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.

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

Background Vaccine-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 presentation A 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. Conclusions Despite 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.

A 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 Coronavirus Disease 2019 vaccine. Many reports of VITT have mostly been in the Caucasian population. Here, we present first reported case from an oriental population.Case presentationA 26-year-old female who 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 level during hospitalization. Serology for antibody against platelet factor 4 was positive on several days with very high optical density readings. Furthermore, we found 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 three weeks follow-up.ConclusionsDespite lower rate of thrombosis, VITT can present in Asian population. Early detection and prompt treatment of VITT can improve patients’ clinical outcome. Thromboprophylaxis of non-heparin anticoagulants also results in prevention of clot formation.

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

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 < 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. Objective. To determine the state of platelet hemostasis in patients with essential hypertension (HT), with concomitant non-alcoholic fatty liver disease (NAFLD). 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. 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<0.001) and in NAFLD with concomitant hypertension (p<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<0.05) higher than in the control cohort, and 2.4% (p<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<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<0.001), in patients with HT II stage. and concomitant NAFLD - 4.1 times (p<0.001). 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.

LOW-INTENSITY LASER RADIATION IN CORRECTION OF BLOOD HEMODYNAMIC AND RHEOLOGICAL PARAMETERS AFTER SURGERY FOR HEAD AND NECK CANCER

Purpose: to study the effect of low-level laser therapy (LLLT) in correction of blood hemodynamic and rheological parameters after surgery for head and neck cancer.Material and Methods. The study included 134 head and neck cancer patients with a median age of 54.24 ± 12.7 years (range: 23–78 years). There were 39 (29.1 %) men and 95 (70.9 %) women. To assess the effectiveness of LLLT, all patients were divided into 3 groups. Group I included 45 patients, who received conventional therapy without the use of LLLT. Group II consisted of 45 patients, who received conventional therapy in combination with LLLT. Group III included 44 patients, who received LLLT alone. Azor 2K-02 was used as a source of LLLT in the red to near infrared spectral range. Characteristics of blood flow in the vessels of the neck and the rheological properties of blood, as well as the parameters of spontaneous platelet aggregation and the content of soluble fibrinmonomer complexes in the blood plasma of patients were studied.Results. The data obtained indicated that the use of LLLT promoted normalization of blood rheological properties in patients, who underwent surgery. Normalization of blood rheological properties occurred faster and lasted for a longer period. In patients, who received conventional therapy in combination with LLLT, the hemodynamic and rheological parameters of blood were improved 2–14 days after therapy, whereas in patients receiving conventional therapy alone, this improvement was seen 30 or more days after therapy.Conclusion. LLLT in combination with conventional therapy was shown to have a positive effect on hemodynamic and rheological parameters after surgery for head and neck cancer.

AGGREGATION AND SIZE OF PLATELETS IN CHILDREN WITH BURN DISEASE

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

The Effect of Oxidant Hypochlorous Acid on Platelet Aggregation and Dityrosine Concentration in Chronic Heart Failure Patients and Healthy Controls

Background and objective: One of the reasons for thrombosis in chronic heart failure (CHF) might be reactive forms of oxygen activating platelets. The aim of this study was to evaluate the effect of oxidant hypochlorous acid (HOCl) on platelet aggregation and dityrosine concentration in CHF patients and healthy controls. Materials and Methods: CHF patients (n = 67) and healthy (n = 31) were investigated. Heart echoscopy, 6-min walking test, complete blood count, platelet aggregation, and dityrosine concentration were performed. Platelet aggregation and dityrosine concentration were measured in plasma samples after incubation with different HOCl concentrations (0.15, 0.0778, and 0.0389 mmol/L). Results: Platelet aggregation without oxidant was lower (p = 0.049) in CHF patients than in controls. The spontaneous platelet aggregation with oxidant added was higher in CHF patients (p = 0.004). Dityrosine concentration was also higher (p = 0.032) in CHF patients. Platelet aggregation was the highest in samples with the highest oxidant concentration in both healthy controls (p = 0.0006) and in CHF patients (p = 0.036). Platelet aggregation was higher in NYHA III group in comparison to NYHA II group (p = 0.0014). Concentration of dityrosine was significantly higher in CHF samples (p = 0.032). The highest concentration of dityrosine was obtained in NYHA IV group samples (p < 0.05). Intensity of platelet aggregation, analyzed with ADP, was correlated with LV EF (r = 0.42, p = 0.007). Dityrosine concentration was correlated with NYHA functional class (r = 0.27, p < 0.05). Conclusions: The increase in platelet aggregation in CHF and healthy controls shows the oxidant effect on platelets. The increase in dityrosine concentration in higher NYHA functional classes shows a higher oxidative stress in patients with worse condition.

