Atypical Presentations of Pediatric-Acquired Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Immune thrombotic thrombocytopenic purpura (iTTP) in children is a rare, severe thrombotic microangiopathy. This condition is characterized by microangiopathic hemolytic anemia, severe thrombocytopenia, and organ ischemia due to reduced activity of the von Willebrand factor-cleaving protease ADAMTS13. METHODS: A retrospective case series evaluating data collected from the medical files of 4 children diagnosed with iTTP. RESULTS: The presented case series depicts a variety of iTTP presentations: 1 case of primary iTTP, 1 case induced by Shiga toxin, 1 associated with RAS-associated autoimmune leukoproliferative disease (RALD), and 1 initial manifestation of systemic lupus erythematosus (SLE). Notably, 2 patients recovered without undergoing plasma exchange. CONCLUSION: Early ADAMTS13 testing in children with unexplained hemolysis or thrombocytopenia is crucial. The diverse underlying causes, including infections and autoimmune disorders, underscore the complexity of iTTP in the pediatric population. These cases highlight the necessity for personalized treatment approaches that consider each patient's unique clinical situation and potential alternatives or modifications to conventional therapeutic regimens.

Special Issue "Genetic, Functional and Therapeutic Aspects of Procoagulant and Anticoagulant Factors".

Pro- and anticoagulant factors are core components of hemostasis [...].

Bleeding phenotype and hemostatic evaluation by thrombin generation in children with Noonan syndrome: A prospective study.

BACKGROUND: This study aimed to evaluate the bleeding phenotype and to conduct a comprehensive hemostatic evaluation in individuals with Noonan syndrome (NS), a dominantly inherited disorder caused by pathogenic variants in genes associated with the Ras/MAPK signaling pathway. METHODS: Children with a genetically confirmed diagnosis of NS underwent clinical evaluation, routine laboratory tests, platelet function testing, and thrombin generation (TG) assessment. RESULTS: The study included 24 children. The most frequently reported bleeding symptoms were easy bruising and epistaxis, while bleeding complications were observed in 15% of surgical procedures. Various hemostatic abnormalities were identified, including platelet dysfunction, von Willebrand disease, and clotting factor deficiencies. Abnormal platelet function was observed in 50% of the patients, and significantly lower TG parameters were found compared to controls. However, no significant correlation was observed between bleeding symptoms and TG results. CONCLUSIONS: The study suggests that the bleeding diathesis in NS is multifactorial, involving both platelet dysfunction and deficiencies of plasma coagulation factors. The potential role of TG assay as an ancillary tool for predicting bleeding tendencies in individuals with NS undergoing surgery warrants further investigation.

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.

Can T-cell and B-cell excision circles predict development of inhibitors in pediatric hemophilia A?

BACKGROUND: Hemophilia A (HA) therapy requires intravenous replacement infusions of factor (F) VIII concentrate. Inhibitors are high-affinity immunoglobulin G that are directed against FVIII and thereby render replacement therapy ineffective. This complication has significant prognostic implications. We aimed to examine the immune system involvement in inhibitor formation specifically T-cell excision circles (TRECs) and B-cell excision circles (KRECs), markers of new T and B cells, respectively, and examine them as surrogate markers for inhibitor formation. METHODS: Blood samples were collected from 35 children with severe HA. Children were divided into two groups: with FVIII inhibitors and without FVIII inhibitors. TRECs and KRECs were measured in peripheral blood. RESULTS: A total of 11 patients with inhibitors and 24 without were evaluated. Children with inhibitors had higher levels of TRECs however not statistically significant (p = 0.085). CjKREC levels were higher in the inhibitor patients (p = 0.003). Moreover, the sj/cjKREC ratio was lower in the inhibitor patients (p = 0.015). CONCLUSIONS: Our findings may add to the notion that inhibitor formation is attributed to humoral immunity due to peripheral B-cell expansion and loss of peripheral tolerance. Improved knowledge regarding the involvement of the immune system in the formation of FVIII inhibitors will enable better therapy tailoring in the era of non-replacement therapies. IMPACT: The etiology of FVIII inhibitor formation is multifactorial, in which the immune system plays a pivotal role. Our findings may add to the notion that inhibitor formation is attributed to humoral immunity due to peripheral B-cell expansion and production of antibodies against FVIII. Improved knowledge regarding the involvement of the immune system in the development of FVIII inhibitors will enable the identification of patients prone to inhibitor development and better therapy tailoring in the new era of non-replacement therapies.

