"Ultrastructural changes of platelets in COVID-19 and chronic viral hepatitis patients ".

Platelet-viral interactions are evolving as a new concern. Coagulation disorder is a major consequence of the COVID-19 infection. In chronic hepatitis virus infections, defect in coagulation factors, thrombocytopenia and platelet function abnormalities are common. A SARS-CoV-2 infection on top of chronic viral hepatitis infection can be common in areas where viral hepatitis is endemic. Here, we investigate the platelet ultrastructural changes and estimate the serum platelet factor-4 (PF-4), ferritin, CRP, and D-dimer in COVID-19 patients (n = 60), COVID-19 patients with associated chronic viral hepatitis (n = 20), and healthy subjects (n = 20). Ultrastructural changes were demonstrated in all test groups, denoting platelet activation. In chronic viral hepatitis patients, Platelet ultrastrustural apoptotic changes were also seen. Significantly high levels of PF-4 were confirmed in moderate and severe COVID-19 patients (P.value <0.001), with a cut off value of 17 ng/ml for predicting disease severity. A positive correlation of PF-4 with the level of serum ferritin, CRP, and D-dimer (p value < 0.001) was noted, while negatively correlated with platelet count and platelet granule count (p value < 0.001). In our study, chronic viral hepatitis patients presented mild COVID-19 signs, and their PF-4 level was comparable with the subgroup of mild COVID-19 infection. The platelet's critical role in COVID-19 coagulopathy and chronic viral hepatitis is evidenced by the ultrastructural changes and the high levels of PF4. Moreover, a dual viral infection poses a substantial burden on the platelets, necessitating close monitoring of the patient's coagulation profile.

Proteomic characterization of esophageal squamous cell carcinoma response to immunotherapy reveals potential therapeutic strategy and predictive biomarkers.

Immunotherapy is the first-line therapy for esophageal squamous cell carcinoma (ESCC), yet many patients do not respond due to drug resistance and the lack of reliable predictive markers. We collected 73 ESCC patients (including discovery cohort and validation cohort) without immune thrombocytopenia and undergoing anti-PD1 immunotherapy. Proteomic and phosphoproteomic analysis of 73 ESCC treatment-naive samples by mass spectrometry-based label-free quantification were applied to explore the potential resistant and sensitive mechanisms, and identify predictive markers of ESCC immunotherapy. Comparative analysis found the pathways related to immune and mitochondrial functions were associated with ESCC immunotherapy sensitivity; while platelet activation bioprocess showed negative correlation with CD8+ T cells and related to ESCC immunotherapy non-sensitivity. Finally, we identified 10 ESCC immunotherapy predictive biomarkers with high accuracy (≥ 0.90) to predict the immunotherapeutic response, which was validated in the independent cohort.

Differential platelet activation through an interaction with spike proteins of different SARS-CoV-2 variants.

COVID-19 disease is associated with an increased risk of thrombotic complications, which contribute to high short-term mortality. Patients with COVID-19 demonstrate enhanced platelet turnover and reactivity, which may have a role in the development of thrombotic events and disease severity. Evidence has suggested direct interaction between SARS-CoV-2 and platelets, resulting in platelets activation. Here, we compare the effect of various SARS-CoV-2 spike variants on platelet activation. Engineered lentiviral particles were pseudotyped with spike SARS-CoV-2 variants and incubated with Platelet Rich Plasma obtained from healthy individuals. The pseudotyped SARS-CoV-2 exhibiting the wild-type Wuhan-Hu spike protein stimulated platelets to increase expression of the surface CD62P and activated αIIbß3 markers by 3.5 ± 1.2 and 3.3 ± 0.7 fold, respectively (P = 0.004 and 0.003). The Delta variant induced much higher levels of platelet activation; CD62P expression was increased by 6.6 ± 2.2 fold and activated αIIbß3 expression was increased by 5.0 ± 1.5 fold (P = 0.005 and 0.026, respectively). The Omicron BA.1 and the Alpha variants induced the lowest level of activation; CD62P expression was increased by 1.7 ± 0.4 and 1.6 ± 0.9 fold, respectively (P = 0.003 and 0.008), and activated αIIbß3 expression by 1.8 ± 1.1 and 1.6 ± 0.8, respectively (P = 0.003 and 0.001). The Omicron BA.2 variant induced an increase of platelets activation comparable to the Wuhan-Hu (2.8 ± 1.2 and 2.1 ± 1.3 fold for CD62P and activated αIIbß3 markers, respectively). The results obtained for various COVID-19 variants are in correlation with the clinical severity and mortality reported for these variants.

