NLRC4-mediated pyroptosis was involved in coagulation disorders of acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. METHODS: RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). RESULTS: Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1ß, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. CONCLUSIONS: NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.

Bleeding disorders in Saudi Arabia, causes and prevalence: a review.

As bleeding disorders are a worldwide health concern, Saudi Arabia is experiencing a notable prevalence of such disorders. Studying the frequency and cause of hemostatic disorders is the key to successful clinical interventions and instigating effective public policies that limit the spread of such disorders. The current review aims to highlight the major findings of the body of literature that has investigated the causes, prevalence, and major challenges associated with bleeding disorders in the country. The current review summarizes the major findings of different studies that have been conducted in Saudi Arabia regarding different bleeding disorders. Multiple causes and symptoms of bleeding disorders have been reported by different studies. Some studies investigated the genetic aspect of bleeding disorders and revealed specific mutations in coagulation factor genes influencing the symptoms of different bleeding disorders. Moreover, rare bleeding disorders such as Glanzmann thrombasthenia and Henoch-Schönlein purpura, have been reported in different regions of Saudi Arabia. Combining clinical presentations, genetic factors, and epidemiological data, the current review of the literature provides a comprehensive insight into bleeding disorders in the kingdom. This will help in advancing the diagnostic capabilities and genetic counseling enhancing management strategies and therapeutic interventions benefiting bleeding disorder patients and the kingdom.

Blood Coagulation Disorders Among the Iranian Population: a Systematic Review.

BACKGROUND: Blood coagulation disorders are one of the causes of mortality. Therefore, the study of coagulation disorders is also important. This systematic review was conducted to investigate blood coagulation disorders in the Iranian population. METHODS: Searches in electronic databases such as Web of Science, PubMed, Scopus, SID, ProQuest, and Magiran from May 10, 1990 to May 10, 2019 were performed according to PRISMA guidelines. Cross-sectional, cohort, experimental, and case-control studies were included according to the inclusion criteria without gender and language restrictions. RESULTS: After screening and selection, 14 studies were selected for data extraction. Accordingly, the most common blood coagulation disorder in the south of Iran was a defect in FXIII (599 of 1,165). C.559T>C (27 of 189) and c.562T>C (20 of 189) mutations had the highest frequency. The most common FXIII polymorphism among the Iranian Azerbaijanis was Val34Leu (203 of 410). The second most common coagulation disorder was FV Leiden (396 of 1,165). Then, c.1691G>A (151 of 396) was the most common mutation. CONCLUSIONS: This study shows that the most critical coagulation disorder among the Iranian population is FXIII deficiency and the most common mutation is c.562T>C.

Genetic Analysis of Hereditary Coagulation Factor V Deficiency in Two Chinese Families Caused by Compound Heterozygous Mutations.

OBJECTIVE: This study aims to provide a preliminary discussion of the molecular basis of FV deficiency caused by compound heterozygous mutations in two Chinese families. METHODS: Relative coagulation index was measured by the one-stage clotting method and the FV:Ag was measured by ELISA. All exons and flanking regions of the F5 gene were amplified by PCR and directly sequenced. ClustalX-2.1-win was used to analyze the conservation of mutations. The online software was used to predict the pathogenicity of mutations. PyMOL was used to analyze the variation in the spatial structure of the FV protein before and after mutations. Calibrated automated thrombogram was used to analyze the function of the mutant protein. RESULTS: Phenotyping suggested that both probands had a simultaneous decrease in FV:C and FV:Ag. Their genetic tests showed that proband A had a missense mutation p.Ser111Ile in exon 3 and a polymorphism p.Arg2222Gly in exon 25. At the same time, the proband B had a missense mutation p.Asp96His in exon 3 and a frame-shift mutation p.Pro798Leufs*13 in exon 13. Meanwhile, the p.Ser111Ile is conserved among homologous species. The bioinformatics and protein model analysis revealed that p.Ser111Ile and p.Pro798Leufs*13 were pathogenic and could affect the structure of the FV protein. The thrombin generation test revealed that the clotting function of proband A and B had been affected. CONCLUSION: These four mutations may be responsible for the reduction of FV levels in two Chinese families. Moreover, the p.Ser111Ile mutation is a novel pathogenic variant that has not been reported.

