Coagulation-related Genes Research Articles

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

MicroRNA-122-5p regulates coagulation and inflammation through MASP1 and HO-1 genes.

MiR-122-5p is a diagnostic and prognostic biomarker of sepsis and is correlated with coagulation abnormalities in sepsis. However, its functional aspects remain unknown. This study applied bioinformatics analysis to evaluate the coagulation-related target genes for miR-122-5p. THP-1, HUVEC, and LO-2 cell lines were used in this study. MiR-122-5p mimics were transfected into the three previously mentioned cell lines, which helped in detecting mRNA and protein levels by qRT-PCR and western blotting, respectively. Serum samples from 84 sepsis patients were collected to evaluate target gene code proteins. The protein and mRNA levels of Heme oxygenase1(HO-1), IL-1β, IL-6, monocyte chemoattractant protein 1(MCP-1), and TNF-α were also evaluated in three cell lines. Mannan binding lectin serine peptidase 1(MASP1) was a direct target gene of miR-122-5p, and levels of MASP1, C3, and C4 were all significantly lower in the sepsis with disseminated intravascular coagulopathy (DIC) group than in the sepsis without DIC group. MiR-122-5p mimics could down-regulate HO-1 in the three cell lines. HO-1, IL-1β, IL-6, MCP-1, and TNF-α gene and protein levels were decreased after miR-122-5p mimics were added. MiR-122-5p regulated coagulation and inflammation through MASP1 and HO-1, respectively.

Epigenetic regulation of inflammatory genes involving histone methylation is associated with hyperimmune response in COVID-19 patients

Abstract One of the leading causes of the severe COVID-19 disease symptoms is the production of massive amounts of pro-inflammatory cytokines. Although the precise mechanisms that trigger such hyperimmune response is unclear, epigenetic mechanisms are known to play important roles in the regulation of cytokine production and inflammation. In this study we examined global gene expression and histone methylation profile in the PBMCs of hospitalized COVID-19 patients. Despite individual variations, the expressions of many inflammation-related genes such as Arginase 1 (ARG1) and IL1 receptor 2 (IL1R2) were significantly upregulated in COVID-19 patients. We also found the expressions of coagulation-related genes, VWF and Protein S, were altered in COVID-19 patients. The expression patterns of some genes such as IL1R2 correlated with their histone methylation marks. Pathway analysis revealed that most of these dysregulated genes were in the TGFb, IL1b, IL6 and IL17 pathway. Interestingly, we found that the majority of those altered genes were in the dexamethasone targeting pathway. We also found that the expression of BMX, a member of TEC family kinases, was significantly increased in the PBMCs of COVID-19 patient at both RNA level and protein level. Interestingly, some TEC kinase inhibitors have been used to treat COVID-19 patients in clinical trials. Overall, this study has identified genes in PBMCs of COVID-19 patients with altered histone mark and gene expression profile, providing important information towards identifying potential biomarkers and therapeutic targets for COVID-19 disease. (Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.) Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.

Ebola virus VP35 hijacks the PKA-CREB1 pathway for replication and pathogenesis by AKIP1 association

Ebola virus (EBOV), one of the deadliest viruses, is the cause of fatal Ebola virus disease (EVD). The underlying mechanism of viral replication and EBOV-related hemorrhage is not fully understood. Here, we show that EBOV VP35, a cofactor of viral RNA-dependent RNA polymerase, binds human A kinase interacting protein (AKIP1), which consequently activates protein kinase A (PKA) and the PKA-downstream transcription factor CREB1. During EBOV infection, CREB1 is recruited into EBOV ribonucleoprotein complexes in viral inclusion bodies (VIBs) and employed for viral replication. AKIP1 depletion or PKA-CREB1 inhibition dramatically impairs EBOV replication. Meanwhile, the transcription of several coagulation-related genes, including THBD and SERPINB2, is substantially upregulated by VP35-dependent CREB1 activation, which may contribute to EBOV-related hemorrhage. The finding that EBOV VP35 hijacks the host PKA-CREB1 signal axis for viral replication and pathogenesis provides novel potential therapeutic approaches against EVD.