Diverticular Disease Worsening Is Associated with Increased Oxidative Stress and Gut Permeability: New Insights by Circulating Biomarkers.

Diverticular disease (DD) management is impaired by its pathogenesis, which is still not completely defined, with an unmet clinical need for improved therapies. Ex vivo DD human models demonstrated the presence of a transmural oxidative imbalance that supports an ischemic pathogenesis. This study aimed to assess, with the use of circulating biomarkers, insights into DD pathogenesis and possible therapeutic targets. Nox2-derived peptide, H2O2, antioxidant capacity, isoprostanes, thromboxanes, TNF-α, LPS and zonulin were evaluated by ELISA in healthy subjects (HS) and asymptomatic and symptomatic DD patients. Compared to HS, DD patients presented low antioxidant capacity and increase in sNox2-dp, H2O2 and isoprostanes paralleled to a TNFα increase, lower than that of oxidative markers. TxB2 production correlated to Nox2 and isoprostanes, suggesting platelet activation. An increase in zonulin and LPS highlighted the role of gut permeability and LPS translocation in DD pathogenesis. The increase of all the markers statistically correlated with DD severity. The present study confirmed the presence of a main oxidative imbalance in DD and provides evidence of platelet activation driven by LPS translocation. The use of circulating biomarkers could represent a new clinical tool for monitoring disease progression and validate therapeutic strategies never tested in DD as antioxidant supplementation.

VASCULAR-PLATELET HEMOSTASIS OF INJURED PATIENTS: PROSPECTIVE OBSERVATIONAL STUDY.

OBJECTIVE: The aim: We study vascular-platelet hemostasis peculiarities in patients with severe trauma. PATIENTS AND METHODS: Materials and methods: We included 50 patients, who were divided into control (n=15) and study (n=35) groups. The control group included patients without traumatic injuries, study group - patients with severe trauma. The study group was divided into the I subgroup (patients received 1 g tranexamic acid IV at the prehospital stage), and the II subgroup (1 g tranexamic acid IV after hospital admission). RESULTS: Results: The main changes in the I subgroup started on the 3rd day, while in the II subgroup - on the 1st day. Patients of both subgroups on the 1st and 3rd days had a normal number of platelets in venous blood, however, on the 3rd day, there was a decreasing level of discocytes whereas the level of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased in comparison with the control group (p<0.05). CONCLUSION: Conclusions: The changes in vascular-platelet hemostasis in patients appeared in the I subgroup on the 3rd day, while in the II subgroup - on the 1st day. For the I subgroup was the decreasing level of discocytes, whereas the level of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased. For the II subgroup on the 1st day, there was an increasing sum of active forms of platelets, on the 3rd day - the level of discocytes was decreased, and levels of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased.

Carbonized Platycladus orientalis Derived Carbon Dots Accelerate Hemostasis through Activation of Platelets and Coagulation Pathways.

Achieving rapid and effective hemostasis remains a multidisciplinary challenge. Here, distinctive functional carbon dots derived from carbonized Platycladus orientalis (CPO-CDs) are developed using one-step hydrothermal method. The negatively charged surface of CPO-CDs retains partial functional groups from CPO precursor, exhibiting excellent water solubility and high biocompatibility. Both rat liver injury model and tail amputation model have confirmed the rapid and effective hemostatic performance of CPO-CDs on exogenous hemorrhage. Further, on endogenous blood-heat hemorrhage syndrome rat model, CPO-CDs could inhibit hemorrhage and alleviate inflammation response. Interestingly, the excellent hemostasis performance of CPO-CDs is ascribed to activate exogenous coagulation pathway and common coagulation pathway. More importantly, metabolomics of rat plasma suggests that the hemostasis effect of CPO-CDs is closely related to platelet functions. Therefore, the designed in vitro experiments are performed and it is discovered that CPO-CDs significantly promote platelets adhesion, activation, and aggregation. Further, the underlying mechanism investigation suggests that Src/Syk signal pathway plays a key role in platelets activation triggered by CPO-CDs. Overall, CPO-CDs with rapid and excellent hemostatic performance are discovered for the first time, which could be an excellent candidate for the treatment of hemorrhagic diseases.

