Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow

BACKGROUND: Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system. METHODS: Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s(-1 )to 1500 s(-1)). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated. RESULTS: Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s(-1 )and 50 s(-1 )(r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 ± 3.3%) and high (117 ± 4.1%) antithrombin activity (1786 ± 516 U vs. 823 ± 331 U, p < 0.05) at low flow rate (13 s(-1), within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4 p = 0.008, respectively). CONCLUSION: It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance

Platelet adhesion onto immobilized fibrinogen under arterial and venous in-vitro flow conditions does not significantly differ between men and women

BACKGROUND: Gender-related differences in incidence of arterial thrombosis have been a focus of interest for years. The platelet integrin αIIbβ3 is primarily responsible for the interaction between platelets and fibrinogen and consecutive thrombus growth. In this study, we evaluated platelet adhesion onto immobilized fibrinogen under venous and arterial flow conditions in men and women. METHODS: Platelets in whole anticoagulated blood were labelled with the fluorescence dye Mepacrine and perfused through the rectangular flow chamber over glass cover slips coated with fibrinogen (shear rates of 50 s(-1), 500 s(-1 )and 1500 s(-1)). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 seconds, 1 and 5 minutes after the start of perfusion. RESULTS: During perfusion, the platelet adhesion linearly increased in regard to exposition time and shear rate. After five minutes of perfusion the platelet adhesion onto immobilized fibrinogen showed no significant gender related difference, neither at 50 s(-1 )nor at 500 s(-1 )and 1500 s(-1 )(p > 0.05), respectively. No significant difference in platelet adhesion onto immobilized fibrinogen, in regard to the menopausal status, was either observed (p > 0.05). CONCLUSION: In our in vitro experimental system, hormonal differences between men and women did not influence platelet adhesion onto immobilized fibrinogen, neither under venous nor under arterial rheological conditions

The screening power of methylenetetrahydrofolate reductase C677T polymorphism versus plasma homocysteine concentration in patients with stenosis of the internal carotid artery

BACKGROUND: Hyperhomocysteinemia is an important and independent risk factor for vascular disease. About 35% of patients with stroke and 47% of patients with peripheral arterial disease have elevated plasma homocysteine (HCY) concentrations. The relationship between plasma HCY and the methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is still unclear, especially in regard to screening/diagnostic power. METHODS: This case-control study was performed on 96 patients, who underwent surgery due to asymptomatic or symptomatic high grade stenosis of the internal carotid artery (ICA), and 96 healthy age and sex-matched, controls. Plasma HCY concentration was determined using a commercial kit for fully automated analysis (AxSYM, Abbott). The C677T polymorphism of the MTHFR-gene was assessed by PCR. RESULTS: The mean plasma HCY concentration was significantly higher in the group with stenosis of ICA compared to the controls, 12.43 ± 6.96 μM and 10.16 ± 3.16 μM, respectively, (p < 0.05). An HCY plasma concentration of 1.5 SD above the mean value of the control group, was defined as cut-off for a pathological versus physiological plasma concentration. The sensitivity and specificity of HCY was 0.27 and 0.94, respectively. The positive predictive value was 0.82. There was no significant difference in the frequency of the MTHFR 677 CT and TT genotype between patients and controls (47% vs. 47% and 8.3% vs. 11.4%, respectively). Carriers of the T-allele (CT and TT genotypes) have significantly higher plasma HCY concentrations than CC patients, 14.1 ± 7.6 μM and 10.29 ± 5.2 μM, respectively, p < 0.05. Sensitivity and specificity of the MTHFR C677T polymorphism (T-allele) were 0.56 and 0.40, respectively. The positive predictive value was 0.48. There was no significant difference in plasma HCY or genotype frequency of the MTHFR C677T polymorphism between asymptomatic and symptomatic patients. CONCLUSION: Our study shows that in a population with a given pretest disease probability of 50%, the determination of plasma HCY concentration, with a positive predictive value of 0.82, is more suitable for screening of patients at risk than analysis of the MTHFR C677T polymorphism

Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding.

INTRODUCTION Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far. METHODS SERT-deficient (Sert -/-) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, Sert -/- and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization. RESULTS After 19 weeks of high-fat diet, Sert -/- and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in Sert -/-. In obese Sert -/- animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese Sert -/- mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, Sert -/- mice fed a control diet did not display adipose tissue inflammation. DISCUSSION Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment

NLRP3 inflammasome assembly in neutrophils is supported by PAD4 and promotes NETosis under sterile conditions

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Muenzer, P., Negro, R., Fukui, S., di Meglio, L., Aymonnier, K., Chu, L., Cherpokova, D., Gutch, S., Sorvillo, N., Shi, L., Magupalli, V. G., Weber, A. N. R., Scharf, R. E., Waterman, C. M., Wu, H., & Wagner, D. D. NLRP3 inflammasome assembly in neutrophils is supported by PAD4 and promotes NETosis under sterile conditions. Frontiers in Immunology, 12, (2021): 683803, https://doi.org/10.3389/fimmu.2021.683803.Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.This work was supported by a grant from National Heart, Lung, and Blood Institute of the National Institutes of Health (grant R35 HL135765) and a Steven Berzin family support to DDW, an Individual Erwin Deutsch fellowship by the German, Austrian and Swiss Society of Thrombosis and Hemostasis Research to RES, a Whitman fellowship (MBL) to DDW, and an Individual Marie Skłodowska-Curie Actions fellowship by the European Commission (796365 - COAGULANT) to PM. ANRW was funded by the Deutsche Forschungsgemeinschaft (TRR156/2 –246807620) and a research grant (We-4195/15-19). CMW was supported by the Division of Intramural Research, NHLBI, NIH

The EHA Research Roadmap:Platelet Disorders

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1 to 2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cellbased Immune Therapies; and Gene Therapy

AluY-mediated germline deletion, duplication and somatic stem cell reversion in <i>UBE2T</i> defines a new subtype of Fanconi anemia

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.</p