Characterization of Altered Gene Expression and Histone Methylation in Peripheral Blood Mononuclear Cells Regulating Inflammation in COVID-19 Patients.

The pandemic of COVID-19 has caused >5 million deaths in the world. One of the leading causes of the severe form of COVID-19 is the production of massive amounts of proinflammatory cytokines. Epigenetic mechanisms, such as histone/DNA methylation, miRNA, and long noncoding RNA, are known to play important roles in the regulation of inflammation. In this study, we investigated if hospitalized COVID-19 patients exhibit alterations in epigenetic pathways in their PBMCs. We also compared gene expression profiles between healthy controls and COVID-19 patients. Despite individual variations, the expressions of many inflammation-related genes, such as arginase 1 and IL-1 receptor 2, were significantly upregulated in COVID-19 patients. We also found the expressions of coagulation-related genes Von Willebrand factor and protein S were altered in COVID-19 patients. The expression patterns of some genes, such as IL-1 receptor 2, correlated with their histone methylation marks. Pathway analysis indicated that most of those dysregulated genes were in the TGF-β, IL-1b, IL-6, and IL-17 pathways. A targeting pathway revealed that the majority of those altered genes were targets of dexamethasone, which is an approved drug for COVID-19 treatment. We also found that the expression of bone marrow kinase on chromosome X, a member of TEC family kinases, was increased in the PBMCs of COVID-19 patients. Interestingly, some inhibitors of TEC family kinases have been used to treat COVID-19. Overall, this study provides important information toward identifying potential biomarkers and therapeutic targets for COVID-19 disease.

SERPINE1 rs6092 Variant Is Related to Plasma Coagulation Proteins in Patients with Severe COVID-19 from a Tertiary Care Hospital.

Simple SummarySevere forms of coronavirus disease 2019 (COVID-19) are related to an alteration in the coagulation process determined by genetic factors. Variability on F5 and SERPINE1 genes has been previously reported as a risk factor for thrombosis associated with different diseases. In this study, we aimed to evaluate if two relevant variants in the F5 and SERPINE1 genes were related to the requirement of invasive mechanical ventilation in hospitalized patients with COVID-19 and/or the levels of coagulation-related proteins. We observed that patients presenting the studied variants in F5 and SERPINE1 exhibit different levels of coagulation-related proteins. Moreover, the levels of these proteins were higher among patients requiring invasive mechanical ventilation when compared to hospitalized patients with no ventilation support. This study contributes to the genetics insight regarding COVID-19 severity and the inter-individual susceptibility for a severe form of the infectious disease.An impaired coagulation process has been described in patients with severe or critical coronavirus disease (COVID-19). Nevertheless, the implication of coagulation-related genes has not been explored. We aimed to evaluate the impact of F5 rs6025 and SERPINE1 rs6092 on invasive mechanical ventilation (IMV) requirement and the levels of coagulation proteins among patients with severe COVID-19. Four-hundred fifty-five patients with severe COVID-19 were genotyped using TaqMan assays. Coagulation-related proteins (P-Selectin, D-dimer, P-selectin glycoprotein ligand-1, tissue plasminogen activator [tPA], plasminogen activator inhibitor-1, and Factor IX) were assessed by cytometric bead arrays in one- and two-time determinations. Accordingly, SERPINE1 rs6092, P-Selectin (GG 385 pg/mL vs. AG+AA 632 pg/mL, p = 0.0037), and tPA (GG 1858 pg/mL vs. AG+AA 2546 pg/mL, p = 0.0284) levels were different. Patients carrying the CT F5-rs6025 genotype exhibited lower levels of factor IX (CC 17,136 pg/mL vs. CT 10,247 pg/mL, p = 0.0355). Coagulation proteins were also different among IMV patients than non-IMV. PSGL-1 levels were significantly increased in the late stage of COVID-19 (>10 days). The frequencies of F5 rs6025 and SERPINE1 rs6092 variants were not different among IMV and non-IMV. The SERPINE1 rs6092 variant is related to the impaired coagulation process in patients with COVID-19 severe.