[Activation of Hippo Pathway Proteins in Thrombin-Stimulated Platelets and Regutates Platelets Activation in Vitro].

OBJECTIVE: To investigate the phosphorylation levels of Hippo pathway proteins in thrombin stimulated platelets and to explore its effects on platelet activation. METHODS: The phosphorylation levels of Hippo pathway proteins - Mammalian STE20-like kinase 1/2 (MST1/2), Nuclear Dbf2 related kinase 1/2 (NDR1/2) and Mps one binder 1(MOB1) in human thrombin stimulated platelets was detected by Western blot. The effect of MST1/2 inhibitor XMU-MP-1 on platelet aggregation was detected by Platelet Aggregometer. The effect of XMU-MP-1 on platelet integrin αIIbß3 activation and CD62p expression was detected by flow cytometry. The effect of XMU-MP-1 on the "outside-in" signal of platelet integrin was detected by blood clot retraction test. The effects of XMU-MP-1 on platelet Hippo pathway proteins and p38 phosphorylation levels was detected by Western blot. RESULTS: The phosphorylation levels of MST1/2, NDR1/2 and MOB1 were significantly increased in thrombin activated platelets; XMU-MP-1 inhibited thrombin-induced platelet aggregation and αIIbß3 activation, but did not affect α-granules release and clot retraction. In addition, thrombin induced phosphorylation of the Hippo proteins were decreased in XMU-MP-1 treated platelets, while the phosphorylation of p38 was not affected. CONCLUSION: In thrombin stimulated platelets, Hippo pathway proteins were activated and contributed to platelets activation.

New potential biomarkers for early chronic kidney disease diagnosis in patients with different glucose tolerance status.

Background: The purpose of the present study was to investigate the role of oxidative stress, platelet activation, and endocan levels in renal dysfunction in normal glucose tolerance (NGT) patients with 1-h plasma glucose values ≥155 mg/dl (NGT ≥ 155), compared to NGT < 155, impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2DM) newly diagnosed subjects. We enlisted 233 patients subjected to an oral glucose tolerance test (OGTT). Materials and methods: The serum levels of platelet activation (glycoprotein VI and sP-selectin), oxidative stress biomarkers (8-isoprostane and Nox-2), and endocan were evaluated using an ELISA test. Results: Among NGT < 155 patients and the T2DM group, there was a statistically significant increase in 8-isoprostane (p < 0.0001), Nox-2 (p < 0.0001), glycoprotein VI (p < 0.0001), and sP-selectin (p < 0.0001) serum levels. Higher serum endocan levels were found with the worsening of metabolic profile (p < 0.0001); specifically, NGT ≥ 155 patients presented higher serum endocan values when compared to NGT < 155 patients (p < 0.0001). From the multivariate linear regression analysis, 1-h glucose resulted in the major predictor of estimated glomerular filtration rate (e-GFR) justifying 23.6% of its variation (p < 0.0001); 8-isoprostane and Nox-2 added respectively another 6.0% (p < 0.0001) and 3.2% (p = 0.001). Conclusion: Our study confirmed the link between 1-h post-load glucose ≥155 mg/dl during OGTT and the possible increased risk for chronic kidney disease (CKD) in newly diagnosed patients. The novelty is that we demonstrated a progressive increase in oxidative stress, platelet activation, and serum endocan levels with the worsening of metabolic profile, which becomes evident early during the progression of CKD.

Arterial Thrombotic Complications in COVID-19: A Case of Renal Infarction.

COVID-19 infection has been associated with thrombotic complications, especially venous thromboembolism. Although arterial thrombotic complications are rarely seen in these patients, we report the case of a 43-year-old patient who developed thrombosis of the main branch of the left renal artery, causing partial infarction of the left kidney associated with severe pain. He had no risk factors for thrombosis except for COVID-19 infection. We excluded any possible condition usually associated with renal artery thrombosis/embolism (i.e., cardiovascular, oncological, hematological, or rheumatic). The thrombosis resolved after a combination of anticoagulant and anti-platelet therapy. This case highlights the importance of the risk of recurrence of thrombosis in patients with a recent history of COVID-19, even after hospital discharge, improvement of the initial thrombotic event, and clearance of SARS-CoV-2 infection.

Anti-thrombotic effects mediated by dihydromyricetin involve both platelet inhibition and endothelial protection.