Missense Variants of von Willebrand Factor in the Background of COVID-19 Associated Coagulopathy.

COVID-19 associated coagulopathy (CAC), characterized by endothelial dysfunction and hypercoagulability, evokes pulmonary immunothrombosis in advanced COVID-19 cases. Elevated von Willebrand factor (vWF) levels and reduced activities of the ADAMTS13 protease are common in CAC. Here, we aimed to determine whether common genetic variants of these proteins might be associated with COVID-19 severity and hemostatic parameters. A set of single nucleotide polymorphisms (SNPs) in the vWF (rs216311, rs216321, rs1063856, rs1800378, rs1800383) and ADAMTS13 genes (rs2301612, rs28729234, rs34024143) were genotyped in 72 COVID-19 patients. Cross-sectional cohort analysis revealed no association of any polymorphism with disease severity. On the other hand, analysis of variance (ANOVA) uncovered associations with the following clinical parameters: (1) the rs216311 T allele with enhanced INR (international normalized ratio); (2) the rs1800383 C allele with elevated fibrinogen levels; and (3) the rs1063856 C allele with increased red blood cell count, hemoglobin, and creatinine levels. No association could be observed between the phenotypic data and the polymorphisms in the ADAMTS13 gene. Importantly, in silico protein conformational analysis predicted that these missense variants would display global conformational alterations, which might affect the stability and plasma levels of vWF. Our results imply that missense vWF variants might modulate the thrombotic risk in COVID-19.

MiR-186-5p Downregulates NAMPT and Functions as a Potential Therapeutic Target for Sepsis-Induced Coagulation Disorders.

Purpose: Present study is aimed to explore the role of miR-186-5p in sepsis-induced coagulation disorders and molecular mechanisms. Methods: Thirty-four sepsis patients and 34 respiratory infection/pneumonia patients were selected in the present study. Polymicrobial sepsis model was created by cecal ligation and puncture (CLP). The mRNA expression was detected by qRT-PCR. Western blot was utilized to measure protein expression. Thromborel S Reagent was applied to measure the prothrombin time (PT). Platelet count of blood was measured via LH 780. ELISA kits were utilized to evaluate the fibrinogen and PAI-1 concentration. Results: MiR-186-5p expression was lower and nicotinamide phosphoribosyltransferase (NAMPT) mRNA expression was higher in sepsis patients in contrast to control group. Coagulation time was markedly prolonged and platelet count was markedly decreased in CLP mice. In addition, fibrinogen concentration was obviously lower and PAI-1 concentration was obviously higher in CLP mice. MiR-186-5p mimic obviously decreased coagulation time and PAI-1 concentration, while raised platelet count and fibrinogen concentration. Targetscan predicted miR-186-5p might directly regulates NAMPT, and luciferase reporter assay verified this prediction. In addition, miR-186-5p mimic obviously inhibited the mRNA expression of NAMPT. Knockdown of NAMPT improved coagulation dysfunction in sepsis. Overexpression of NAMPT reversed the improvement effect of miR-186-5p on coagulation dysfunction. MiR-186-5p mimic markedly inhibited NF-κB pathway. Conclusion: MiR-186-5p inhibited sepsis-induced coagulation disorders via targeting NAMPT and inactivating NF-κB pathway.

Hemorrhagic shock and encephalopathy syndrome in a patient with a de novo heterozygous variant in KIF1A.