A Coagulation-Related Gene-Based Prognostic Model for Invasive Ductal Carcinoma

Background: Invasive ductal carcinoma (IDC) is the most common type of metastatic breast cancer. Due to the lack of valuable molecular biomarkers, the diagnosis and prognosis of IDC remain a challenge. A large number of studies have confirmed that coagulation is positively correlated with angiogenesis-related factors in metastatic breast cancer. Therefore, the purpose of this study was to construct a COAGULATION-related genes signature for IDC using the bioinformatics approaches.Methods: The 50 hallmark gene sets were obtained from the molecular signature database (MsigDB) to conduct Gene Set Variation Analysis (GSVA). Gene Set Enrichment Analysis (GSEA) was applied to analyze the enrichment of HALLMARK_COAGULATION. The COAGULATION-related genes were extracted from the gene set. Then, Limma Package was used to identify the differentially expressed COAGULATION-related genes (DECGs) between ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) samples in GSE26340 data set. A total of 740 IDC samples from The Cancer Genome Atlas (TCGA) database were divided into a training set and a validation set (7:3). The univariate and multivariate Cox regression analyses were performed to construct a risk signature, which divided the IDC samples into the high- and low-risk groups. The overall survival (OS) curve and receiver operating characteristic (ROC) curve were drawn in both training set and validation set. Finally, a nomogram was constructed to predict the 1-, 2-, 3-, 4-, and 5-year survival rates of IDC patients. Quantitative real-time fluorescence PCR (qRT-PCR) was performed to verify the expression levels of the prognostic genes.Results: The “HALLMARK_COAGULATION” was significantly activated in IDC. There was a significant difference in the clinicopathological parameters between the DCIS and IDC patients. Twenty-four DECGs were identified, of which five genes (SERPINA1, CAPN2, HMGCS2, MMP7, and PLAT) were screened to construct the prognostic model. The high-risk group showed a significantly lower survival rate than the low-risk group both in the training set and validation set (p=3.5943e-06 and p=0.014243). The risk score was demonstrated to be an independent predictor of IDC prognosis. A nomogram including risk score, pathological_stage, and pathological_N provided a quantitative method to predict the survival probability of 1-, 2-, 3-, 4-, and 5-year in IDC patients. The results of decision curve analysis (DCA) further demonstrated that the nomogram had a high potential for clinical utility.Conclusion: This study established a COAGULATION-related gene signature and showed its prognostic value in IDC through a comprehensive bioinformatics analysis, which may provide a potential new prognostic mean for patients with IDC.

Proposing a molecular classification associated with hypercoagulation in ovarian clear cell carcinoma

BackgroundAlthough ovarian clear cell carcinoma (CCC) is associated with high incidence of thromboembolism, the clinicopathological and biological significance of hypercoagulable status in CCC remains unclear. Materials and methodsWe retrospectively analyzed pretreatment D-dimer levels, thromboembolic status, and clinical outcome of 125 CCCs in the discovery set and 143 CCCs in two other independent validation sets. Next, we performed RNA sequencing of 93 CCCs and compared coagulation-related gene profiles with 2492 pan-cancer data. We investigated differences in molecular characteristics of CCC subclasses based on coagulation status. ResultsIn the discovery dataset, D-dimer elevation above the normal range was significantly associated with shorter progression-free and overall survival, irrespective to thromboembolic status. Multivariate analysis identified D-dimer elevation and clinical stage as an independent prognostic factors. We confirmed the prognostic significance of D-dimer elevation in the validation sets. Tissue factor and IL6, which are considered key elements of cancer-induced hypercoagulation, were highly expressed in CCC than in other cancers regardless of D-dimer level. Higher activity of various oncogenic pathways was observed in CCC with compared to without D-dimer elevation. Moreover, hierarchical cluster analysis divided 57 CCCs with D-dimer elevation into immunologically hot and cold tumor subtypes. Hot tumors were characterized by enrichment of T-cell inflamed phenotype, inflammation, the epithelial-mesenchymal transition, and high serum levels of CRP, and cold tumors by enrichment of cell cycle and MYC pathways. ConclusionsCCC represents hypercoagulable disease and elevate D-dimer is a prognostic factor for decreased survival in CCC. D-dimer high CCC has distinct molecular characteristics into the inflammatory-driven pathway (hot tumor) and the immune-suppressive pathway (cold tumor). Treatment implication of our proposed molecular classification merits further investigation.