Anti-TFPI for hemostasis induction in patients with rare bleeding disorders, an ex vivo thrombin generation (TG) guided pilot study.

BACKGROUND: Rare bleeding disorders (RBD) are inherited coagulopathies, whose hemostatic control is based upon replacement therapy. Marstacimab (PF-06741086) is a human monoclonal IgG that targets the Kunitz2 domain of tissue factor pathway inhibitor [TFPI]. Marstacimab is currently in development for bleeding prophylaxis in patients with hemophilia. OBJECTIVES: To assess the potential impact of Marstacimab upon thrombin generation (TG) in RBD patients' plasma samples. RESULTS: Our cohort included 18 RBD patients, with severe deficiencies: 5 Von Willebrand Disease (VWD) type 3, 4 FVII, 3 FXI, 2 FXIII deficiency and 1 patient with: FX, FV + FVIII, Fibrinogen, combined vitamin K dependent factors' deficiency. Citrated samples from RBD patients were collected and spiked with Marstacimab, TG was measured by calibrated automated thrombogram. Among all patients a reduced baseline TG was observed as compared to controls. Improvement of median (lag time, peak and ETP was observed in Marstacimab spiked samples from 8 min, 99 nM, 1116 nMx min to 5.5 min, 194 nM,1614 nMx min, respectively. None of the values measured among RBD patients exceeded normal controls. CONCLUSION: These in vitro data suggest that Marstacimab may serve as a promising approach for restoring the hemostatic balance in various RBD, though potential clinical implications should be further investigated.

Pediatric severe factor XI deficiency: A multicenter study.

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive bleeding disorder. Only scarce publications address its clinical features in children. The increased prevalence of FXI deficiency in Israel enabled data collection for this large multicenter cohort study. OBJECTIVE: Some hemostatic challenges may be unique or more common in children, such as bleeding in the neonatal period or trauma-related injury. The current study was designed to explore the potential impact of these differences in children with severe FXI deficiency. METHODS: Medical files of all children with FXI level under 15% followed at five tertiary centers were evaluated. The retrieved data comprised demographic and clinical characteristics, including bleeding episodes, surgical interventions, treatment strategies, as well as laboratory features. RESULTS: Sixty children, whose median age at diagnosis was 4.2 years and their median FXI level was 4%, were included. Three children experienced triggered intracranial hemorrhage (ICH) and two children had major bleeds. No bleeding complications occurred in surgeries in which hemostatic treatment consisting mostly of tranexamic acid or fresh frozen plasma was applied (n = 45). In contrast, excessive bleeding was noted in 25% of surgical procedures performed without hemostatic preparation (p = .002). CONCLUSION: This study's findings confirm the generally favorable outcome of this rare bleeding disorder, with no spontaneous bleeds or cases of perinatal ICH. Nonetheless, proper diagnosis and adequate hemostasis in the surgical setting are imperative. Unlike previous studies in adults, our pediatric study suggests an association between the severity of FXI deficiency and bleeding tendency.

Molecular Mechanisms of Skewed X-Chromosome Inactivation in Female Hemophilia Patients-Lessons from Wide Genome Analyses.

