Platelet Microparticles Research Articles

Platelet Microparticle Controversial Role in Cancer.

Platelet-derived microparticles (PMPs) are a group of micrometer-scale extracellular vesicles released by platelets upon activation that are responsible for the majority of microvesicles found in plasma. PMPs’ physiological properties and functions have long been investigated by researchers. In this regard, a noticeable area of studies has been devoted to evaluating the potential roles and effects of PMPs on cancer progression. Clinical and experimental evidence conflictingly implicates supportive and suppressive functions for PMPs regarding cancer. Many of these functions could be deemed as a cornerstone for future considerations of PMPs usage in cancer targeted therapy. This review discusses what is currently known about PMPs and provides insights for new and possible research directions for further grasping the intricate interplay between PMPs and cancer.

Evaluation of Coagulation and Inflammatory Markers in Pediatric Patients on Extracorporeal Membrane Oxygenation (ECMO)

Background: Bleeding and thrombosis remain the primary complications related to the use of extracorporeal membrane oxygenation (ECMO). To date, no single test or parameter has been identified to accurately predict the risk of these hemostatic complications. Thrombin generation may be the key marker for both thrombosis and bleeding, and may be influenced by inflammation. The aims of this study were to evaluate if novel laboratory tests including thrombin generation assay, neutrophil extracellular traps (NETs), microparticles (MPs), and red blood cell (RBC) membrane fragility/adhesion could better predict patients at risk for bleeding and/or thrombosis compared to routine laboratory tests. We hypothesized that ECMO related thrombosis is associated with increased thrombin generation and ECMO related bleeding is associated with decreased thrombin generation. Methods: An IRB approved prospective pilot study of patients requiring ECMO was conducted at Children's Hospital of Michigan. Patients were enrolled within 24 hours of ECMO cannulation after informed consent. Patients aged 0-17 years placed on venoarterial (VA) or venovenous (VV) ECMO were included. Patient demographics, baseline laboratory, clinical data, and novel assays were collected. Presented is the preliminary data of the first 16 patients enrolled. SPSS was used for data analysis. Results: Of the 16 patients, there were 9 males and 7 females with the majority being cannulated for primary respiratory etiologies. Median age was 8.5 months with 6 patients being neonates (38%). Twelve patients (75%) survived the ECMO run, and 11 patients (69%) survived to hospital discharge. Laboratory data was grouped based on "no event" (N=94), "bleeding event" (N=13) and "clotting event" (N=21) days. There were no differences in the number of RBC, fresh frozen plasma, or cryoprecipitate transfusions between events. Platelet transfusions were used at higher volumes in bleeding events, p=0.011. Platelet counts did not differ between groups, whereas fibrinogen levels were significantly lower on days of bleeding events, p≤0.001 (Figure 1A). There were no differences seen in the activated partial thromboplastin time (aPTT) or activated clotting time (ACT) which are routinely used for monitoring anticoagulation. However, the prothrombin time (PT) was significantly prolonged in bleeding events with a median of 16 seconds (p=0.001), which may be related to the lower fibrinogen levels (Figure 1B). Thrombin generation was assessed using the maximum rate of thrombin generation (MRTG) value from the thromboelastograph (TEG) generated velocity curve (Figure 1D). Bleeding events had a significantly lower median MRTG of 7.93 mm/min compared to 12.36 mm/min in the no event group and 12.14 mm/min in the clotting event group (p=0.004). The thrombin generation assay results are in process. NETs were analyzed using an ELISA to detect citrullinated histone-3 (CitH3) levels (ng/mL). There were no significant differences seen in our population, however there is a trend toward increased levels in thrombotic events (Figure 1E). Both absolute MPs and platelet MPs were significantly increased in clotting events compared to bleeding events, p=0.003 and p≤0.001 respectively (Figure 1C). In our population, there was no evidence that hemolysis played a role in thrombosis as assessed by flow based RBC mechanical fragility testing. Of note, flow based RBC adhesion showed a non-significant trend toward increased adhesion to P-selectin. This finding requires further investigation because P-selectin is a primary site for adhesion on endothelial cells and plays a crucial role in coagulation (Figure 1F). Conclusions: Bleeding events on ECMO are associated with increased mortality. Our findings suggest that lower fibrinogen levels, prolonged PT and decreased thrombin generation (as assessed by the TEG) are associated with an increased risk for major bleeding. Increased levels of NETs, MPs and RBC adhesion have a trend towards increased thrombotic events. Thus, although preliminary, our results suggest that novel laboratory tests were effective in identifying bleeding events, and the trends seen in thrombotic events may allow for the use of new medications to reduce clotting in ECMO. The results illustrate the challenges in monitoring ECMO anticoagulation and additional study of novel tests are needed to identify and avoid clinical complications. Disclosures Tarasev: Functional Fluidics:Current Employment, Current equity holder in private company.Hines:Functional Fluidics:Current equity holder in private company.Chitlur:Biovertiv:Honoraria;Pfizer:Honoraria;Novo Nordisk:Consultancy, Honoraria;Takeda:Honoraria;Agios Pharmaceuticals:Research Funding.