Distinct Hepatic Gene-Expression Patterns of NAFLD in Patients With Obesity.

Approaches to manage nonalcoholic fatty liver disease (NAFLD) are limited by an incomplete understanding of disease pathogenesis. The aim of this study was to identify hepatic gene‐expression patterns associated with different patterns of liver injury in a high‐risk cohort of adults with obesity. Using the NanoString Technologies (Seattle, WA) nCounter assay, we quantified expression of 795 genes, hypothesized to be involved in hepatic fibrosis, inflammation, and steatosis, in liver tissue from 318 adults with obesity. Liver specimens were categorized into four distinct NAFLD phenotypes: normal liver histology (NLH), steatosis only (steatosis), nonalcoholic steatohepatitis without fibrosis (NASH F0), and NASH with fibrosis stage 1‐4 (NASH F1‐F4). One hundred twenty‐five genes were significantly increasing or decreasing as NAFLD pathology progressed. Compared with NLH, NASH F0 was characterized by increased inflammatory gene expression, such as gamma‐interferon‐inducible lysosomal thiol reductase (IFI30) and chemokine (C‐X‐C motif) ligand 9 (CXCL9), while complement and coagulation related genes, such as C9 and complement component 4 binding protein beta (C4BPB), were reduced. In the presence of NASH F1‐F4, extracellular matrix degrading proteinases and profibrotic/scar deposition genes, such as collagens and transforming growth factor beta 1 (TGFB1), were simultaneously increased, suggesting a dynamic state of tissue remodeling. Conclusion: In adults with obesity, distinct states of NAFLD are associated with intrahepatic perturbations in genes related to inflammation, complement and coagulation pathways, and tissue remodeling. These data provide insights into the dynamic pathogenesis of NAFLD in high‐risk individuals.

Correlation between the Polymorphism of Coagulation-Related Genes and Lower Extremity Deep Venous Thrombosis.

Objective To investigate the correlation between the polymorphism of 4 coagulation-related genes, rs1799963 （coagulation factor V gene Leiden）, rs6025 （prothrombin gene G20210A）, rs1042579 （thrombomodulin protein gene c.1418C>T） and rs1801131 （methylenetetrahydroflate reductase gene） and lower extremity deep venous thrombosis （LEDVT）. Methods The 4 genotypes mentioned above of 150 LEDVT patients and 153 healthy controls were detected by the kompetitive allele specific polymerase chain reaction （KASP）, then related blood biochemical indicators were collected, binary Logistic regression was established to screen the independent risk factors of LEDVT, and the correlation between polymorphism of 4 coagulation-related genes and LEDVT and its indicators under different genetic modes after adjusting confounding factors were analyzed. Results Five variables, D-dimer, fibrinogen degradation product, homocysteine, sex and age might be the risk factors of LEDVT. These variables were put into 4 genetic inheritance models, and adjusted in binary Logistic regression. The results suggested that the mutations of rs1042579 were correlated with LEDVT under dominant inheritance mode. Conclusion The gene polymorphism of rs1799963, rs6025 and rs1801131 has no significant correlation with the formation of LEDVT. The gene polymorphism of rs1042579 plays a role under dominant inheritance mode, and might be an independent risk factor for formation of LEDVT.

Regulatory changes in China on xenotransplantation and related products.

Given the persistence and the worldwide shortage of organs from both the deceased and living donors for clinical transplantation, pig organs or tissues hold immense promises for the patients on the waiting list, and xenotransplantation is deemed as one of the solutions to the organ shortage crisis. Indeed, the emerging gene editing technologies, such as CRISPR/Cas9, have led to tremendous progress in the generation of genetically modified pigs to overcome many barriers associated. We presented a description of the xenotransplantation regulations in China and the related products. Several groups in China have successfully generated transgenic pigs with the elimination of immune rejection or coagulation-related genes, and both pre-clinical and clinical studies have been reported. However, the pre-clinical evaluation and clinical application of porcine xenotransplantation raises ethical and regulatory considerations. Herein, in this review, we will summarize and discuss the progress in xenotransplantation in China and xenotransplantation-related products from the regulatory perspective. There has been remarkable progress in both the genetically modified pigs and pre-clinical studies in China, and China will be the first country to successfully fulfill the xenotransplantation from pig organ to human in the near future.

