coronary syndrome, ischemic stroke, peripheral arterial occlusion, is proposed by the employees of the Department of Technology of Medications of the Zaporizhzhia State Medical University. The aim of the work is to investigate the specific activity of rectal suppositories with clopidogrel. Materials and methods. Experimental suppositories with clopidogrel 0,075 g for rectal administration were used as the objects in pre-line studies. The ability of clopidogrel to reduce the inhibitory effect of ADP on adenylate cyclase activity and decrease the number of binding sites for 2-methylthio-ADP (analogue of ADP) without altering the receptor topography is used as a base of the method of comparative study of its specific activity in the rectal dosage form (suppository) and in comparison with the reference medication Plavix (SANOFI WINTHROP INDUSTRIE, France) in the tablet form. Studies were performed on white non-linear rats of different sexes with different mass 150–210 g, aged 3.5–5.0 months. Results. Statistically significant differences in the inhibition of induced platelet aggregation were registered after 6 hours of rectal administration of clopidogrel and gastric administration of the reference medication, indicating the effectiveness of the rectal applicative transmucosal route for this active pharmaceutical ingredient (API). On the 5th day of administration, inhibition of induced platelet aggregation significantly increased, and that is supported by literature on the cumulative effect of clopidogrel in daily life. When comparing the digital material of the table, it is obvious that the rectal administration of clopidogrel is more effective in comparison with oral route which is probably due to the rapid delivery of API to the liver and the formation of an active metabolite of clopidogrel inhibiting the induction and spontaneous aggregation of platelets in human’s and animal’s blood. Obtained data provide with sound arguments for development of rectal suppositories with clopidogrel because in clinical care faster achievement of an anti-aggregation effect in patients with acute coronary syndrome is the primary task of clinical pharmacology and pharmacy. Conclusions. Using the biochemical model of pathology, it was established that the applicative semisolid medication of clopidogrel in the form of rectal suppository exhibited inductive anti-aggregation activity and its administration didn’t reveal any side effects and undesirable events. Rectal suppositories with clopidogrel have been shown to exhibit faster reliable anti-aggregation effect in comparison with intragastric administration.

Effect of type 2 diabetes mellitus and mutations of the cyp2c19 gene on the platelets functional activity of patients with ischemic heart heart disease

Abstract Goal To establish sensitivity to clopidogrel in carriers of various G681A alleles of the CYP2C19 gene on the basis of a comprehensive assessment of platelet hemostasis. Materials and methods We examined 120 patients with various forms of coronary heart disease (CHD), including: 60 patients with acute coronary syndrome (ACS), 60 patients with chronic coronary syndrome (CCS). All patients were treated with clopidogrel. Results Genotyping of patients revealed that 73.3% of the examined cohorts of patients with ACS and 86.4% of patients with CCS were dominated by the native genotype G/G. In the cohort of patients with ACS, the degree of spontaneous platelet aggregation (SPA) among patients with G/A-A/A genotype exceeded the control values by 4.6 times (p&amp;lt;0.001). When compared with the corresponding indicator in patients with G/G genotype, it was found that the presence of a mutated A-allele had no significant effect on the increase in SPA (OR=2,450; 95% CI: 0.462–12.982; p=0.480). The degree and rate of arachidonic acid (AA)-induced platelet aggregation was significantly lower than the control values in all subgroups, but did not differ between subgroups of patients with different polymorphisms (OR=0.625; 95% CI: 0.065–6.090; p=0.912). Studying the degree and rate of ADP-induced platelet aggregation did not reveal statistically significant changes either in comparison with the control group or in the intergroup analysis. There was also no significant association between CYP2C19 gene polymorphisms and the degree of ADP aggregation (OR=0.795; 95% CI: 0.216–2.928; p=0.983). In patients with stable CHD, despite the use of clopidogrel in all patients, the level of SPA was significantly higher than the control values and depended on the polymorphism of the CYP2C19 gene. To evaluate the effect of diabetes on the effectiveness of inhibition of platelet functional activity by clopidogrel in carriers of different allelic variants of the CYP2C19 gene, we compared the G/G and G/A-A/A genotypes. The group of isolated coronary heart disease consisted of 48 patients with G/G genotype (80%) and 12 patients with mutated A-allele (20%). Patients with pathology of carbohydrate metabolism were distributed as follows: 47 patients had G/G genotype (78%), and 13 - G/A-A/A (22%). It was found that when carrying the G/A-A/A genotype, the intergroup difference was 72.6% (57.65 [49.34; 67.8] vs. 33.4 [32.47; 37.99], p&amp;lt;0.01). The chance of encountering a high degree of ADP-induced aggregation among carriers of the A-allele of the CYP2C19 gene with diabetes was almost 17 times significantly higher than the group of patients with isolated coronary heart disease (OR=16,667 (2,273–122,218), p=0.009). Conclusion The data obtained in our study showed that the patients with CHD and concomitant diabetes mellitus who had a mutated A-allele CYP2C19 gene was less sensitive to clopidogrel, which was manifested in a higher degree of ADP-induced platelet aggregation. Funding Acknowledgement Type of funding sources: None.