INTRODUCTION: KIF1A, a gene that encodes a neuron-specific motor protein, plays important roles in cargo transport along neurites. Variants in KIF1A have been described in three different disorders, and neurodegeneration and spasticity with or without cerebellar atrophy or cortical visual impairment syndrome (NESCAVS) is the severest phenotype. CASE REPORT: A 3-year-old girl was born at term with a birth weight of 2590 g. At five months of age, she visited our hospital due to developmental delay. An EEG showed multiple epileptic discharge, and a nerve conduction study showed severe axonopathy of both motor and sensory nerves. We performed exome sequencing and identified a de novo heterozygous missense variant in KIF1A (NM_001244008.1: c. 757G > A, p.E253K). At six months of age, she developed acute encephalopathy, multiple organ failure and disseminated intravascular coagulation, necessitating intensive care. Her brain CT showed severe brain edema, followed by profound brain atrophy. We diagnosed hemorrhagic shock and encephalopathy syndrome (HSES) according to the clinico-radiological features. Currently, she is bed-ridden, and requires gastrostomy because of dysphagia. CONCLUSION: The clinical course of our case confirmed that p.E253K is associated with severe neurological features. Severe KIF1A deficiency could cause thermoregulatory dysfunction and may increase the risk of acute encephalopathy including HSES.

A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients.

BACKGROUND: The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors. METHODS: Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography. RESULTS: Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors. Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls. CONCLUSION: Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level.

Factor V east Texas variant causes bleeding in a three-generation family.

BACKGROUND: The factor V east Texas bleeding disorder (FVETBD) is caused by increased plasma tissue factor pathway inhibitor-α (TFPIα) concentration. The underlying cause is a variant in F5 causing alternative splicing within exon 13 and producing FV-short, which tightly binds the C-terminus of TFPIα, prolonging its circulatory half-life. OBJECTIVES: To diagnose a family presenting with variable bleeding and laboratory phenotypes. PATIENTS/METHODS: Samples were obtained from 17 family members for F5 exon 13 sequencing. Plasma/platelet TFPI and platelet FV were measured by ELISA and/or western blot. Plasma thrombin generation potential was evaluated using calibrated automated thrombography. RESULTS: The FVET variant was identified in all family members with bleeding symptoms and associated with elevated plasma TFPIα (4.5- to 13.4-fold) and total TFPI (2- to 3-fold). However, TFPIα and FV-short were not elevated in platelets. TF-initiated thrombin generation in patient plasma was diminished but was restored by a monoclonal anti-TFPI antibody or factor VIIa. TFPIα localized within vascular extracellular matrix in an oral lesion biopsy from an affected family member. CONCLUSIONS: Factor V east Texas bleeding disorder was diagnosed in an extended family. The variant was autosomal dominant and highly penetrant. Elevated plasma TFPIα, rather than platelet TFPIα, was likely the primary cause of bleeding. Plasma FV-short did not deplete TFPIα from extracellular matrix. In vitro thrombin generation was restored with an anti-TFPI antibody or factor VIIa suggesting effective therapies may be available. Increased awareness of, and testing for, bleeding disorders associated with F5 exon 13 variants and elevated plasma TFPI are needed.

Contribution of factor VII polymorphisms to coagulopathy in patients with isolated traumatic brain injury.

BACKGROUND: Coagulopathy is a severe complication of traumatic brain injury (TBI) and can cause secondary injuries and death. Decrease of FVII activity contributes to the coagulopathy and progressive hemorrhagic injury (PHI) in patients with isolated TBI. Some polymorphic loci of coagulation factor VII (FVII) are shown to be essential for FVII activity. However, the relationship between FVII gene polymorphisms and coagulopathy in patients with isolated TBI is still unknown. Therefore, the present study aimed to investigate the relationship between FVII gene polymorphisms and plasma FVIIa levels, and assess whether FVII polymorphisms were associated with TBI-related coagulopathy, PHI, and 6 months GOS in patients with isolated TBI. METHODS: One-hundred-forty-nine patients with isolated TBI (from East of China) admitted to Huashan Hospital's Neurological Trauma Center from March 2012 to March 2016 were enrolled in this study. The Polymorphism-Polymerase Chain Reaction (PCR) method was used to analyze the five FVII polymorphism loci (-323P0/P10, R353Q, -401G/T, -402G/A, and -670A/C) of these patients. Patients' blood was collected to test the activated partial thromboplastin time, international normalized ratio, platelet, and FVIIa concentrations. Other clinical characteristics were also recorded. RESULTS: The minor alleles of three genotypes of -323 P0/P10, R353Q, and -401G/T each independently associated with 23.3%, 28.6%, and 27.6% lower FVIIa levels, respectively. These polymorphisms explained 21% of the total variance of FVIIa levels (adjusted R2:0.206). The genotype of -323P0/P10 was an independent risk factor for coagulopathy (OR = 2.77, p = 0.043) and PHI (OR = 3.47, p = 0.03) after adjustment for confounding factors in the logistic regression model. Polymorphisms of FVII were not independently associated with 6 months Glasgow Outcome Scale (GOS) of isolated TBI patients. CONCLUSION: -323P0/P10, R353Q, and -401 G/T genotypes were associated with FVIIa levels. -323P0/P10 genotype was independently associated with traumatic coagulopathy and PHI in isolated TBI patients.