SPONTANEOUS PLATELET AGGREGATION IN THIRD TRIMESTER PREGNANCY AT ADAM MALIK HOSPITAL, MEDAN

Spontaneous Platelet Aggregation (SPA) is a condition when spontaneously platelet aggregate in plasma left in a tube at room temperature for 2-3 hours without adding any agonists. It is not entirely sure why this phenomenon happens. Pregnant females have a more tendency of hypercoagulability. Nevertheless, SPA demonstrates hyper aggregation of platelet and not the coagulation pathway. So far, there is no report regarding this phenomenon in pregnant females. This study aimed to find out whether pregnant females, especially in the third-trimester of the pregnancy, had the tendency of SPA. This cross-sectional study was established in the Department of Clinical Pathology and Department of Obstetrics-Gynecology, Adam Malik General Hospital in Medan between January–March 2016. Twenty-six females were included. Ultrasonography (USG) was used to know the age of pregnancy. From 26 females in this study, 10 were pregnant females in the third-trimester of pregnancy while 16 others were normal non-pregnant females as controls, with a mean age of 29.2±2.78 years old and 36.5±4.63 years old. There were significant differences between platelet aggregation using ADP agonist in pregnant females and controls (p <0.01). In the median Tmax 10%, there were no significant differences between platelet aggregation without ADP agonist in pregnant females and controls after 3 hours (p >0.05). Hyper aggregation and hypercoagulability in the third-trimester pregnancy tend to develop SPA. Even though there are only mild signs and symptoms, it can be an arising risk of thrombus.

PLATELET AGGREGATION ACTIVITY IN PATIENTS WITH ISCHEMIC STROKE, CARRIERS OF A GENETIC POLYMORPHISM OF SOME TOLL - LIKE RECEPTORS

Цель - изучение генетического полиморфизма толл-подобных рецепторов при ишемическом инсульте и его влияние на агрегационную активность тромбоцитов. Методика. В исследовании приняли участие 115 больных ишемическим инсультом, в возрасте 55-65 лет и 94 сопоставимых по возрасту здоровых респондента. Материалом для молекулярно-генетического анализа служили образцы ДНК, выделенные из лейкоцитов периферической крови. Первичный гемостаз (спонтанная и индуцированная агрегатометрия) изучался на двухканальном лазерном анализаторе агрегации тромбоцитов АЛАТ-2. Результаты показали, что предикторами ишемического инсульта являются аллели: 753Arg TLR2, 249Pro TLR6, 1237C TLR9 и генотипы 753Arg/Arg TLR2, 299Asp/Asp TLR4, 249Pro/Pro TLR6 с отношением шансов от 1,49 до 3,35. Для развития инсульта имеет значение не только качество полиморфных маркеров, но и количество предикторных аллельных вариантов. У больных ишемическим инсультом в острейший период и в период формирования инфаркта мозга отмечается увеличение скорости и степени спонтанной агрегации тромбоцитов с последующим снижением к концу острого периода. При внесении АДФ (5 и 1,25 мкг/мл) и коллагена наблюдается снижение скорости и степени агрегации на 1-е и 10-е сут инсульта с увеличением таковых к 21-м сут. У больных - обладателей двух и более предиктивных аллелей полиморфизма толл-подобных рецепторов в геноме в динамике инсульта отмечаются сравнительно низкие показатели степени и скорости индуцированной агрегации тромбоцитов с увеличением радиуса агрегатов. Aim. To study genetic polymorphism of toll-like receptors in ischemic stroke and the polymorphism effect on the platelet aggregation ability. Methods. The study involved 115 patients with ischemic stroke aged 55-65 and 94 age-matched healthy respondents. DNA samples isolated from peripheral blood leukocytes were used for molecular genetic analysis of TLR2 (Arg753Gln), TLR3 (Phe412Leu), TLR4 (Asp299Gly), TLR6 (Ser249Pro), TLR9 (T - 1237 C). Primary hemostasis (spontaneous and induced aggregatometry) was studied using an ALAT-2 two-channel laser analyzer of platelet aggregation. Results. The study showed that predictors of ischemic stroke included the 753Arg TLR2, 249Pro TLR6, 1237C, and TLR9 alleles and the -753Arg/Arg TLR2, 299Asp/Asp TLR4, and 249Pro/Pro TLR6 genotypes with odds ratios from 1.49 to 3.35. Not only the quality of polymorphic markers but also the number of predictive allelic variants are important for the development of stroke. In patients with ischemic stroke in the acute phase and during the formation of cerebral infarction, increased rate and degree of spontaneous platelet aggregation were observed with a subsequent decrease by the end of acute period. Addition of ADP (5 and 1.25 mg/ml) and collagen decreased the rate and degree of platelet aggregation on days 1 and 10 of stroke with an increase in both by day 21. In patients carrying two or more predictive alleles of the toll-like receptor polymorphism in the genome, relatively low rate and degree values of induced platelet aggregation with increased radius of the aggregates were observed in the dynamics of stroke