Successful venous thromboprophylaxis in a patient with vaccine-induced immune thrombotic thrombocytopenia (VITT): a case report of the first reported case in Thailand

BackgroundVaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but fatal complication of the Coronavirus Disease 2019 vaccine. The many reports of VITT have mostly been in the Caucasian population. Here, we present the first reported case in an Asian population.Case presentationA 26-year-old female had severe headache and severe thrombocytopenia 8 days after administration of the ChAdOx1 nCoV-19 vaccine developed by AstraZeneca. Although no thrombosis was demonstrated by imaging studies, she had very highly elevated d-dimer levels during hospitalization. Serology for antibodies against platelet factor 4 was positive on several days with very high optical density readings. We found that the antibody could induce spontaneous platelet aggregation without the presence of heparin. We decided to treat her with intravenous immunoglobulin, high-dose dexamethasone, and a prophylactic dose of apixaban. She improved rapidly and was discharged from the hospital 6 days after admission. Neither thrombocytopenia nor thrombosis was subsequently detected at the three-week follow-up.ConclusionsDespite the lower rate of thrombosis, VITT can occur in the Asian population. Early detection and prompt treatment of VITT can improve the patient’s clinical outcome. Thromboprophylaxis with nonheparin anticoagulants also prevents clot formation.

Агрегационная способность тромбоцитов у пациентов с гипертонической болезнью в сочетании с неалкогольной жировой болезнью печени на фоне ожирения

Актуальность. Активация тромбоцитов и их агрегации являются важнейшими процессами в патофизиологии ишемической болезни сердца. Зависимость смертности от сердечно-сосудистой заболеваемости может усугубляться присутствием других сопутствующих сердечно-сосудистых факторов риска, в том числе гипертонической болезнью (ГБ), ожирением, неалкогольной жировой болезнью печени (НАЖБП). Цель работы: изучить наличие тромбофилических изменений крови у пациентов с гипертонической болезнью и сопутствующим ожирением и неалкогольной жировой болезнью печени путем определения агрегационной способности тромбоцитов. Материалы и методы. Обследован 91 пациент. Средний возраст — (57,9 ± 9,4) года. Выделено три группы пациентов: I — 36 больных ГБ II стадии с сопутствующей НАЖБП, II — 28 пациентов с НАЖБП без ГБ, III группа — 27 пациентов с ГБ II стадии без сопутствующей НАЖБП. Результаты. Выявлено достоверное увеличение степени спонтанной агрегации у пациентов во всех группах по сравнению с группой контроля: у пациентов с ГБ II ст. — в 3 раза (p &lt; 0,05), у пациентов с НАЖБП — в 4,2 раза (p &lt; 0,001), у пациентов с НАЖБП + ГБ II ст. — в 7 раз (p &lt; 0,01). Степень АДФ-индуцированной агрегации тромбоцитов достоверно возрастала во всех группах пациентов: в группе ГБ II ст. увеличивалась на 116 % (p &lt; 0,05), в группе НАЖБП + ГБ II ст. — на 93 % (р &lt; 0,001), при самостоятельной НАЖБП — на 71,7 % (p &lt; 0,05). Степень агрегации, индуцированной арахидоновой кислотой, достоверно снижалась на 8,3 % (p &lt; 0,05) в группе пациентов с НАЖБП + ГБ II ст. Выводы. При гипертонической болезни наблюдалось увеличение функциональной активности тромбоцитов, выраженность которой значительно возрастала при присоединении НАЖБП. У пациентов с изолированной НАЖБП также наблюдалось статистически значимое усиление спонтанной агрегации тромбоцитов, что позволяет рассматривать НАЖБП как один из факторов риска тромбофилических изменений первичного звена гемостаза.

