Biology of factor XI.

ABSTRACT: Unique among coagulation factors, the coagulation factor XI (FXI) arose through a duplication of the gene KLKB1, which encodes plasma prekallikrein. This evolutionary origin sets FXI apart structurally because it is a homodimer with 2 identical subunits composed of 4 apple and 1 catalytic domain. Each domain exhibits unique affinities for binding partners within the coagulation cascade, regulating the conversion of FXI to a serine protease as well as the selectivity of substrates cleaved by the active form of FXI. Beyond serving as the molecular nexus for the extrinsic and contact pathways to propagate thrombin generation by way of activating FIX, the function of FXI extends to contribute to barrier function, platelet activation, inflammation, and the immune response. Herein, we critically review the current understanding of the molecular biology of FXI, touching on some functional consequences at the cell, tissue, and organ level. We conclude each section by highlighting the DNA mutations within each domain that present as FXI deficiency. Together, a narrative review of the structure-function of the domains of FXI is imperative to understand the etiology of hemophilia C as well as to identify regions of FXI to safely inhibit the pathological function of activation or activity of FXI without compromising the physiologic role of FXI.

Microfluidic devices for studying coagulation biology.

The ability to study the behavior of cells, proteins, and cell-cell or cell-protein interactions under dynamic forces such as shear stress under fluid flow, provides a more accurate understanding of the physiopathology of hemostasis. This review touches upon the traditional methods for studying blood coagulation and platelet aggregation and provides an overview on cellular and protein response to shear stress. We also elaborate on the biological aspects of how cells recognize mechanical forces and convert them into biochemical signals that can drive various signaling pathways. We give a detailed description of the various types of microfluidic devices that are employed to study the complex processes of platelet aggregation and blood coagulation under flow conditions as well as to investigate endothelial shear-response. We also highlight works mimicking artificial vessels as platforms to study the mechanisms of coagulation, and finish our review by describing anticipated clinical uses of microfluidics devices and their standardization.

The effects of coagulation factors on the risk of endometriosis: a Mendelian randomization study.

BACKGROUND: Endometriosis is recognized as a complex gynecological disorder that can cause severe pain and infertility, affecting 6-10% of all reproductive-aged women. Endometriosis is a condition in which endometrial tissue, which normally lines the inside of the uterus, deposits in other tissues. The etiology and pathogenesis of endometriosis remain ambiguous. Despite debates, it is generally agreed that endometriosis is a chronic inflammatory disease, and patients with endometriosis appear to be in a hypercoagulable state. The coagulation system plays important roles in hemostasis and inflammatory responses. Therefore, the purpose of this study is to use publicly available GWAS summary statistics to examine the causal relationship between coagulation factors and the risk of endometriosis. METHODS: To investigate the causal relationship between coagulation factors and the risk of endometriosis, a two-sample Mendelian randomization (MR) analytic framework was used. A series of quality control procedures were followed in order to select eligible instrumental variables that were strongly associated with the exposures (vWF, ADAMTS13, aPTT, FVIII, FXI, FVII, FX, ETP, PAI-1, protein C, and plasmin). Two independent cohorts of European ancestry with endometriosis GWAS summary statistics were used: UK Biobank (4354 cases and 217,500 controls) and FinnGen (8288 cases and 68,969 controls). We conducted MR analyses separately in the UK Biobank and FinnGen, followed by a meta-analysis. The Cochran's Q test, MR-Egger intercept test, and leave-one-out sensitivity analyses were used to assess the heterogeneities, horizontal pleiotropy, and stabilities of SNPs in endometriosis. RESULTS: Our two-sample MR analysis of 11 coagulation factors in the UK Biobank suggested a reliable causal effect of genetically predicted plasma ADAMTS13 level on decreased endometriosis risk. A negative causal effect of ADAMTS13 and a positive causal effect of vWF on endometriosis were observed in the FinnGen. In the meta-analysis, the causal associations remained significant with a strong effect size. The MR analyses also identified potential causal effects of ADAMTS13 and vWF on different sub-phenotypes of endometrioses. CONCLUSIONS: Our MR analysis based on GWAS data from large-scale population studies demonstrated the causal associations between ADAMTS13/vWF and the risk of endometriosis. These findings suggest that these coagulation factors are involved in the development of endometriosis and may represent potential therapeutic targets for the management of this complex disease.

