The Prothrombin-Prothrombinase Interaction.

The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with exposure of tissue factor from the damaged endothelium and culminates with conversion of prothrombin to thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca2+, and phospholipids. This cofactor-dependent activation is paradigmatic of analogous reactions of the blood coagulation and complement cascades, which makes elucidation of its molecular mechanism of broad significance to the large class of trypsin-like zymogens to which prothrombin belongs. Because of its relevance as the most important reaction in the physiological response to vascular injury, as well as the main trigger of pathological thrombotic complications, the mechanism of prothrombin activation has been studied extensively. However, a molecular interpretation of this mechanism has become available only recently from important developments in structural biology. Here we review current knowledge on the prothrombin-prothrombinase interaction and outline future directions for the study of this key reaction of the coagulation cascade.

Effects of dabigatran, rivaroxaban, and apixaban on fibrin network permeability, thrombin generation, and fibrinolysis.

INTRODUCTION: There are important pharmacological differences between direct oral anticoagulants (DOAC) and a deeper knowledge of how they influence different aspects of hemostasis in patients on treatment is desirable. MATERIALS AND METHODS: Blood samples from patients on dabigatran (n = 23), rivaroxaban (n = 26), or apixaban (n = 20) were analyzed with a fibrin network permeability assay, a turbidimetric clotting and lysis assay, the calibrated automated thrombogram (CAT), plasma levels of thrombin-antithrombin complex (TAT) and D-dimer, as well as DOAC concentrations, PT-INR and aPTT. As a comparison, we also analyzed samples from 27 patients on treatment with warfarin. RESULTS: Patients on dabigatran had a more permeable fibrin network, longer lag time (CAT and turbidimetric assay), and lower levels of D-dimer in plasma, compared with patients on rivaroxaban- and apixaban treatment, and a more permeable fibrin network than patients on warfarin. Clot lysis time was slightly longer in patients on dabigatran than in patients on rivaroxaban. Warfarin patients formed a more permeable fibrin network than patients on apixaban, had longer lag time than patients on rivaroxaban (CAT assay), and lower peak thrombin and ETP compared to patients on treatment with both FXa-inhibitors. CONCLUSIONS: Results from this study indicate dabigatran treatment is a more potent anticoagulant than apixaban and rivaroxaban. However, as these results are not supported by clinical data, they are probably more related to the assays used and highlight the difficulty of measuring and comparing the effect of anticoagulants.

Thromboelastography in acute immunologic reactions: a prospective pilot study.

Background: Biomarkers of fibrinolysis are elevated during acute immunologic reactions (allergic reactions and angioedema), although it is unclear whether fibrinolysis is associated with disease severity. Objectives: We investigated a possible association between maximum lysis (ML) measured by thromboelastography and the severity of acute immunologic reactions. Methods: We recruited patients with acute immunologic reactions at a high-volume emergency department. Clinical disease severity at presentation and at the end of the emergency department stay was assessed using a 5-grade scale, ranging from local symptoms to cardiac arrest. We determined ML on admission by thromboelastography (ROTEM's extrinsic [EXTEM], and aprotinin [APTEM] tests), expressed as ML%. Hyperfibrinolysis was defined as an ML of >15% in EXTEM, which was reversed by adding aprotinin (APTEM). We used exact logistic regression to investigate an association between ML% and disease severity (grades 1 and 2 [mild] vs 3-5 [severe]) and between hyperfibrinolysis and disease severity. Results: We included 31 patients (71% female; median age, 52 [IQR, 35-58] years; 10 [32%] with a severe reaction). ML% was higher in patients with severe symptoms (21 [IQR, 12-100] vs 10 [IQR, 4-17]). Logistic regression found a significant association between ML% and symptom severity (odds ratio, 1.07; 95% CI, 1.01-1.21; P = .003). Hyperfibrinolysis was detected in 6 patients and found to be associated with severe symptoms (odds ratio, 17.59; 95% CI, 1.52-991.09; P = .02). D-dimer, tryptase, and immunoglobulin E concentrations increased with the severity of immunologic reactions. Conclusion: ML, quantified by thromboelastography, is associated with the severity of acute immunologic reactions.

Using microfluidic shear to assess transfusion requirements in trauma patients.