INTRODUCTION: Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency or dysfunction due to F8 gene mutations. HA carriers are usually asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals. However, in rare cases, a carrier may exhibit symptoms of moderate to severe HA primarily due to skewed inactivation of her non-hemophilic X chromosome. AIM: The aim of the study was to investigate X-chromosome inactivation (XCI) patterns in HA carriers, with special emphasis on three karyotypically normal HA carriers presenting with moderate to severe HA phenotype due to skewed XCI, in an attempt to elucidate the molecular mechanism underlying skewed XCI in these symptomatic HA carriers. The study was based on the hypothesis that the presence of a pathogenic mutation on the non-hemophilic X chromosome is the cause of extreme inactivation of that X chromosome. METHODS: XCI patterns were studied by PCR analysis of the CAG repeat region in the HUMARA gene. HA carriers that demonstrated skewed XCI were further studied by whole-exome sequencing (WES) followed by X chromosome-targeted bioinformatic analysis. RESULTS: All three HA carriers presenting with the moderate to severe HA phenotype due to skewed XCI were found to carry pathogenic mutations on their non-hemophilic X chromosomes. Patient 1 was diagnosed with a frameshift mutation in the PGK1 gene that was associated with familial XCI skewing in three generations. Patient 2 was diagnosed with a missense mutation in the SYTL4 gene that was associated with familial XCI skewing in two generations. Patient 3 was diagnosed with a nonsense mutation in the NKAP gene that was associated with familial XCI skewing in two generations. CONCLUSION: Our results indicate that the main reason for skewed XCI in our female HA patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation on the silenced X chromosome, thus complicating the phenotype of a monogenic X-linked disease. Based on our study, we are currently offering the X inactivation test to symptomatic hemophilia carriers and plan to expand this approach to symptomatic carriers of other X-linked diseases, which can be further used in pregnancy planning.

Women with Hemophilia: Case Series of Reproductive Choices and Review of Literature.

Aim Very little is known regarding reproductive choices, pregnancy, and delivery of women with moderate to severe hemophilia. Our aim was to describe our experience with three hemophiliac women and their journey to achieve motherhood. Methods Medical charts of women with moderate to severe hemophilia A treated at our center were evaluated. Data regarding choices of conception, pregnancy course, mode of delivery, and pregnancy outcomes were obtained. Results Three women are presented. Whereas patient 1 chose to adopt her first child and later had twins through egg donations and a surrogate mother, patient 2 underwent spontaneous pregnancy and delivered via cesarean section. Patient 3 preferred in vitro fertilization and preimplantation genetic diagnosis to avoid hemophilia and hemophilia carriership in her offspring. Conclusion The appropriate means to achieve parenthood for women with moderate to severe hemophilia should be individualized and requires support of a comprehensive multidisciplinary team.

Novel mutation in coagulation factor VII (Carmel mutation): Identification and characterization.

BACKGROUND: Measurement of factor VII (FVII) activity does not enable prediction of bleeding tendency in individuals with inherited FVII deficiency. OBJECTIVE: To characterize the molecular and functional features of FVII in a family with FVII deficiency and correlate them with the bleeding tendency. PATIENTS/METHODS: We studied 7 family members with very low FVII activity using prothrombin time (PT), activated factor VII (FVIIa), FVII activity level, and thrombin generation. The factor 7 gene was sequenced and the mutation was analyzed by prediction software. RESULTS: The proband has very low FVII activity (0%-4%), with PT ranging between 5% to 18% depending on the tissue factor (TF) origin. Direct sequencing demonstrated a single homozygous nucleotide substitution G > A in exon 6, predicting a novel missense mutation Cys164Tyr. Three members of the family were found to be heterozygous carriers of this mutation. One of them was a compound heterozygote, carrying both the Cys164Tyr and Ala244Val mutation (linked to Arg353Gln polymorphism). Her FVII activity and antigen levels were 3%-7% and 8%, respectively. The other heterozygous carriers demonstrated FVII activity of 41%-54%, FVII antigen of 46%-66%, and FVIIa activity of 30%. FVIIa was undetectable in the homozygous and compound heterozygous subjects. Thrombin generation was normal in the presence of calcium, but no response to TF addition was observed in the homozygous proband, and a reduced response was observed in the compound heterozygous subject. CONCLUSION: The patient homozygous for the "Carmel" mutation has mild clinical manifestations despite very low FVII activity, which correlates with thrombin generation results.

Maternal Anti-HPA-1a Antibodies Increase Endothelial Cell Apoptosis and Permeability.