Thrombosis in Patients with Immune Thrombocytopenia ,Review of Literature

Introduction ITP is characterized by low platelet count due to immune mediated destruction and bleeding tendency. However, during last few decades thromboembolic events have been reported in patients with ITP. This review is done to study the reported cases of thromboembolic phenomenon in patient with ITP in an attempt to assess the patient characteristics and to understand the underlying mechanism. Methods We searched google Scholar, PubMed about cases with ITP and thrombosis the summary is presented in the following table (Table 1). Results Around 30 reported cases of ITP with thrombotic events were identified and a total of 36 events were recognized in last 10 years. The ages ranged from 3 years to 81 years with a mean of 51 years. Most of the patients were young and middle aged (18-65 years of age), meanwhile around 9 patients were elderly (age > 65 years). Only 3 cases were observed in pediatric age. Almost equal incidence in both genders was recognized. Half of the patient had chronic ITP while in the rest it was diagnosed less than a year. 20 out of 36 (55.6%) events happened at platelet count less than 100*10^9. While 16 events were reported with platelet count higher than this or unknown. Majority of the patients (around 64%) developed arterial events while fewer developed venous thrombosis. For treatment, most of the patients (44%) were not receiving any particular treatment for ITP at the time of thrombotic event. While 6 events (17%) happened while being treated with IVIG and 10 events (28%') happened while on TPO-RA. Only 3 patients were treated with corticosteroids prior to the event. In patients treated with TPO-RAs arterial and venous events were almost similar (57% vs 43% respectively) while majority of the events happened at lower than normal platelet count (7/10 events). Almost half of the patients had one or more underlying risk factor predisposing to atherosclerosis and thrombosis. Most of the patients were treated appropriately for the events with either antiplatelet agents or anticoagulation while simultaneously treatment for ITP was given. Corticosteroids were most frequently used for ITP during the episode followed by IVIG (52% and 28% of total treated patients respectively). Only 1 patient was treated with TPO-RA after the event for low platelet counts while others received other treatments (Rituximab, Danazol and splenectomy). Discussion Thrombosis is a complex process involving arteries and veins. Accelerated atherosclerosis and plaque rupture is the underlying event for arterial thrombosis. While in venous thrombosis immobility and procoagulant states are the main factors. Immune thrombocytopenia is characterized by immune mediated destruction and impaired production of platelets predisposing to bleeding mostly. However, it is a unique pathological process that is linked to both bleeding and thrombosis. Multiple factors predispose patients to thrombosis in ITP. The patients with chronic and active disease are particularly at risk of paradoxical thrombosis due to accelerated atherosclerosis as in other autoimmune conditions, predisposing to arterial thrombotic events. Active disease is also characterized by increased turnover of platelets in bone marrow and higher levels of circulating platelets microparticles (PMPs) which promote thrombin formation and promote venous thrombosis. The patients treated with IVIG and TPO-RA are at higher risk as compared to other forms of treatment. IVIG is used in acute states as it prevents the destruction of platelets but simultaneously promotes thrombosis by increasing blood viscosity and thrombin production. TPO-RAs are agents which mimic the action of thrombopoietin on megakaryocytes promoting their growth and differentiation and increasing platelet production. Increasing platelet count above the normal target might contribute to thrombosis however megakaryocyte activation itself leads to increased risk of thrombosis, despite low platelet count. In patients with ITP and thrombotic events, judicious use of antiplatelet therapy and anticoagulation is indicated along with simultaneous therapy directed at improving platelet count. Conclusion Patient with active ITP are predisposed to thrombosis in addition to bleeding. A treating physician needs to be vigilant to diagnose early the events and then to institute proper use of antiplatelets and anticoagulation along with therapy directed at ITP. Figure Disclosures No relevant conflicts of interest to declare.