CHAPLE syndrome uncovers the primary role of complement in a familial form of Waldmann's disease.

Primary intestinal lymphangiectasia (PIL) or Waldmann's disease was described in 1961 as an important cause of protein-losing enteropathy (PLE). PIL can be the sole finding in rare individuals or occur as part of a multisystemic genetic syndrome. Although genetic etiologies of many lymphatic dysplasia syndromes associated with PIL have been identified, the pathogenesis of isolated PIL (with no associated syndromic features) remains unknown. Familial cases and occurrence at birth suggest genetic etiologies in certain cases. Recently, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (the CHAPLE syndrome) has been identified as a monogenic form of PIL. Surprisingly, loss of CD55, a key regulator of complement system leads to a predominantly gut condition. Similarly to other complement disorders, namely paroxysmal nocturnal and hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), CHAPLE disease involves pathogenic cross-activation of the coagulation system, predisposing individuals to severe thrombosis. The observation that complement system is overly active in CHAPLE disease introduced a novel concept into the management of PLE; anti-complement therapy. While CD55 deficiency constitutes a treatable subgroup in the larger pool of patients with isolated PIL, the etiology remains to be identified in the remaining patients with intact CD55.

Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Molecular basis of anticoagulant and anticomplement activity of the tick salivary protein Salp14 and its homologs.

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodesscapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding of mannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag time of thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin-associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here using NMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain-like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.

Domain Evolution of Vertebrate Blood Coagulation Cascade Proteins.

Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus.

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

A pan-cancer analysis of the human tumor coagulome and its link to the tumor immune microenvironment.

OBJECTIVE: Solid tumors often establish a procoagulable state that can lead to venous thromboembolism (VTE). Although some of the key genes involved in this process are known, no previous study has compared the "coagulome", i.e., the expression of coagulation/fibrinolysis genes, across different primary tumor types. It is also unclear whether the coagulome is associated with specific characteristics of the tumor microenvironment (TME). We aimed to address this question. METHODS: We analyzed the expression of the genes F3, PLAU, PLAT, PLAUR, SERPINB2, and SERPINE1 in 32 cancer types using data from The Cancer Genome Atlas (TCGA) and other freely available resources. RESULTS: We identified specific expression patterns of procoagulant and fibrinolytic genes. The expression of the Tissue Factor (F3) was found to be tumor type dependent, with the highest expression in glioblastoma (GBM), a highly procoagulable tumor type. Conversely, high expression of the fibrinolysis gene cluster PLAU, PLAUR, SERPINE1 was consistently linked to the characteristics of the TME (monocytic infiltration) and high expression of important checkpoints of the immune response, such as PD-L2 and CD276/B7-H3. CONCLUSION: These tumor-specific patterns of expression might partially explain the differences in VTE risk among tumor types. We propose that biomarkers of coagulation fibrinolysis might provide valuable information about the TME in cancer patients.

Molecular pathways in patients with systemic lupus erythematosus revealed by gene-centred DNA sequencing.

OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE. METHODS: We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS). RESULTS: We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes. CONCLUSIONS: Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients.

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

Significance of coagulation and fibrinolysis markers for benign and malignant soft tissue tumors.