Molecular genetic diagnosis by next-generation sequencing in a cohort of Mexican patients with haemophilia and report of novel variants

IntroductionMolecular analysis in haemophilia is currently used in the diagnosis, treatment and prognosis of this disease. Hispanic populations in Latin America have been of interest to researchers due to the reportedly high prevalence of inhibitors in these patients. AimTo perform next-generation sequencing (NGS) in a cohort of Mexican patients with HA and HB and correlate with clinical phenotypes. MethodsPatients with Haemophilia A (HA) or haemophilia B (HB), were evaluated using NGS with an Ion AmpliSeq Custom Panel. Odds ratios (ORs) for associations between F8 variants and inhibitors were obtained. ResultsA total of 85 patients (60 with HA and 25 with HB) were included. Pathogenic variants in F8 were found in 93.3% of HA patients and in F9 in 96% of HB patients. Twelve novel potentially pathogenic variants were found. Inhibitors were observed in 20% of patients with severe HA. Four patients clinically diagnosed with HA were negative for F8 variants. ConclusionOverall detection rate of pathogenic variants in F8 and F9 genes was 94.6%. We identified 12 non previously reported variants and pathogenic variants in other coagulation related genes. Molecular diagnosis of HA and HB permits better options for management, assessment and genetic counseling.

Effects of Actr2 gene Variants on Mouse Viability, Gene Expression, and Cytoskeletal Dynamics

Venous thromboembolism (VTE) is a prevalent human disease that exhibits significant heritability. Factor V Leiden (F5L) is the most common known VTE risk factor, but modifier genes significantly influence VTE development. We recently identified a p.R258G missense mutation in the Actr2 gene (Actr2+/G, ARP2 protein) as a genetic suppressor of lethal thrombosis in mice homozygous for F5L (F5L/L) and hemizygous for tissue factor pathway inhibitor (Tfpi+/-). However, the antithrombotic mechanism of Actr2+/G is unknown. We used genetic and genomic techniques to investigate the phenotypic effects of the Actr2G variant on cells and mice. We previously analyzed progeny from Actr2+/G x Actr2+/G (N7 backcross generations to C57BL6/J) to investigate the effects of Actr2G on mouse survival. The progeny deviated from Mendelian frequencies, as only ~12% were Actr2G/G mice (N=303; p<9x10-7) and these mice have a significantly reduced lifespan of ~six months (N=131; p<0.0001). The Actr2+/G mice, when compared to wildtype mice, were morphologically indistinguishable and were born at the anticipated frequency. In the context of F5L, a F5L/LActr2+/G x F5L/LActr2+/G cross yielded only 3 F5L/LActr2G/G, which also significantly departed from expected frequencies (N=66; p<0.01). The F5L/LActr2+/G mice were born at the anticipated frequency. To determine if the mouse embryos were produced at normal frequencies, we examined pups at embryonic day 14.5 of this F5L/LActr2+/G x F5L/LActr2+/G cross. We observed normal frequencies at embryonic day 14.5, with 5 F5L/LActr2G/G embryos out of 18 total. Taken together, our results suggest that Actr2G exhibits gene dosage lethality, with Actr2G/G homozygosity detrimental for late stage embryonic development/early adulthood. We next studied the mechanism(s) of Actr2G thrombosis suppression by gene expression studies. RT-qPCR for multiple coagulation genes was performed in liver samples from Actr2+/+ and Actr2+/G mice. Serpine2 mRNA (Protease Nexin-1 (PN-1)) was significantly increased in Actr2+/G mice (N=3; p<0.0001). We also observed an increase in plasma PN-1 by quantitative Western Blot analysis. To investigate the Actr2G regulation of PN-1 expression, mouse N2a cells containing Actr2+/G or V5-tagged PN-1 were generated using CRISPR/Cas9. Genomic analysis revealed homology directed repair (HDR)-mediated knock-in frequencies of 24.3% (9/37) for Actr2+/G and 5.6% (1/18) for PN-1-V5. We then attempted to generate PN-1-V5 cells carrying mutant and/or Myc-tagged ARP2 (GMyc2). For the GMyc2 lines, the HDR efficiency for double knock-in was 15.6% (7/45). Analysis of all of our cell lines revealed however, that indels were also present within both the endogenous Actr2 and Serpine2 loci. Subsequent analysis of our GMyc2 cells revealed 5 Actr2 alleles. This suggests that cells avoid the loss of Actr2 function and highlights the difficulty with creating an Actr2 mutant cell model. To further investigate the effects of the Actr2G mutation, we isolated mouse embryonic fibroblasts (MEFs) from Actr2+/+ and Actr2G/G mice. The Actr2G/G MEFs grew poorly in culture, and displayed a significant reduction in cell surface area 20 minutes after plating on fibronectin when compared to Actr2+/+ (q=0.0003). Further analysis revealed F-actin aggregation in the root of cellular protrusions of Actr2G/G MEFs. These results indicate cytoskeletal remodeling defects in Actr2G/G cells. In summary, our studies indicate that mutations affecting ARP2 function can have profound effects on mouse and cell survival, as well as pronounced effects on specific physiological processes like blood coagulation and the transcriptional regulation of coagulation related genes. Disclosures No relevant conflicts of interest to declare.