Increased Pro-Thrombotic Platelet Activity Associated with Thrombin/PAR1-Dependent Pathway Disorder in Patients with Secondary Progressive Multiple Sclerosis.

Epidemiological studies confirm the high risk of ischemic events in multiple sclerosis (MS) that are associated with increased pro-thrombotic activity of blood platelets. The most potent physiological platelet agonist is thrombin, which activates platelets via cleavage of specific protease-activated receptors (PARs). Our current study is aimed to determine the potential genetics and proteomic abnormalities of PAR1 in both platelets and megakaryocytes, which may have thromboembolic consequences in the course of MS. The obtained results were correlated with the expression level of platelet and megakaryocyte transcripts for APOA1 and A2M genes encoding atherosclerosis biomarkers: apolipoprotein A1 (ApoA1) and α-2-macroglobulin (α2M), respectively. Moreover, PAR1 functionality in MS platelets was assessed by flow cytometry, determining the level of platelet–platelet and platelet–leukocyte aggregates, platelet microparticles and surface expression of P-selectin. As a PAR1 agonist, the synthetic TRAP-6 peptide was used, which made it possible to achieve platelet activation in whole blood without triggering clotting. Comparative analyses showed an elevated level of platelet activation markers in the blood of MS patients compared to controls. The mRNA expression of gene coding α2M was upregulated, whilst ApoA1 was down-regulated, both in platelets and megakaryocytes from MS patients. Furthermore, we observed an increase in both mRNA expression and surface density of PAR1 in platelets and megakaryocytes in MS compared to controls. Both the level of platelet activation markers and PAR1 expression showed a high correlation with the expression of transcripts for APOA1 and A2M genes.

Improved placental vascular repair in a rat preeclampsia model by implantation of endothelial progenitor cells treated with platelet microparticles

ABSTRACT Objective: To detect the role of endothelial progenitor cells (EPCs) treated with platelet microparticles (PMPs) in preeclampsia. Methods: EPCs treated with/without PMPs were labeled and injected to PE rats. The differentiation of EPCs, the change of endothelial nitric oxide synthase (eNOS), blood pressure and proteinuria were measured. The blood pressure and proteinuria increased in each of PE groups, and were improved by EPCs which was strengthened by PMPs. Transplantation of EPCs increased placental angiogenesis. The trend of change of NO was the same as blood pressure. Conclusion: Transplantation of EPCs treated with PMPs improved blood pressure and proteinuria more effectively.

Platelet activation in rabbits with decompression sickness

Platelets are the most easily altered type of peripheral blood cell in decompression sickness (DCS), which can feature decreased platelet count and the appearance of platelet microparticles in plasma. We hypothesized that DCS results in platelet activation in the bloodstream. The present study was carried out on 45 rabbits. The platelet count and concentration of plasma platelet markers were determined in 35 rabbits; the platelet shape was observed under scanning electron microscope in 10 rabbits. All indexes were collected at two points: 24 hours before the simulated dive and 30 minutes after the simulated dive. Platelet count decreased noticeably after DCS, from 380.10 ± 73.61 (G/L) to 330.23 ± 115.72 (G/L), a change of approximately -13.49 ± 25.57 (%). Platelet count was further decreased in the severe DCS group (a change of -45.99 ± 18.57%). Platelet count after DCS was proportional to the survival time of the rabbits after DCS. The concentration of two plasma platelet markers (PF4 and BTG) did not demonstrate statistically significant change at 30 minutes after DCS. However, platelet shape was changed, and the following features were observed: oblong, distortion, flattening shape, sticking together, mixing of membrane, and abundance of pseudopods with a 100- to 200-nm diameter. We conclude there is platelet activation in the bloodstream in cases of DCS.