BACKGROUND: The intimate relationship between coagulation and fibrinolysis in malignant tumors is a well-known phenomena, with the malignant phenotype enhancing coagulation and fibrinolysis. We hypothesized that soft tissue sarcoma (STS) affects the expression of coagulation and fibrinolysis markers, which could be used to distinguish STS from benign soft tissue tumors. We analyzed the correlations between plasma levels of D-dimer (DD), plasmin-α2 plasmin inhibitor complex (PIC), soluble fibrin (SF), and thrombin-antithrombin III complex (TAT) in benign soft tissue tumors and STS to elucidate whether these markers can be used to predict STS. METHODS: Plasma DD, PIC, SF and TAT levels in primary soft tissue tumors (benign 67, STS 68) were measured before biopsy or treatment. The marker levels were analyzed and compared to various clinicopathological parameters. RESULTS: In malignancy (STS), the average DD, PIC and SF levels were significantly higher than in benign tumors. Multivariate logistic analysis of continuous variables indicated that only PIC exhibited a significant difference (OR: 24.5, 95%CI: 3.55-170, p = 0.0012). Receiver operating characteristic curve analysis produced area under the curve values for DD: 0.691, PIC: 0.784, SF: 0.734 and TAT: 0.588. Youden's index was used to establish thresholds of 0.37 (DD), 0.80 (PIC), 0.90 (SF) and 0.82 (TAT). Threshold values for PIC and SF indicated high specificity (0.881, 0.791) and high positive predictive value (0.818, 0.745), respectively. The highest accuracy value among the markers was observed for PIC (0.704). Significant differences in multivariate analysis of binary variables were demonstrated by categorizing low and high groups based on their threshold, PIC (≥0.80) (OR: 3.36, 95%CI: 1.19-9.43, p = 0.0212) and SF (≥0.90) (OR: 2.63, 95%CI: 1.04-6.66, p = 0.0404) . CONCLUSIONS: Of the coagulation and fibrinolysis markers studied, increased PIC levels were related to STS and over 0.80 PIC was the most suitable for the prediction of STS, which, along with other diagnostic tools, represents a helpful subsidiary tool for the prediction of STS.

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

BACKGROUND: Although 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 METHODS: We 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. RESULTS: In 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. CONCLUSIONS: CCC 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.

Identification of a Plasma MicroRNA Profile Associated With Venous Thrombosis.

OBJECTIVE: Venous thrombosis (VT) is a complex condition with a highly heritable genetic component that predisposes one to its development. Certain microRNAs (miRNAs) might be used as biomarkers of VT, but few studies have examined miRNA expression in this respect. The aim of the present work was to identify a plasma miRNA profile associated with VT. Approach and Results: miRNAs were analyzed by quantitative polymerase chain reaction in plasma samples from members of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) population (n=935). A discovery phase involving the screening of 752 miRNAs from a subset of 104 GAIT-2 subjects was followed by an internal validation phase in which the selected miRNAs were quantified in the whole GAIT-2 population. In the discovery phase, 16 miRNAs were selected, including 9 associated with VT and 7 that correlated with an intermediate phenotype of VT. In the next phase, 4 miRNAs were validated as differentially expressed (false discovery rate, <0.1) in VT: hsa-miR-126-3p, hsa-miR-885-5p, hsa-miR-194-5p, and hsa-miR-192-5p. The 4 miRNAs each returned a significant (P<0.05) odds ratio for VT (range of 1.3-1.8). A risk model including the 4 miRNAs, age, and sex returned an area under the receiver operating characteristic curve of 0.77. Moreover, all 4 miRNAs showed significant correlations with intermediate phenotypes of VT (eg, protein S and factor VII). The targets of the miRNAs in the blood coagulation pathway and their interactions are also discussed. CONCLUSIONS: The present results suggest a 4-miRNA plasma profile associated with VT is of potential use in predicting the risk of this condition.

The role of haematological traits in risk of ischaemic stroke and its subtypes.