Classical antithrombotics and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention. Dihydromyricetin (DHM), the most abundant flavonoid in Ampelopsis grossedentata, has unique vasoprotective effects. This study aims to characterize the antithrombotic potential of DHM. The effects of DHM on the activation of platelets and endothelial cells were evaluated in vitro. Calcium mobilization and activation of mitogen-activated protein kinases (MAPKs) were examined as the potential targets of DHM based on molecular docking analysis. The in vivo effects of DHM were determined in FeCl3-injured carotid arteries and laser-injured cremasteric arterioles. The results showed that DHM suppressed a range of platelet responses including aggregation, secretion, adhesion, spreading and integrin activation, and inhibited exocytosis, phosphatidylserine exposure and tissue factor expression in activated endothelial cells. Mechanistically, DHM attenuated thrombin-induced calcium mobilization and phosphorylation of ERK1/2 and p38 both in platelets and endothelial cells. Intravenous treatment with DHM delayed FeCl3-induced carotid arterial thrombosis. Furthermore, DHM treatment inhibited both platelet accumulation and fibrin generation in the presence or absence of eptifibatide in the laser injury-induced thrombosis model, without prolonging ex vivo plasma coagulation or tail bleeding time. DHM represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation as a result of endothelial protection.

Platelet and immune characteristics of immune thrombocytopaenia patients non-responsive to therapy reveal severe immune dysregulation.

Multifactorial mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) might condition the ability of patients with ITP to respond to treatments. Examining their platelet and immune features, we aimed to detect singular characteristics of patients with ITP who do not respond to any treatment. We studied patients with chronic primary ITP who had been without treatment, or untreated (UT-ITP), for at least six months; included were responders to agonists of thrombopoietin receptors (TPO-RA), patients who showed no response to first- and second-line treatments (NR-ITP), and healthy controls. Platelets from NR-ITP patients exposed a reduced amount of sialic acid residues. Increased loss of platelet surface sialic acid residues was associated with increased platelet apoptosis. NR-ITP patients had an increased fraction of naive lymphocyte (L) B cells and a reduced LTreg (Lymphocyte T-regulator) subset. They also presented an anomalous monocyte and NK (Natural Killer) cells distribution. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls. In conclusion, our results indicate a severe deregulation of the immune system of NR-ITP. The inverse correlation between loss of sialic acid and LTreg count suggests a potential relationship between glycan composition on the platelet surface and immune response, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP.

Simulation of thrombosis in a stenotic microchannel: The effects of vWF-enhanced shear activation of platelets.

This study was undertaken to develop a numerical/computational simulation of von Willebrand Factor (vWF) - mediated platelet shear activation and deposition in an idealized stenosis. Blood is treated as a multi-constituent mixture comprised of a linear fluid component and a porous solid component (thrombus). Chemical and biological species involved in coagulation are modeled using a system of coupled convection-reaction-diffusion (CRD) equations. This study considers the cumulative effect of shear stress (history) on platelet activation. The vWF activity is modeled as an enhancement function for the shear stress accumulation and is related to the experimentally-observed unfolding rate of vWF. A series of simulations were performed in an idealized stenosis in which the predicted platelets deposition agreed well with previous experimental observations spatially and temporally, including the reduction of platelet deposition with decreasing expansion angle. Further simulation indicated a direct relationship between vWF-mediated platelet deposition and degree of stenosis. Based on the success with these benchmark simulations, it is hoped that the model presented here may provide additional insight into vWF-mediated thrombosis and prove useful for the development of more hemo-compatible blood-wetted devices in the future.

Platelets Are at the Nexus of Vascular Diseases.

Platelets are important actors of cardiovascular diseases (CVD). Current antiplatelet drugs that inhibit platelet aggregation have been shown to be effective in CVD treatment. However, the management of bleeding complications is still an issue in vascular diseases. While platelets can act individually, they interact with vascular cells and leukocytes at sites of vascular injury and inflammation. The main goal remains to better understand platelet mechanisms in thrombo-inflammatory diseases and provide new lines of safe treatments. Beyond their role in hemostasis and thrombosis, recent studies have reported the role of several aspects of platelet functions in CVD progression. In this review, we will provide a comprehensive overview of platelet mechanisms involved in several vascular diseases.

Tubulin in Platelets: When the Shape Matters.