Oncogenes and Clotting Factors: The Emerging Role of Tumor Cell Genome and Epigenome in Cancer-Associated Thrombosis.

There are emerging linkages between biological and genetic aspects of cancer progression and the mechanisms of cancer-associated thrombosis. It is argued that reciprocal influences between cancer cells, their associated vascular stroma, and the hemostatic system may shape the mechanism of coagulopathy. In this regard, glioblastoma multiforme offers a paradigm where the prevalent occurrence of local microthrombosis and peripheral venous thromboembolism can be linked to the profiles of oncogenic driver mutations and their impact on the expression of coagulation-related genes (coagulome). These relationships can be recapitulated in cellular models of glioblastoma, where the expression of tissue factor, podoplanin, and the release of procoagulant microparticles (extracellular vesicles) remains under the control of oncogenic pathways (epidermal growth factor receptor variant III, isocitrate dehydrogenase 1). These pathways define molecular subtypes of glioblastoma that express differential coagulomes. Moreover, single-cell sequencing of glioblastoma samples reveals a combinatorial rather than common profile of both subtype markers and coagulation-related genes. Based on these emerging observations, the authors suggest that cancers may operate as coagulant composites, where individual cells and their dominant populations express different procoagulant phenotypes, resulting in the net impact on the hemostatic system. They suggest that relating these mechanisms to clinical presentations of thrombosis may facilitate a more causality-based, personalized, and possibly cancer-specific thromboprophylaxis and treatment.

Screening for inherited thrombophilia and genome sequencing

To screening coagulation and anticoagulation genes and mutations in Chinese patients with venous thromboembolism suspected of inhered thrombophilia. Patients with venous thromboembolism caused by inherited thrombophilia were collected from the second Affiliated Hospital, Zhejiang University School of Medicine during June 2015 and December 2017. Gene sequencing was conducted to screen the anticoagulant-related genes PROC, PROS1, SERPINC1 and coagulation-related genes F5, F2, F8, CBS, MTHFR. The thousands of human genome database, ESP6500 database, Genoma database, HGMD mutation database were used to compare the mutation sites, and the pathogenic mutation sites were predicted according to SIFT, Polyphen, MutationTaster and CADD databases. Among 25 patients screened, eight patients with PROC mutations, two patients with PROS1 mutations, three patients with SERPINC1 mutations; one patient with F5 mutation, one patient with F2 mutation, one patient with F8 mutation, two patient with CBS mutations, and one patient with MTHFR mutation. The second generation gene sequencing helps to diagnose inherited thrombophilia and guide the decision of anticoagulation duration.