Intracranial hemorrhage (ICH) associated with fetal/neonatal alloimmune thrombocytopenia (FNAIT) is attributed mainly to endothelial damage caused by binding of maternal anti-HPA-1a antibodies to the αvß3 integrin on endothelial cells (ECs). We examined the effect of anti-HPA-1a antibodies on EC function using 2 EC lines from different vascular beds, HMVEC of dermal origin and hCMEC/D3 of cerebral origin. Anti-HPA-1a sera significantly increased apoptosis in both HMVEC and hCMEC/D3 cells and permeability in hCMEC/D3 cells only. This increase in both apoptosis and permeability was significantly inhibited by a monoclonal anti-ß3 antibody (SZ21) binding to the HPA-1a epitope. Our results indicate that (1) maternal anti-HPA-1a antibodies impair EC function by increasing apoptosis and permeability and (2) ECs from different vascular beds vary in their susceptibility to pathological effects elicited by maternal anti-HPA-1a antibodies on EC permeability. Examination of maternal anti-HPA-1a antibodies for their effect on EC permeability may predict potential ICH associated with FNAIT.

Mutations in RASGRP2 gene identified in patients misdiagnosed as Glanzmann thrombasthenia patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AIM: We aimed to study the genetic cause of IPFD mimicking GT. METHODS: During 2017-2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. RESULTS: Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbß3 and αvß3, as well as normal integrin αIIbß3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DISCUSSION: Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. CONCLUSION: Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.

The potential role of emicizumab prophylaxis in severe von Willebrand disease.

BACKGROUND: Severe von Willebrand disease (VWD) may be associated with chronic joint damage and may require prophylactic therapy. Emicizumab is a humanized bispecific antibody, which mimics the function of coagulation factor VIII (FVIII), and it has been approved for prophylaxis in hemophilia A. METHODS: This is the first study assessing the potential future role of emicizumab as an alternative prophylactic treatment in patients with severe VWD, based upon a thrombin generation (TG) ex vivo analysis. We report 51 weeks of successful off label emicizumab prophylaxis in a child with severe VWD and recurrent hemarthroses and progressive arthropathy despite adherence to previous prophylaxis with replacement therapy. RESULTS AND CONCLUSIONS: Our work demonstrated that ex vivo spiking with emicizumab increased TG in plasma from patients with type 3 VWD. Similar TG results were observed in our treated patient, whose therapy was well tolerated without any adverse events. Both in vitro and ex vivo TG data support sufficient hemostasis without exceeding the range seen in healthy volunteers. Further collaborative studies on the efficacy and safety of emicizumab prophylaxis in severe VWD is warranted.

Severe Protein C Deficiency due to Novel Biallelic Variants in PROC and Their Phenotype Correlation.

Severe protein C deficiency due to biallelic PROC mutations is an extremely rare thrombophilia, most commonly presenting during the neonatal period as purpura fulminans. Despite treatment, severe morbidity and mortality are frequent. The current study reports 3 unrelated patients harboring novel homozygous PROC mutations and their clinical phenotypes. We discuss how the cytoprotective activity of protein C and its role in the stabilization of endothelial barriers may account for the unique symptoms of this thrombophilia.

The effect of civil and military flights on coagulation, fibrinolysis and blood flow: insight from a rat model.

BACKGROUND: Air travel thrombosis continues to be a controversial topic. Exposure to hypoxia and hypobaric conditions during air travel is assumed a risk factor. The aim of this study is to explore changes in parameters of coagulation, fibrinolysis and blood flow in a rat model of exposure to hypobaric conditions that imitate commercial and combat flights. METHODS: Sixty Sprague-Dawley male rats, aged 10 weeks, were divided into 5 groups according to the type and duration of exposure to hypobaric conditions. The exposure conditions were 609 m and 7620 m for 2 and 12 h duration. Blood count, thrombin- antithrombin complex, D-dimer, interleukin-1 and interleukin-6 were analyzed. All rats went through flight angiography MRI at day 13-post exposure. RESULTS: No effect of the various exposure conditions was observed on coagulation, fibrinolytic system, IL-1 or IL-6. MRI angiography showed blood flow reduction in lower limb to less than 30% in 50% of the rats. The reduction in blood flow was more pronounced in the left vessel than in the right vessel (p = 0.006, Wilcoxon signed rank test). The extent of occlusion differed across exposure groups in the right, but not the left vessel (p = 0.002, p = 0.150, respectively, Kruskal-Wallis test). However, these differences did not correlate with the exposure conditions. CONCLUSION: In the present rat model, no clear correlation between various hypobaric conditions and activation of coagulation was observed. The reduction in blood flow in the lower limb also occurred in the control group and was not related to the type of exposure.