Platelets are anuclear cells with a short lifespan that play an essential role in many pathophysiological processes, including haemostasis, inflammation, infection, vascular integrity, and metastasis. Billions of platelets are produced daily from megakaryocytes (platelet precursors). Despite this high production, the number of circulating platelets is stable and, under resting conditions, they maintain their typical discoid shape thanks to cytoskeleton proteins. The activation of platelets is associated with dynamic and rapid changes in the cytoskeleton. Two cytoskeletal polymer systems exist in megakaryocytes and platelets: actin filaments and microtubules, based on actin, and α- and ß-tubulin heterodimers, respectively. Herein, we will focus on platelet-specific tubulins and their alterations and role of the microtubules skeleton in platelet formation (thrombopoiesis). During this process, microtubules mediate elongation of the megakaryocyte extensions (proplatelet) and granule trafficking from megakaryocytes to nascent platelets. In platelets, microtubules form a subcortical ring, the so-called marginal band, which confers the typical platelet discoid shape and is also responsible for changes in platelet morphology upon activation. Molecular alterations in the gene encoding ß1 tubulin and microtubules post-translational modifications may result in quantitative or qualitative changes in tubulin, leading to altered cytoskeleton reorganization that may induce changes in the platelet number (thrombocytopenia), morphology or function. Consequently, ß1-tubulin modifications may participate in pathological and physiological processes, such as development.

Hemin-induced platelet activation and ferroptosis is mediated through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin.

Reactive oxygen species (ROS) are capable of inducing cell death or apoptosis. Recently, we demonstrated that lipid-ROS can mediate ferroptosis and activation of human platelets. Ferroptosis is an intracellular iron-mediated cell death, distinct from classical apoptosis and necrosis, which is mediated through the accumulation of ROS, lipid peroxides and depletion of cellular GSH. Lately, we demonstrated that hemoglobin degradation product hemin induces ferroptosis in platelets via ROS and lipid peroxidation. In this study, we demonstrate that hemin-induced ferroptosis in platelets is mediated through ROS-driven proteasome activity and inflammasome activation, which were mitigated by Melatonin (MLT). Although inflammasome activation is linked with pyroptosis, it is still not clear whether ferroptosis is associated with inflammasome activation. Our study for the first time demonstrates an association of platelet activation/ferroptosis with proteasome activity and inflammasome activation. Although, high-throughput screening has recognized ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent ferroptosis inhibitors, having an endogenous antioxidant such as MLT as ferroptosis inhibitor is of high interest. MLT is a well-known chronobiotic hormone that regulates the circadian rhythms in vertebrates. It also exhibits potent antioxidant and ROS quenching capabilities. MLT can regulate fundamental cellular functions by exhibiting cytoprotective, oncostatic, antiaging, anti-venom, and immunomodulatory activities. The ROS scavenging capacity of MLT is key for its cytoprotective and anti-apoptotic properties. Considering the anti-ferroptotic and anti-apoptotic potentials of MLT, it could be a promising clinical application to treat hemolytic, thrombotic and thrombocytopenic conditions. Therefore, we propose MLT as a pharmacological and therapeutic agent to inhibit ferroptosis and platelet activation.

The Main Determinants of Diabetes Mellitus Vascular Complications: Endothelial Dysfunction and Platelet Hyperaggregation.

Diabetes mellitus is a common disease that affects 3⁻5% of the general population in Italy. In some countries of northern Europe or in North America, it can even affect 6⁻8% of the population. Of great concern is that the number of cases of diabetes is constantly increasing, probably due to the increase in obesity and the sedentary nature of the population. According to the World Health Organization, in the year 2030 there will be 360 million people with diabetes, compared to 170 million in 2000. This has important repercussions on the lives of patients and their families, and on health systems that offer assistance to patients. In this review, we try to describe in an organized way the pathophysiological continuity between diabetes mellitus, endothelial dysfunction, and platelet hyperaggregation, highlighting the main molecular mechanisms involved and the interconnections.

Mean platelets volume and neutrophil to lymphocyte ratio as predictors of stroke.