Thrombosis and platelet activation play a central role in stroke pathogenesis, and antiplatelet and anticoagulant therapies are central to stroke prevention. However, whether haematological traits contribute equally to all ischaemic stroke subtypes is uncertain. Furthermore, identification of associations with new traits may offer novel treatment opportunities. The aim of this research was to ascertain causal relationships between a wide range of haematological traits and ischaemic stroke and its subtypes. We obtained summary statistics from 27 published genome-wide association studies of haematological traits involving over 375 000 individuals, and genetic associations with stroke from the MEGASTROKE Consortium (n = 67 000 stroke cases). Using two-sample Mendelian randomization we analysed the association of genetically elevated levels of 36 blood cell traits (platelets, mature/immature red cells, and myeloid/lymphoid/compound white cells) and 49 haemostasis traits (including clotting cascade factors and markers of platelet function) with risk of developing ischaemic (AIS), cardioembolic (CES), large artery (LAS), and small vessel stroke (SVS). Several factors on the intrinsic clotting pathway were significantly associated (P < 3.85 × 10-4) with CES and LAS, but not with SVS (e.g. reduced factor VIII activity with AIS/CES/LAS; raised factor VIII antigen with AIS/CES; and increased factor XI activity with AIS/CES). On the common pathway, increased gamma (γ') fibrinogen was significantly associated with AIS/CES. Furthermore, elevated plateletcrit was significantly associated with AIS/CES, eosinophil percentage of white cells with LAS, and thrombin-activatable fibrinolysis inhibitor activation peptide antigen with AIS. We also conducted a follow-up analysis in UK Biobank, which showed that amongst individuals with atrial fibrillation, those with genetically lower levels of factor XI are at reduced risk of AIS compared to those with normal levels of factor XI. These results implicate components of the intrinsic and common pathways of the clotting cascade, as well as several other haematological traits, in the pathogenesis of CES and possibly LAS, but not SVS. The lack of associations with SVS suggests thrombosis may be less important for this stroke subtype. Plateletcrit and factor XI are potentially tractable new targets for secondary prevention of ischaemic stroke, while factor VIII and γ' fibrinogen require further population-based studies to ascertain their possible aetiological roles.

Age-related gene expression and DNA methylation changes in rhesus macaque.

Aging is a very complicated biological process that can change gene expressions. The Chinese rhesus macaque (Macaca mulatta lasiota; CR) is closely related to humans. We explored gene expression with increasing age and DNA methylation changes in young and old CRs. Results showed blood transcriptome and DNA methylome significantly changed from young to old CRs. The age-associated differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were associated with age-related biological features, such as immunity, blood coagulation, and biosynthetic process. The measurements of coagulation indicators confirmed old CRs had shorter coagulation time than young CRs, and the activities of coagulation factor II (FII) and factor VIII (FVIII) were enhanced in old CRs. Humans and CRs exhibited the same enhanced blood coagulation with age phenotype. Our study found aging is a critical factor affecting gene expression in CRs, and also provided new insights into the blood coagulation changes in non-human primates.

Extension of the Human Fibrinogen Database with Detailed Clinical Information-The αC-Connector Segment.

Fibrinogen, an abundant plasma glycoprotein, is involved in the final stage of blood coagulation. Decreased fibrinogen levels, which may be caused by mutations, are manifested mainly in bleeding and thrombotic disorders. Clinically relevant mutations of fibrinogen are listed in the Human Fibrinogen Database. For the αC-connector (amino acids Aα240-410, nascent chain numbering), we have extended this database, with detailed descriptions of the clinical manifestations among members of reported families. This includes the specification of bleeding and thrombotic events and results of coagulation assays. Where available, the impact of a mutation on clotting and fibrinolysis is reported. The collected data show that the Human Fibrinogen Database reports considerably fewer missense and synonymous mutations than the general COSMIC and dbSNP databases. Homozygous nonsense or frameshift mutations in the αC-connector are responsible for most clinically relevant symptoms, while heterozygous mutations are often asymptomatic. Symptomatic subjects suffer from bleeding and, less frequently, from thrombotic events. Miscarriages within the first trimester and prolonged wound healing were reported in a few subjects. All mutations inducing thrombotic phenotypes are located at the identical positions within the consensus sequence of the tandem repeats.

Confocal Blood Flow Videomicroscopy of Thrombus Formation over Human Arteries and Local Targeting of P2X7.

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.