Impact of Epicatechin on the Procoagulant Activities of Microparticles

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 μM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.

Platelet Microparticles Regulate Neutrophil Extracellular Traps in Acute Pancreatitis.

This study aimed to explore the clinical significance of the increase of platelet microparticles (PMPs) in acute pancreatitis (AP). Clinical data and plasma samples from patients with AP were collected, and healthy subjects were controls. The PMPs were detected by flow cytometry; meanwhile, the ability to promote neutrophil extracellular traps (NETs) formation was investigated. Neutrophils from healthy subjects were co-cultured with PMPs from AP patients. The NETs were visualized by confocal laser scanning microscopy. In the supernatant of cell co-culture, myeloperoxidase, neutrophil elastase, and histone H3 were detected by enzyme-linked immunosorbent assay. Patients with AP had elevated plasma levels of PMPs compared with controls; moreover, there were significantly higher PMPs levels in severe AP than mild AP and moderately severe AP. Healthy subjects' neutrophils were stimulated with PMPs from AP patients to release NETs. It was observed that NETs formed in AP group, but not in the controls. Correspondingly, there were higher levels of myeloperoxidase, neutrophil elastase, and histone H3 in AP group than in controls. The level of PMPs is a positive correlation with AP severity, which may be related to PMPs-NETs interaction. Platelet microparticles may be a potential predictor of severe AP and promising novel therapeutic target for AP.

Platelet-Derived Microparticles Bearing PF4 and Anti-GAGS Immunoglobulins in Patients with Sepsis.

PF4 is a megakaryocyte-derived cationic chemokine that plays a part in innate immunity through its activity on the macrophages. In bacterial sepsis, PF4 binds to glycosaminoglycans (GAGs) on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of anti-PF4 IgG-IgA-IgM. This triggers the immune response in patients receiving heparin therapy who develop heparin-induced thrombocytopenia (HIT). These antibodies have also been identified in patients with chronic Gram-negative infections. Given the complexity of this innate immune response network, our study on 45 patients with sepsis focused on the immune response mediated by platelet PF4. We analyzed the role of IgG-IgA-IgM against PF4-GAGs, and the presence of specific PF4-bearing platelet microparticles (PMPs). Anti-GAGs/PF4 IgG-IgA-IgM levels were significantly higher in septic patients than in control groups (healthy controls or acute patients without sepsis, p < 0.001). PF4-bearing PMP levels were only significantly higher in septic patients (p < 0.001). The occurrence of IgG-IgA-IgM against PF4-GAGs and PF4+ PMPs correlated with an improvement in patients’ sepsis. In conclusion, we demonstrated that, in the course of bacterial sepsis, platelet activation leads to the formation of specific PF4-bearing PMPs. These specific microparticles bind to polyanionic sequences on the surface of aerobic bacteria, giving rise to an antigenic complex that induces the early formation of IgG-IgA-IgM against PF4-GAGs as an innate immune response to infection.

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Advanced chronic kidney disease is associated with higher serum concentration of monocyte microparticles

Advanced chronic kidney disease is associated with high rates of cardiovascular disease. Considering the crucial role of capillaries in renal function, our study aimed to evaluate microparticles related to vascular physiology examining the link between stages of chronic kidney disease with circulating endothelial (EMP), platelet (PMP) and monocytic (MMP) microparticles. Cross-sectional study with blinded endpoints included subjects of both sexes, aged 40–75 years (n = 247), with established cardiovascular disease (coronary heart disease, ischemic stroke, or peripheral artery disease). They were stratified 1:1 by the presence or absence of decreased glomerular filtration rate (GFR < 60 mL/min/1.73 m2) estimated by the CKD-EPI criteria, and according to the stages of CKD. Microparticles were quantified by flow-cytometry using specific antibodies to identify endothelial, platelet, and monocytic derived microparticles. Higher percentages of circulating MMP (p = 0.036), but not for EMP or PMP, were observed in subjects with reduced GFR. Circulating MMP were also related to the stages of chronic kidney disease (trend analysis across renal stages, p = 0.038). Higher percentages of circulating MMP were found in subjects with reduced GFR, and their percentages were progressively higher according to the stage of chronic renal function.