F5-Atlanta: A novel mutation in F5 associated with enhanced East Texas splicing and FV-short production.

BACKGROUND: Elucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B-domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half-life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls. OBJECTIVE: We identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5-Atlanta (F5-ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression. METHODS & RESULTS: Assessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV-short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5-ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells. CONCLUSIONS: These findings support a mechanistic model consisting of cis-acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV-short and TFPIα levels.

Whole Blood Thromboelastometry by ROTEM and Thrombin Generation by Genesia According to the Genotype and Clinical Phenotype in Congenital Fibrinogen Disorders.

The outcome of congenital fibrinogen defects (CFD) is often unpredictable. Standard coagulation assays fail to predict the clinical phenotype. We aimed to assess the pheno- and genotypic associations of thrombin generation (TG) and ROTEM in CFD. We measured fibrinogen (Fg) activity and antigen, prothrombin fragments F1+2, and TG by ST Genesia® with both Bleed- and ThromboScreen in 22 patients. ROTEM was available for 11 patients. All patients were genotyped for fibrinogen mutations. Ten patients were diagnosed with hypofibrinogenemia, nine with dysfibrinogenemia, and three with hypodysfibrinogenemia. Among the 17 mutations, eight were affecting the Fg γ chain, four the Fg Bß chain, and five the Fg Aα chain. No statistical difference according to the clinical phenotypes was observed among FGG and FGA mutations. Median F1+2 and TG levels were normal among the different groups. Fg levels correlated negatively with F1+2 and peak height, and positively with lag time and time to peak. The pheno- and genotypes of the patients did not associate with TG. FIBTEM by ROTEM detected hypofibrinogenemia. Our study suggests an inverse link between low fibrinogen activity levels and enhanced TG, which could modify the structure-function relationship of fibrin to support hemostasis.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

Diagnosis of rare bleeding disorders.

Rare bleeding disorders result in significant morbidity but are globally underdiagnosed. Advances in genomic testing and specialist laboratory assays have greatly increased the diagnostic armamentarium. This has resulted in the discovery of new genetic causes for rare diseases and a better understanding of the underlying molecular pathology.

Genetics of equine bleeding disorders.

Genetic bleeding disorders can have a profound impact on a horse's health and athletic career. As such, it is important to understand the mechanisms of these diseases and how they are diagnosed. These diseases include haemophilia A, von Willebrand disease, prekallikrein deficiency, Glanzmann's Thrombasthenia and Atypical Equine Thrombasthenia. Exercise-induced pulmonary haemorrhage also has a proposed genetic component. Genetic mutations have been identified for haemophilia A and Glanzmann's Thrombasthenia in the horse. Mutations are known for von Willebrand disease and prekallikrein deficiency in other species. In the absence of genetic tests, bleeding disorders are typically diagnosed by measuring platelet function, von Willebrand factor, and other coagulation protein levels and activities. For autosomal recessive diseases, genetic testing can prevent the breeding of two carriers.