BACKGROUND: Mean platelets volume (MPV) is a marker, which indicates platelet function, and is a potential link between inflammation and thrombosis. Previous studies have found a relation between high MPV levels and high risk of stroke. Another factor that has been associated with the risk of stroke is neutrophil to lymphocyte ratio (NLR). Several studies have reported an association between increased NLR and increased risk of cerebrovascular disease. It was found that NLR levels have a relation to the prognosis as well. Since both NLR and MPV have been associated with increased risk of cardiovascular disease, together they may predict the risk of stroke and the prognosis with higher sensitivity and specificity. METHODS: This is a descriptive retrospective study. Data were gathered from medical records of patients who applied the Ziv medical center and were diagnosed with stroke. Stroke severity was evaluated using the NIHSS (national institutes of health stroke scale). MPV and NLR levels of patients with stroke were compared to those of 30 healthy individuals. RESULTS: Neutrophil to lymphocyte ratio levels were found significantly higher in patients with stroke compared with healthy individuals. NLR was also found higher in patients with moderate/severe stroke compared with those with minor stroke. No association was found between MPV level, the risk of stroke, and stroke prognosis. Moreover, an interaction effect between MPV and NLR level was not found. CONCLUSION: Neutrophil to lymphocyte ratio is a good predictive factor of stroke and stroke prognosis. Further prospective studies are needed to establish the relationship between the MPV level and the risk of stroke. NLR and MPV interaction effect can be tested again in the future after establishing the association between MPV, the risk of stroke, and stroke prognosis.

Neuroprotective effect of salvianolic acid B against cerebral ischemic injury in rats via the CD40/NF-κB pathway associated with suppression of platelets activation and neuroinflammation.

Neuroinflammation plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury. Activated platelets are increasingly regarded as initiators and/or amplifiers of inflammatory processes in cerebral I/R injury. Salvianolic acid B (SAB) is the most abundant bioactive compound of Salviae miltiorrhizae, a well-known Chinese herb used to promote blood circulation and eliminating blood stasis. S. miltiorrhizae has been used clinically in Asia for the treatment of ischemic cerebrovascular diseases. In the present study, a rat model of transient middle cerebral artery occlusion (tMCAO) was established to investigate the neuroprotective effects and mechanisms of SAB treatment against focal cerebral I/R insult. The results showed that SAB treatment (3mg/kg, 6mg/kg and 12mg/kg, i.p.) dose-dependently decreased I/R-induced neurological deficits at 24, 48, and 72h after reperfusion and decreased plasma-soluble P-selectin and soluble CD40 ligand as early as 6h after onset of I/R insult. At 24h after reperfusion, SAB treatment significantly reduced neuronal and DNA damage in the hippocampal CA1 region and decreased neural cell loss in the ischemic core. The I/R-induced pro-inflammatory mediator mRNA and protein overexpression in the penumbra cortex, including ICAM-1, IL-1ß, IL-6, IL-8, and MCP-1, were significantly inhibited by SAB in a dose-dependent manner. Further studies suggested SAB treatment attenuated CD40 expression and NF-κB activation, which involved NF-κB/p65 phosphorylation and IκBα phosphorylation and degradation. In conclusion, our findings indicated that the neuroprotective effects of SAB post cerebral I/R injury are associated with the inhibition of both platelets activation and production of pro-inflammatory mediators and the downregulation of the CD40/NF-κB pathway.

Association of frequent genetic variants in platelet activation pathway genes with large-vessel ischemic stroke in Polish population.

Platelets are critically involved in the development of cerebral ischemia. Our study aimed to establish an association between frequent (minor allele frequency (MAF) > 5%) genetic polymorphisms in 84 candidate genetic loci previously linked to platelet reactivity by the use of next-generation sequencing of exons from pooled DNA samples in Polish patients with a history of large-vessel ischemic stroke. Genetic analysis was performed on blood samples obtained from 500 patients (diagnosed with acute non-cardioembolic ischemic stroke with coexisting large-artery atherosclerosis) and age/sex/history of smoking matching 500 controls of Polish origin with high risk of cardiovascular disease. Sequencing of 10 pools (five for each ischemic and control groups) was performed on the Ilumina HiSeq2500 sequencer which generated an average of 36.1 (22.7-45.9 range) million pair-end 101 bp reads and 5.3 (3-7 range) Gbp per pooled sample consisting of 100 subjects. In total, we observed 789 frequent polymorphisms in the sequenced 84 genes (703 of single-nucleotide polymorphism (SNP) type and 86 indels). When the MAF between control and stroke groups was compared, only two intronic polymorphisms (1 SNP and 1 indel) in RGS7 (rs127445 36) and ANKS1B (rs398098426) genes, respectively, show statistically significant differences, which persisted after individual genotyping of the variants and adjustment for potential confounding factors. From the remaining variants, 35 polymorphisms displayed various degrees of nominal significance (from 0.6.3 × 10-5 to 5 × 10-2) and 754 polymorphisms did not show any statistical significance when comparison was evaluated for differences in MAF between the study groups. In conclusion, the results of the study demonstrate statistically significant differences in two frequent intronic genetic variants (in RGS7 and ANKS1B) that could be associated with the platelet function between ischemic stroke patients with coexisting large-vessel atherosclerosis and control patients having high vascular risk.