Background: Viscoelastic assays have widely been used for evaluating coagulopathies but lack the addition of shear stress important to in vivo clot formation. Stasys technology subjects whole blood to shear forces over factor-coated surfaces. Microclot formation is analyzed to determine clot area (CA) and platelet contractile forces (PCFs). We hypothesize the CA and PCF from this novel assay will provide information that correlates with trauma-induced coagulopathy and transfusion requirements. Methods: Blood samples were collected on adult trauma patients from a single-institution prospective cohort study of high-level activations. Patient and injury characteristics, transfusion data, and outcomes were collected. Thromboelastography, coagulation studies, and Stasys assays were run on paired samples collected at admission. Stasys CA and PCFs were quantified as area under the curve calculations and maximum values. Normal ranges for Stasys assays were determined using healthy donors. Data were compared using Kruskal-Wallis tests and simple linear regression. Results: From March 2021 to January 2023, 108 samples were obtained. Median age was 37.5 (IQR 27.5-52) years; patients were 77% male. 71% suffered blunt trauma, 26% had an Injury Severity Score of ≥25. An elevated international normalized ratio significantly correlated with decreased cumulative PCF (p=0.05), maximum PCF (p=0.05) and CA (p=0.02). Lower cumulative PCF significantly correlated with transfusion of any products at 6 and 24 hours (p=0.04 and p=0.05) as well as packed red blood cells (pRBCs) at 6 and 24 hours (p=0.04 and p=0.03). A decreased maximum PCF showed significant correlation with receiving any transfusion at 6 (p=0.04) and 24 hours (p=0.02) as well as transfusion of pRBCs, fresh frozen plasma, and platelets in the first 6 hours (p=0.03, p=0.03, p=0.03, respectively). Conclusions: Assessing coagulopathy in real time remains challenging in trauma patients. In this pilot study, we demonstrated that microfluidic approaches incorporating shear stress could predict transfusion requirements at time of admission as well as requirements in the first 24 hours. Level of evidence: Level II.

Mass spectrometry-based proteomic profiling of extracellular vesicle proteins in diabetic and non-diabetic ischemic stroke patients: a case-control study.

Acute ischemic stroke is the most common cause of neurologic dysfunction caused by focal brain ischemia and tissue injury. Diabetes is a major risk factor of stroke, exacerbating disease management and prognosis. Therefore, discovering new diagnostic markers and therapeutic targets is critical for stroke prevention and treatment. Extracellular vesicles (EVs), with their distinctive properties, have emerged as promising candidates for biomarker discovery and therapeutic application. This case-control study utilized mass spectrometry-based proteomics to compare EVs from non-diabetic stroke (nDS = 14), diabetic stroke (DS = 13), and healthy control (HC = 12) subjects. Among 1288 identified proteins, 387 were statistically compared. Statistical comparisons using a general linear model (log2 foldchange ≥0.58 and FDR-p≤0.05) were performed for nDS vs HC, DS vs HC, and DS vs nDS. DS vs HC and DS vs nDS comparisons produced 123 and 149 differentially expressed proteins, respectively. Fibrinogen gamma chain (FIBG), Fibrinogen beta chain (FIBB), Tetratricopeptide repeat protein 16 (TTC16), Proline rich 14-like (PR14L), Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKE), Biorientation of chromosomes in cell division protein 1-like 1 (BD1L1), and protein PR14L exhibited significant differences in the DS group. The pathway analysis revealed that the complement system pathways were activated, and blood coagulation and neuroprotection were inhibited in the DS group (z-score ≥2; p ≤ 0.05). These findings underscore the potential of EVs proteomics in identifying biomarkers for stroke management and prevention, warranting further clinical investigation.

Biochemical Behavior, Influence on Cell DNA Condition, and Microbiological Properties of Wool and Wool-Copper Materials.

The paper presents the study concerning the preparation and physio-chemical and biological properties of wool-copper (WO-Cu) materials obtained by the sputter deposition of copper onto the wool fibers. The WO-Cu material was subjected to physio-chemical and biological investigations. The physio-chemical investigations included the elemental analysis of materials (C, N, O, S, and Cu), their microscopic analysis, and surface properties analysis (specific surface area and total pore volume). The biological investigations consisted of the antimicrobial activity tests of the WO-Cu materials against colonies of Gram-positive (Staphylococcus aureus) bacteria, Gram-negative (Escherichia coli) bacteria, and fungal mold species (Chaetomium globosum). Biochemical-hematological tests included the evaluation of the activated partial thromboplastin time and pro-thrombin time. The tested wool-copper demonstrated the ability to interact with the DNA in a time-dependent manner. These interactions led to the DNA's breaking and degradation. The antimicrobial and antifungal activities of the WO-Cu materials suggest a potential application as an antibacterial/antifungal material. Wool-copper materials may be also used as customized materials where the blood coagulation process could be well controlled through the appropriate copper content.

Is anticoagulative therapy in systemic sclerosis to be reconsidered?