Characterization of hemostasis in mice lacking the novel thrombosis susceptibility gene Slc44a2

IntroductionRecent genome wide association studies (GWAS) identified a novel susceptibility locus for thrombosis, harbouring the SLC44A2 gene which encodes the Solute Carrier Family 44 Member 2 protein (SLC44A2). Thus far, SLC44A2 has not been studied in the context of thrombosis, and may be a unique contributor to thrombotic disease. Here we utilize mice lacking SLC44A2 (Slc44a2−/−) to evaluate a possible role of SLC44A2 in hemostasis. MethodsSlc44a2−/− mice were evaluated in key aspects of normal hemostasis including a challenge of vascular damage by applying laser induced injury to the cremaster muscle arteriole. ResultsSlc44a2−/− mice had comparable levels of thrombin generation and gene expression of coagulation related genes, as compared to littermate wild type controls. Lower levels of circulating plasma Von Willebrand factor (VWF) were measured in Slc44a2−/− mice, while no difference in VWF multimerization or vascular localization was detected. Upon in vivo laser injury of the cremaster arterioles, we detected an impairment of clot formation for Slc44a2−/− mice. ConclusionsAlthough mice lacking SLC44A2 are normal for several hemostasis parameters, we do observe a reduction of plasma VWF levels and an altered response upon vascular damage, which suggests that SLC44A2 contributes to hemostasis upon injury. These findings are in line with the reported GWAS data and support further research on SLC44A2 in thrombosis.

Histological and transcriptomic responses of two immune organs, the spleen and head kidney, in Nile tilapia (Oreochromis niloticus) to long-term hypersaline stress

Hyperosmotic stress can adversely affect fish immunity, but little is known about the histological and transcriptomic responses of immune organs in fish in a hyperosmotic environment. This study evaluated the effects of long-term hypersaline conditions (16‰) on the growth, histology and transcriptomics of the two main immune organs, the spleen and head kidney, in Nile tilapia Oreochromis niloticus relative to those reared in freshwater for eight weeks. No differences in weight gain and specific growth rate were found between fish reared under these two salinities. Hyperosmotic stress induced a congestive or enlarged spleen. Platelet- and coagulation-related gene expression was significantly decreased in tilapia at 16‰. The red cell distribution width and value of the mean corpuscular hemoglobin were significantly greater in fish at 16‰ salinity than in control fish in freshwater. A large volume of melano-macrophages in the spleen and pigment deposition in both the spleen and head kidney were observed in the histological sections in fish at 16‰ salinity. Transmission electron microscopic results showed abnormal macrophages with deposition granules in the spleen and head kidney and more neutrophils in the head kidney of fish at 16‰ than in control fish. In total, 772 and 502 genes were annotated for significantly different expression in the spleen and head kidney, respectively, and corresponded to five and one significantly changed immune system pathways, respectively. The complement pathway in the spleen was significantly down-regulated at 16‰. This study indicates that long-term exposure of Nile tilapia to a hyperosmotic environment can induce splenomegaly, reduce coagulation function, enhance phagocytic activity and down-regulate the complement pathway in the spleen. The spleen is a more sensitive organ for immune responses to chronic ambient salinity stress than the head kidney in Nile tilapia.

Transcript Profiling of Hevea brasiliensis during Latex Flow.

Latex exploitation enhances latex regeneration in rubber trees. The latex exploitation-caused latex flow lasts from 10 min to a few hours, which is convenient for exploring the transcript profiling of latex metabolism-related genes at the different stages of latex flow. In the present study, the expression pattern of 62 latex metabolism-related genes involved in water transportation, carbohydrate metabolism, natural rubber biosynthesis, hormone signaling, ROS generation and scavenging, and latex coagulum across three stages of latex flow between rubber tree clones CATAS7-33-97 and CATAS8-79 were comparatively analyzed by quantitative real-time PCR. The two clones show differences in latex regeneration and have a different duration of latex flow. The results showed that the expression levels of 38 genes were significantly higher in CATAS8-79 latex than in CATAS7-33-97 during latex regeneration, while 45 genes had a notably higher expression level in CATAS8-79 latex during latex flow. Together with the activation of the MEP pathway and jasmonate pathway in CATAS8-79 latex, HbPIP1;3, HbPIP1;4, HbSUT3, HbSus3, HbHMGS1-2, HbMK should contribute to the high latex regeneration ability. The up-regulation of ethylene signaling and Hb44KD and the down-regulation of latex coagulation-related genes in CATAS8-79 latex might contribute to its longer latex flow duration. This study provides some cues for revealing the regulation of latex metabolism in rubber trees.