Avaliação da relação entre produção plaquetária e expressão de proteínas do sistema apoptótico plaquetário em diferentes graus de plaquetopenia da Trombocitopenia Imune (PTI) / Assessment of ratio between platelet production and apoptosis in Immune Thrombocytopenia (ITP) at different degrees of thrombocytopenia

A Trombocitopenia imune (PTI) é uma doença imuno mediada adquirida de adultos e crianças caracterizada por plaquetopenia transitória ou persistente, onde o grau de plaquetopenia aumenta o risco de sangramento. Geralmente, os pacientes apresentam manifestações clínicas apenas em plaquetopenias abaixo de 50x103/mm3, e contagem de plaquetas entre 100 e 150 x103/mm3, se estável por mais de 6 meses, necessariamente não indica uma condição patológica. Tem sido sugerido diferentes processos fisiopatológicos relacionados às plaquetas de acordo com a intensidade da plaquetopenia, e que alterações na megacariocitopoiese e diminuição da sobrevida plaquetária são eventos determinantes na PTI. Contagem de plaquetas reticuladas em citometria de fluxo é um teste muito útil para avaliação da plaquetopenia, pois reflete a atividade megacariocitopoiética, destruição das plaquetas e a própria contagem de plaquetas. Tanto as plaquetas quanto os megacariócitos apresentam a via intrínseca da apoptose. A atividade dos principais mediadores da apoptose intrínseca, como Bax a Bak, é regulada por proteínas anti-apoptótica da família Bcl-2, tais como Bcl-xL. Um balanço entre Bcl-xL e Bax regula a sobrevivência plaquetária. Pacientes de PTI apresentam aumento da ativação plaquetária e da formação de micropartículas derivadas de plaquetas (MPP). Nosso objetivo foi avaliar a relação entre produção e apoptose plaquetária, e associá-la com a ativação plaquetária e a formação de MPP nos diferentes graus de intensidade da PTI. Os pacientes recrutados foram diagnosticados para trombocitopenia imune (PTI) primária, "idiopática", não esplenectomizados, acompanhados regularmente no ambulatório de Doenças Hemorrágicas e Trombóticas, do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e foram divididos grupos de acordo com contagem de plaquetas: < 50x103/mm3 (n=7); entre 50-100x103/mm3 (n=7); > 100x103/mm3 (n=7). Como grupo...

Immune thrombocytopenic (ITP) is an immune-mediated acquired disease of adults and children characterized by transient or persistent decrease of the platelet count and, depending upon the degree of thrombocytopenia, increased risk of bleeding. ITP usually presents with clinical manifestations only in platelet counts below 50x103/mm3, and counts between 100 and 150x103/mm3 if they have been stable for more than 6 months do not necessarily indicate a pathologic condition. It has been suggested the existence of different pathophysiological processes involving platelets according to the severity of thrombocytopenia, and that alterations in the megakaryopoiesis and reduced platelet lifespan play a key role in ITP. The flow cytometric analysis of reticulated platelets is useful for evaluating thrombocytopenia that reflect the activity megakaryopoiesis, platelets destruction, plataelet count, and age. Platelets and megakaryocytes contain intrinsic pathway of apoptosis. The activity of key mediators of intrinsic apoptosis, Bak and Bax, is tightly controlled by anti-apoptotic Bcl-2 family members, of which Bcl-xL, wich have been shown to coordinately regulate platelet survival. ITP patients have a larger amount of activated platelets that express P-selectin as well as an increase of platelets-derived microparticles (PMP). We suggest a relationship between platelet production and Bcl-xL and Bax expression in ITP patients from different degrees of thrombocytopenia, that has relate with platelet activation and PMP. Our aim was assessment of ratio between platelets production and apoptosis, and it associate with platelets activation and PMP in differents thrombocytopenia degree in ITP. Patients were considered eligible for the study if they were on regular follow-up at the clinic of haemorrhagic and thrombotic diseases of the Hematology Service of Clinics Hospital of University of São Paulo Medicine School diagnosed with primary immune thrombocytopenia, "idiopathic"....