Tao-Hong-Si-Wu Decoction promotes angiogenesis after cerebral ischaemia in rats via platelet microparticles

Platelet microparticles (PMPs) are membrane particles derived from the platelet membrane that enter into the blood circulation. We sought to explore the therapeutic effects of Tao-Hong-Si-Wu Decoction (THSWD) on angiogenesis in a rat model of cerebral ischaemia-reperfusion (I/R). The protective effect of THSWD on I/R rats was observed morphologically by immunohistochemical expression of VEGF and CD34, along with immunofluorescence results of co-expression of BrdU and vWF. Then, PMPs from different groups of rats were extracted, and cytokine array analysis was used to screen for angiogenesis associated proteins. The results showed that THSWD can promote the expression of VEGF, CD34, BrdU and vWF. Cytokine array analysis revealed the changes in the expression of 29 related angiogenic proteins in the total protein of PMPs, which involved the Notch signalling pathway. Compared with model group, the expression levels of NICD and Hes-1 in the THSWD group were significantly increased. In the context of I/R, the angiogenesis-related proteins of PMPs are different. THSWD may involve the promotion of activation of the Notch signalling pathway to achieve therapeutic effects on cerebral ischaemia.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin β3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Platelet microparticles-derived miR-25-3p promotes the hepatocyte proliferation and cell autophagy via reducing B-cell translocation gene 2.

Platelets are critical regulators of liver regeneration, but the mechanisms are still not fully understood. Platelets have been shown to contain a wide variety of microRNAs (miRNAs) and play an important role in many diseases. However, the mechanism that how the platelet microparticles (PMPs)-derived miRNA regulate the hepatocyte proliferation is not very clear. In this study, we have successfully isolated and identified PMPs. We also found that PMPs, which could be well integrated into the HHL-5 cells, could upregulate the level of miR-25-3p in HHL-5 cells. Meanwhile, we found that PMPs-derived miR-25-3p promoted HHL-5 cells proliferation by accelerating cells into the S phase, and enhanced the autophagy by increasing the LC3II expression and reducing the P62 expression. Then, we proved that the miR-25-3p could target the B-cell translocation gene 2 (BTG2) and downregulate the expression levels of the BTG2 gene in HHL-5 cells. In addition, the overexpression of BTG2 significantly inhibited the proliferation and autophagy abilities of HHL-5 cells, while cotransfected miR-25-3p mimics or PMPs could partially rescue HHL-5 cells proliferation and autophagy. Furthermore, we proved that PMPs accelerated hepatocyte proliferation by regulating autophagy pathways. Therefore, PMPs-derived miR-25-3p promoted HHL-5 cell proliferation and autophagy by targeting BTG2, which may be a new therapeutic method for liver regeneration.

Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release.

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

Морфология тромбоцитов новорожденных детей по данным электронной микроскопии

Objective - to study the morphological features of platelets of newborns by using electron microscopy. Materials and methods. Peripheral blood platelets of healthy newborns were studied by means of transmission electron microscopy (TEM) method. The sample included 50 healthy babies from the Clinical Perinatal Center of Saratov region born at a gestational age of 37-42 weeks with an assessment on the Apgar scale (1952) at the first minute of at least 7 points. After blood sampling, platelet-rich plasma (PRP) was obtained by centrifugation. Results. As a result of the study, 3 forms of platelets were identified: 1. Inactive ones, rounded or more often oval-shaped, in which it is possible to see almost all intracellular structures being typical for platelets; 2. Activated forms that have long processes (pseudopods) and usually have no granules; 3. Transitional forms - apparently, platelets at the initial stages of activation: elements of their open tubular system are already expanding, and this leads to the appearance of vacuoles. However, they do not yet have a distinct rearrangement of the cytoskeleton, and, accordingly, long processes and a significant change in shape. Platelets of newborns are heterogeneous in morphofunctional state: most of them are represented by inactive forms 82(80-85)%, the smaller part includes activated 15(13-17)% and transitional forms 3(2-4)%. Platelet microparticles (PM) are platelet derivatives and are also variable in size and structure. They can be represented by simple vesicles without internal contents, or they can have secretory granules involved in the transmission of signaling information to other platelets and endothelial cells. In our study, several mechanisms of formation and separation of PM from “precursor platelets” were discovered, but their meaning is not yet fully understood and requires further study. Conclusion. This study demonstrates the heterogeneity of the platelet population of newborns associated with their morphofunctional state. For convenience of description, three morphological classes of platelets have been devised. The hypothesis about possible mechanisms of PM formation has been formulated.

Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation.

Background and objectiveFor many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test.ResultsMonocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0–200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine.ConclusionMicroparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Thrombin Generation and other hemostatic parameters in patients with atrial fibrillation in use of warfarin or rivaroxaban.

Patients with atrial fibrillation (AF) present hyperactivation of both platelets and coagulation leading to a hypercoagulable state which contributes to an increased risk of thromboembolism. Therefore, one of the main strategies for treatment of AF is prevention of these events through the use of oral anticoagulants (OAC). The aim of this study was to evaluate hemostasis as a whole in patients with non-valvular AF undergoing warfarin or rivaroxaban by thrombin generation test (TGT), in addition to monocyte-platelet aggregates (MPA), glycoprotein IIb/IIIa (GPIIb/IIIa), and platelet (PMP) and endothelium (EMP) microparticles, compared to age and sex matched controls. PT/INR for OAC use was also determined. In patients taking OAC, compared to control group, a decrease in TGT (p = 0.000 for all parameters) were observed. Patients taking warfarin showed to be more hypocoagulable, presenting lower levels of ETP (p = 0.000) and peak (p = 0.002) than patients using rivaroxaban. Patients on warfarin use with INR > 3 had also lower levels of ETP (p = 0.01) and peak (p = 0.006). A decrease in ETP (p = 0.03) and peak (p = 0.02) values was also observed in patients using rivaroxaban with PT > 21.4s. Patients using warfarin (p = 0.000) and rivaroxaban (p = 0.000) presented lower levels of MPA in relation to control group. It was also observed in patients using warfarin, lower GPIIb/IIIa levels in relation to control group (p = 0.011). Patients taking rivaroxaban (p = 0.003) and warfarin (p = 0.001) had higher PMP levels compared to control group. There was no difference in levels of EMP between the groups (p = 0.0536). The present study reinforces the usefulness of OAC in AF, which decisively contribute to a better management of the disease preventing possible complications.

The association between urinary levels of 1,3-butadiene metabolites, cardiovascular risk factors, microparticles, and oxidative stress products in adolescents and young adults

1,3-Butadiene (BD) is a synthetic colorless gas used in the production of synthetic rubber and polymers. Exposure to BD has been reported to increase oxidative stress and accelerate atherosclerosis in vitro and in animal studies. In occupational studies, BD exposure has been linked to cardiovascular disease (CVD). However, no previous research has been reported on whether BD exposure is associated with CVD risk factors and oxidative stress in the general population. We recruited 853 young participants to study the correlation between urinary levels of the BD metabolite, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), CVD risk factors, serum levels of endothelial microparticles and platelet microparticles, and the urinary levels of 8-hydroxydeoxyguanosine (8-OHdG). The results showed the DHBMA levels were positively correlated with low-density lipoprotein-C, carotid intima-media thickness (CIMT), CD31+/CD42a − counts (endothelial apoptosis markers), and urinary 8-OHdG levels. Moreover, DHBMA levels were negatively correlated with CD62 P counts (platelet activation marker). The correlation between DHBMA, CIMT, and 8-OHdG was more evident when the levels of CD31+/CD42a − or CD62 P were above 50%. In conclusion, we reported that the urinary levels of DHBMA were associated with the lipid profile, CIMT, microparticles, and marker of oxidative stress in this young population. Future studies on BD exposure and atherosclerosis are needed.