Single Low Dose of rFVIIa Combined with Antifibrinolytic Agent is a Simple and Safe Treatment for Factor XI-Deficient Patients undergoing Surgery.

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal bleeding disorder. The rarity of spontaneous bleeding and absence of optimal tools to predict the bleeding risk in FXI-deficient patients hamper the standardization of prophylactic treatment enabling them to undergo major surgeries without blood products. OBJECTIVES: We explored the effectiveness of a single and very low dose of recombinant factor VIIa (rFVIIa) along with tranexamic acid (TXA) as prophylactic treatment for FXI-deficient patients undergoing various types of surgery at various sites of injury. We studied the potential use of thrombin generation (TG) as a surrogate tool for predicting thrombogenicity. PATIENTS AND METHODS: Our cohort consisted of 10 patients with severe FXI deficiency undergoing 12 interventions. Patients received a single dose of 10 to 15 µg/kg rFVIIa at the end of surgery in addition to TXA initiated 2 hours before surgery at the dose of 4 g/day for 3 to 5 days. TG was tested before and 30 minutes after rFVIIa administration. RESULTS: All operations were uneventful and none of the patients bled excessively or required blood products. No thrombotic event was reported, and the postoperative hospitalization duration was comparable to that of patients without bleeding disorders. TG performed at the peak of rFVIIa was below the curve of healthy controls, thus confirming that the administered dose was not thrombogenic. CONCLUSION: A single very low dose of rFVIIa along with TXA is a simple and safe treatment to control hemostasis in severe FXI-deficient patients undergoing diverse type of surgical procedure at various sites.

Activated protein C induces suppression and regression of choroidal neovascularization- A murine model.

Activated protein C (APC) exerts diverse cell signaling pathways which results in multiple distinct cytoprotective actions. These include anti-apoptotic and anti-inflammatory activities and stabilization of endothelial and epithelial barriers. We studied the ability of APC to inhibit the leakage and the growth of newly formed as well as pre-existing choroidal neovascularization (CNV) and examined the ability of APC to stabilize the Retinal Pigmented Epithelium (RPE). We explored the contribution of Tie2 receptor to the protective effects of APC. CNV was induced by laser photocoagulation in C57BL/6J mice. APC was injected intravitreally immediately or 7 days after CNV induction. Neovascularization was evaluated on RPE-choroidal flatmounts using FITC-dextran perfusion and CD31 immunofluorescence. CNV leakage was measured by fluorescein angiography (FA). The ability of APC to stabilize the RPE barrier was evaluated in-vitro by dextran permeability and zonula occludens 1 (ZO1) immunostaining. Tie2 blocking was induced in-vivo by intraperitoneal injection of Tie2 kinase inhibitor and in-vitro by incubation with anti Tie2 antibodies. APC treatment dramatically inhibited the generation of newly formed CNV leakage sites and reversed leakage in 85% of the pre-existing CNV leaking sites. In RPE cell culture, APC induced translocation of ZO1 to the cell membrane, accompanied by reduction in permeability of the monolayer. Inhibition of Tie2 significantly decreased APC protective activities in both the mouse model and the RPE cell culture. Our results show that APC treatment significantly inhibits the leakage and growth of newly formed, as well as pre-existing CNV, and its protective activities are partially mediated via the Tie2 receptor. The data suggest that APC should be further investigated as a possible effective treatment for CNV.