Associations between polymorphisms in coagulation-related genes and venous thromboembolism: A meta-analysis with trial sequential analysis.

Recently, several studies showed that the polymorphisms in the coagulation-related genes might be associated with venous thromboembolism (VTE); however, the results were still controversial. We performed a meta-analysis with trial sequential analysis to investigate the associations between the endothelial cell-activated protein C receptor (EPCR) rs9574, F11 rs2289252, F11 rs2036914, FGG rs2066865, FGG rs1049636, CYP4V2 rs13146272, SERPINC1 rs2227589, and GP6 rs1613662 polymorphisms with the risk of VTE. We searched both the common English-language databases and the Chinese literature databases. Two authors selected studies according to inclusion and exclusion criteria. Crude odds ratios with 95% confidence intervals (CI) were calculated to estimate the strength of this association. Between-study heterogeneity was assessed with the chi-square-based Q test and the I statistic. Overall, a total of 20 studies were included. The meta-analysis revealed that the F11 rs2289252, F11 rs2036914, FGG rs2066865, and CYP4V2 rs13146272 polymorphisms were closely related to the development of VTE in the white race under the best genetic models after multiple testing adjustments. The EPCR rs9574, FGG rs1049636, SERPINC1 rs2227589, and GP6 rs1613662 polymorphisms might be potential candidates in the pathogenesis of VTE, but trial sequential analyses and sensitivity analyses indicated that the evidences were limited. Larger scale studies were demanded to avoid false-positive outcomes. Finally, our study demonstrated the important role of rs2289252, rs2036914, rs2066865, and rs13146272 polymorphisms in the development of VTE in the white race. Rs9574, rs1049636, rs2227589 and rs1613662 polymorphisms might be risk factors of VTE. However, more studies involving diverse races are needed to probe the ethnic difference and the underlying mechanisms of significant associations.

Determinants of acquired activated protein C resistance and D-dimer in breast cancer

BackgroundWe have previously reported acquired activated protein C (APC) resistance and elevated plasma D-dimer levels in breast cancer patients. Here, we aimed to identify phenotypic and genetic determinants that contribute to the acquired APC resistance and increased D-dimer levels in breast cancer. Healthy controls served as reference. We also addressed whether higher APC resistance or D-dimer levels could be potential markers of clinicopathological breast cancer characteristics. Materials and methods358 breast cancer patients and 273 healthy controls were enrolled and hemostatic plasma parameters were determined; factor (F) V, FVIII, FIX, FX, fibrinogen, von Willebrand factor (VWF), normalized APC sensitivity ratio (n-APC-sr), protein C, protein S, antithrombin, tissue factor pathway inhibitor (TFPI), and D-dimer. Common single nucleotide polymorphisms were genotyped in coagulation-related genes in the breast cancer patients. ResultsThe phenotypic hemostatic factors explained 25% and 31% of the variability in acquired APC resistance and D-dimer levels, respectively, in the breast cancer patients. Fibrinogen (β=−0.35, P<0.001), protein C (β=0.28, P<0.001), and FIX (β=0.22, P=0.026) were identified as determinants of n-APC-sr (in FV Leiden non-carriers), whereas TFPI (β=0.28, P<0.001), antithrombin (β=−0.25, P<0.001), and FX (β=0.15, P=0.040) were the major determinants of D-dimer. Moreover, borderline higher APC resistance (>75th percentile) was found in patients with triple negative tumors (odds ratio (OR) 1.97, 95% CI 0.99–3.90). ConclusionsThis study reports phenotypic hemostatic parameters that determine acquired APC resistance and D-dimer levels in breast cancer patients. The explanatory power was modest, however, our findings are hypothesis generating and may contribute to further understand the background for cancer associated-coagulopathy and thrombosis.