Spontaneous platelet aggregation in patients with chronic heart failure with arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: None. Aim of study. To study spontaneous platelet aggregation in patients with arterial hypertension with different functional classes (FC) of CHF with preserved systolic function not taking antiplatelet therapy. Material and Methods 51 patients with AH with different types of CHF, mean age 55.4 ± 5.9 years, with preserved left ventricular systolic function according to ECHO-CS were studied. The patients were divided into 4 groups depending on FC of CHF: Group 1 - 0 FC of CHF (n = 11), Group 2 - 1 FC of CHF (n = 13), Group 3 - 2 FC of CHF (n = 14), and Group 4 - 3 FC of CHF (n = 13). Spontaneous platelet aggregation was determined on a KFK-2MP photoelectrocolorimeter (Russia). Results. During the study, the spontaneous platelet aggregation was 1.08 ± 0.31 in patients with CHF class 0 (1 gr), and 1.22 ± 0.25 in patients with CHF class 1, with no statistical difference in comparison with 1 gr (p = 0.233). In group 3 (2FC CHF), spontaneous platelet aggregation was 1.33 ± 0.27, with a statistically significant difference with spontaneous platelet aggregation in patients with type 0 (p = 0.042), but with group 2 (1FC CHF), no statistically significant difference was found (p = 0.281). Spontaneous platelet aggregation in group 4 (3FC CHF) was 1,62 + 0,32, with statistically significant difference in comparison with all groups, so with group 1 - p &amp;lt; 0,001, with group 2 - p = 0,016 and with group 3 - p = 0,017. Conclusion. Patients with arterial hypertension with preserved systolic function showed an increase in spontaneous platelet aggregation with increasing functional class of CHF, and the most significant increase in platelet aggregation was seen in patients with class 3 CHF.

THE STATE OF PLATELET HEMOSTASIS IN PATIENTS WITH HYPERTENSIVE DISEASE COMBINED WITH NON-ALCOHOLIC FAT LIVER DISEASE

Relevance. Platelet activation and platelet aggregation are central processes in the pathophysiology of coronary heart disease and thrombosis. The relationship between cardiovascular morbidity and mortality varies with the presence of other concomitant cardiovascular risk factors.&#x0D; Objective. To determine the state of platelet hemostasis in patients with essential hypertension (HT), with concomitant non-alcoholic fatty liver disease (NAFLD).&#x0D; Materials and methods. 152 patients were examined: 72 men and 80 women. Three groups were identified: I - 46 patients with stage II HT without concomitant NAFLD, II - 54 patients with NAFLD without HT, group III - 52 patients with HT and concomitant NAFLD. A study of total platelet count, mean platelet volume (MPV), platelet distribution width (PDW), platelet count (PCT) and spontaneous platelet aggregation was performed.&#x0D; Results. The level of mean platelet volume (MPV) in both groups of patients with hepatic steatosis exceeded control values equally - by 6%, both in patients with NAFLD (p&lt;0.001) and in NAFLD with concomitant hypertension (p&lt;0.01). In patients of the NAFLD group and hypertension, the relative width of the platelet distribution by volume (PDW) had high values - 2% (p&lt;0.05) higher than in the control cohort, and 2.4% (p&lt;0.05) than in patients with isolated HT. An increase in the degree of spontaneous aggregation in patients of all surveyed groups compared to controls. So in patients with HT II stage. spontaneous aggregation increased 2.2 times (p&lt;0.001), while in both groups of patients with hepatic steatosis, the increase in spontaneous platelet activity was twice as high: in patients with NAFLD - 4.3 times (p&lt;0.001), in patients with HT II stage. and concomitant NAFLD - 4.1 times (p&lt;0.001).&#x0D; Conclusion. NAFLD is accompanied by an increased in MPV, the size of which correlates with their functional activity. In patients with isolated NAFLD, a statistically significant increase in spontaneous platelet aggregation is also observed, which allows considering NAFLD as one of the risk factors for thrombophilic changes in the primary hemostasis.