[HEMOPHILIA - A ROYAL DISEASE IN THE HOLY LAND].

INTRODUCTION: Hemophilia is a hereditary congenital hemorrhagic diathesis caused by mutations in blood coagulation factor VIII (FVIII) or IX (FIX) genes, causing hemophilia A and B, respectively. Most cases are familial but a significant minority is sporadic. OBJECTIVES: To examine the presenting symptoms of patients with hemophilia in Israel and identify causes for delay in diagnosis. METHODS: Retrospective analysis of data from medical files of newly diagnosed patients with hemophilia during the period from 1st January 2010 to 31s December 2017. RESULTS: During the study period 104 children had been diagnosed with hemophilia. Fifteen percent were diagnosed with hemophilia B and 85% with hemophilia A. In most familial cases the diagnosis was established by examination of aPTT and the level of the relevant clotting factor shortly after birth. Diagnosis of sporadic cases (40 cases) was performed due to suggestive clinical symptoms. Perinatal complications were observed in 6 newborns. The most common presenting symptom was disproportionate bleeding following circumcision. In 6/21 patients who experienced excessive bleeding following circumcision there was a delay in diagnosis. Severe bleeding requiring intensive care admission was observed in twelve neonates. DISCUSSION: Diagnosis of hemophilia in Israel is made at an earlier age than in Europe. This is probably due to the performance of ritual circumcision during the neonatal period. In recent years a reduction in familial cases of severe hemophilia has been noted due to genetic counseling of hemophilia carriers. In cases of excessive bleeding following circumcision aPTT and PT should be examined promptly.

Alternative treatment options for pediatric hemophilia B patients with high-responding inhibitors: A thrombin generation-guided study.

Little is known about the challenging treatment of pediatric patients with hemophilia B and inhibitors due to disease rarity. We describe three patients diagnosed in childhood and followed up to 9 years. All three had allergic reactions to Factor IX, but two were later safely treated for bleeding episodes with activated prothrombin complex concentrates (APCC = FEIBA). The third was given only recombinant activated Factor VIIa. Based on ex vivo thrombin generation analysis, a new alternative treatment of combined bypassing agents was administered for bleeding episodes and several minor surgical procedures with no treatment-associated adverse events or thrombosis.

Possible involvement of NETosis in inflammatory processes in the eye: Evidence from a small cohort of patients.

Purpose: To evaluate whether NETosis is involved in cytokine-induced ocular inflammation and to track neutrophil extracellular traps (NET) complexes in patients with proliferative diabetic retinopathy (PDR). Methods: For the animal model, the eyes of C57BL/6J mice were intravitreally injected with interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), or saline. Histology and immunofluorescence staining for CD11b, neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone 3 (H3Cit), and net-like structure were performed. Vitreous samples were collected from patients with PDR; the PDR1 group had no need for repeated surgical intervention, and the PDR2 group had repeated vitreous bleeding or other complication and controls. Levels of MPO, H3Cit-MPO, and NE-MPO complex were measured with enzyme-linked immunosorbent assay (ELISA). Results: Massive influx of CD11+ inflammatory cells, involving the anterior and posterior chambers, was observed in the murine eyes 24 h after the IL-8 or TNF-α injections. Cells excreted to their surroundings an extracellular net-like structure positive for NE, MPO, and H3Cit. H3Cit staining was abolished with the DNase I treatment, indicating the presence of extracellular DNA in the net-like structures. The vitreous samples of the patients with PDR2 contained statistically significantly higher levels of MPO (173±230) compared to those of the patients with PDR1 (12.0±33.0, p<0.05) or the controls (0.00, p<0.01). The levels of H3Cit-MPO and NE-MPO complexes were also statistically significantly higher in the patients with PDR2 (776.0±1274, 573.0±911.0, respectively) compared to those in the patients with PDR1 (0, p<0.05) and the controls (0, p<0.05). Conclusions: This study showed the existence of NETosis in cytokine-induced ocular inflammation in a mouse model and human samples. Furthermore, the extent of NET complex formation was higher in a subset of patients who exhibited more complicated PDR.