Characterization of a Nanobody That Binds Von Willebrand Factor and Prevents Von Willebrand Factor Cleavage By Plasmin

Introduction: Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adherence to exposed subendothelial collagen at sites of vascular injury. To prevent spontaneous platelet aggregation, accumulation of high molecular weight vWF multimers is prevented by ADAMTS13-mediated cleavage in the vWF A2 domain. Moreover, emerging data demonstrates plasmin-mediated cleavage of vWF in its open conformation in the A1-A2 linker, suggesting a role for plasmin in regulation of vWF multimer composition as a back up for ADAMTS13.Aim: We recently identified a llama-derived anti-vWF nanobody recognizing vWF in a conformation sensitive to plasmin cleavage. Our aim was to characterize this nanobody in terms of binding to- and interference with vWF functionality.Methods and results: The nanobody was produced by immunization of llamas with wildtype vWF, followed by preparation of cDNA from isolated peripheral blood lymphocyte-derived RNA and selection of the vWF A1 domain specific nanobody via phage display. To study recognition of vWF by the nanobody and possible modulating factors we developed an ELISA assay based on a nanobody-coated microtiter plate. Plasmin pre-treatment of normal pooled plasma (NPP) significantly increased nanobody recognition of vWF, but only after subjecting the plasma to shear by vortexing. Ristocetin also increased recognition by the nanobody; however, addition of plasmin to ristocetin-opened vWF in NPP did not further enhance nanobody binding. This suggests that, in contrast to ristocetin, vortexing was not sufficient to fully expose all vWF A1 domains, but the remaining hidden A1 domains were exposed by plasmin cleavage, allowing additional vWF to bind to the nanobody.Ellipsometry was used to assess competition of the nanobody and plasmin binding to vWF. Polyclonal anti-human vWF antibodies were coated to silicon slides, followed by addition of vWF concentrate (Haemate) or recombinant vWF R1306W, plasmin and the nanobody in varying order. We found that plasmin binds much more efficiently to R1306W vWF than to globular vWF. If the nanobody was bound to the R1306W vWF first, plasmin could not bind anymore. Conversely, after plasmin binding, only a small proportion of the nanobody still recognizes the plasmin-sensitive conformation of vWF, suggesting plasmin occupied the majority but not all binding sites and a possible difference in affinity for this conformation of vWF.A flow-cytometry based platelet-activation assay was performed to study the effect of plasmin on the ability of vWF in its plasmin-sensitive conformation to bind to washed platelets. Fluorescent detection of vWF binding was mediated by FITC-labeled monoclonal anti-vWF antibodies or biotin-conjugated labeled nanobody in combination with streptavidin-FITC. Addition of plasmin increased ristocetin-modified vWF binding to platelets by 3.7-fold. This effect of plasmin was abrogated in the presence of heparin, consistent with work by Brophy et al. (2017) showing that heparin prevents cleavage of vWF by plasmin. Hence, it appears that plasmin-cleaved vWF has increased binding affinity to platelets. Of note, a similar pattern in ristocetin-activated vWF to platelet binding upon addition of plasmin (and heparin) was detected by the nanobody. This demonstrates that the nanobody does not interfere with platelet binding of vWF. Moreover, binding of plasmin-cleaved vWF resulted in reduced platelet activation, as determined from P-selectin expression.Discussion: In conclusion, we have identified an anti-vWF nanobody capable of blocking plasmin binding to- and subsequent cleavage of vWF. This nanobody is unable to bind globular vWF in solution or vWF immobilized by polyclonal anti-vWF antibodies, but efficiently interacts with shear- and ristocetin-activated vWF and recombinant vWF comprising the type 2B R1306W mutation. The nanobody epitope on vWF is expected to overlap with- or lie in close proximity to the plasmin binding site in the A1-A2 linker, possibly resulting in steric hindrance. Current studies focus on providing more direct evidence of the vWF binding site of the nanobody. From a clinical perspective, the sensitivity of R1306W vWF to plasmin-mediated cleavage may explain the bleeding diathesis in VWD type 2B patients with this mutation. DisclosuresNo relevant conflicts of interest to declare.

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.

The state of platelets and their functional activity in patients with arterial hypertension in combination with non-alcoholic fatty liver disease

Abstract Background According to modern concepts of hypertension (HT) and obesity, the hemostatic system in these diseases is characterized by prothrombogenic changes. Since the liver is the site of the formation of many factors of hemostasis, the concomitant non-alcoholic fatty liver disease (NAFLD), which often accompanies the course of both of these diseases, is an actual problem. Purpose To improve the efficiency of early diagnosis of thrombophilic blood changes in hypertensive patients with concomitant obesity and NAFLD by determining the state of platelets and their functional activity. Materials and methods We examined 167 patients (80 men and 87 women). The average patient age 55.5 [47.0; 61.0] years. Patients were divided into three groups: I - 46 patients with hypertension without NAFLD, II - 54 patients with NAFLD without hypertension, III group - 52 hypertensive patients with NAFLD. The control group consisted of 15 healthy subjects matched for age and sex. Platelet count and mean platelet volume (MPV) was performed on an automated analyzer, spontaneous and induced platelet aggregation was evaluated by laser aggregometer. Results A significant increase in the degree of spontaneous aggregation of platelet has been found in patients in all groups compared to the control group: I group - 2.2-fold increasing in aggregation (p&amp;lt;0.05), II group to 4.2-fold (p&amp;lt;0.05), III group had increasing by 4.1-fold (p&amp;lt;0.05). ADP-induced platelet aggregation was the same in I group and control, but it was 39% higher (p&amp;lt;0.001) in II cohort and by 22.6% (p&amp;lt;0.01) in III group. Adrenalin-induced platelet activity increased in all groups versus control: I group – 2.1-fold (p&amp;lt;0.001), II group – 2.3-fold (p&amp;lt;0.001), III group – 1.6-fold (p&amp;lt;0.01) elevation. Arachidonic acid-induced aggregation elevated by 64,2% (p&amp;lt;0.001) in I group and decreased by 56.3% (p&amp;lt;0.01) in II and by 43% (p&amp;lt;0.05) III cohorts. Collagen-induced activity had not significant difference between groups. MPV was increased in both groups with NAFLD by 5.9, but in II group significance level was higher - p&amp;lt;0.01 than in group III - p&amp;lt;0.001. MPV had not significant changes in I group versus control. Conclusion Spontaneous platelet aggregation is increased in hypertensive patients and it is significantly enhanced in combination with NAFLD. Thus in patients with isolated NAFLD also observed a statistically significant increase in spontaneous aggregation of platelets. That is possibly explained by an increase in MPV which can be considered as one of the NAFLD risk factors thrombophilic changes in primary hemostasis. An analysis of induced platelet aggregation showed that patients with NAFLD may have a lower sensitivity to acetylsalicylic acid therapy. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Bogomolets National Medical University