Systemic sclerosis is a rare disease with a high mortality rate. It is a multisystem connective tissue disease due to endothelial autoimmune activation along with tissue and vascular fibrosis, inducing vasculopathy, with an angiogenesis wasting. The endothelial damage provokes platelet activation and immune cell adhesion. The detachment of endothelial cells leads to the interaction of platelets and collagen present in the exposed subendothelial layer. This provokes the activation of several coagulative factors, inducing a pro-thrombotic condition by thrombin generation, which converts fibrinogen into fibrin. Moreover, thrombin has other functions, such as the induction of hyperplasia in smooth muscle cells and fibroblasts, thereby favouring fibrosis. An increased risk of venous thromboembolism has been found in systemic sclerosis, whereas pulmonary hypertension may be due to the obstruction of small pulmonary arteries. Pulmonary veno-occlusive disease may also occur. Warfarin showed inconsistent results, while the outcomes of a randomised, placebo-controlled clinical trial on apixaban versus placebo are still awaited. A new anticoagulation strategy based on anti-factor XI drugs is being developed, with the aim of achieving optimal anticoagulation along with a low risk of bleeding. The molecule types under investigation in this category include monoclonal antibodies, small molecules, natural inhibitors, antisense oligonucleotides, and aptamers. Patients with systemic sclerosis may be ideal candidates for clinical trials planned to analyse the efficacy and safety of these molecules.

Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain.

BACKGROUND: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. OBJECTIVES: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. METHODS: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy. RESULTS: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII-/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. CONCLUSION: These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.

Design of Apoptotic Cell-Inspired Particles as a Blood Coagulation Test.

The blood coagulation test is an indispensable test for monitoring the blood coagulation and fibrinolysis functions. Currently, activated partial thromboplastin time (APTT) is the most widely used approach to coagulation testing. However, APTT reagents need to be optimized due to the fact that they are unstable, highly variable, and cannot be easily controlled. In this study, we created apoptotic cell-inspired methacryloyloxyethyl phosphorylserine (MPS) particles for blood coagulation as an alternative to conventional APTT reagents. Particle size could be controlled by changing the concentration of the polymer. The blood coagulation ability of particles was stable at different environmental temperatures. Moreover, the procoagulant activity could be enhanced by increasing the concentration to 0.06 mg/mL and reducing the size of the particles to around 900 nm. Fibrin clotted by particles showed no significant difference from that formed by APTT regent Actin FSL. We propose that MPS particles are a potential alternative to Actin FS for the application of blood coagulation tests.

Modeling the distribution of enzymes on lipid vesicles: A novel framework for surface-mediated reactions in coagulation.

Blood coagulation is a network of biochemical reactions wherein dozens of proteins act collectively to initiate a rapid clotting response. Coagulation reactions are lipid-surface dependent, and this dependence is thought to help localize coagulation to the site of injury and enhance the association between reactants. Current mathematical models of coagulation either do not consider lipid as a variable or do not agree with experiments where lipid concentrations were varied. Since there is no analytic rate law that depends on lipid, only apparent rate constants can be derived from enzyme kinetic experiments. We developed a new mathematical framework for modeling enzymes reactions in the presence of lipid vesicles. Here the concentrations are such that only a fraction of the vesicles harbor bound enzymes and the rest remain empty. We call the lipid vesicles with and without enzyme TF:VIIa+ and TF:VIIa- lipid, respectively. Since substrate binds to both TF:VIIa+ and TF:VIIa- lipid, our model shows that excess empty lipid acts as a strong sink for substrate. We used our framework to derive an analytic rate equation and performed constrained optimization to estimate a single, global set of intrinsic rates for the enzyme-substrate pair. Results agree with experiments and reveal a critical lipid concentration where the conversion rate of the substrate is maximized, a phenomenon known as the template effect. Next, we included product inhibition of the enzyme and derived the corresponding rate equations, which enables kinetic studies of more complex reactions. Our combined experimental and mathematical study provides a general framework for uncovering the mechanisms by which lipid mediated reactions impact coagulation processes.

Three-dimensional morphological characterization of blood droplets during the dynamic coagulation process.

In this study, we employed a method integrating optical coherence tomography (OCT) with the U-Net and Visual Geometry Group (VGG)-Net frameworks within a convolutional neural network for quantitative characterization of the three dimensional whole blood during the dynamic coagulation process. VGG-Net architecture for the identification of blood droplets across three distinct coagulation stages including drop, gelation, and coagulation achieves an accuracy of up to 99%. In addition, the U-Net architecture demonstrated proficiency in effectively segmenting uncoagulated and coagulated portions of whole blood, as well as the background. Notably, parameters such as volume of uncoagulated and coagulated segments of the whole blood were successfully employed for the precise quantification of the coagulation process, which indicates well for the potential of future clinical diagnostics and analyses.

FXII contact activation is dependent on binding through two unique patches in its EGF-1 domain and can be prevented by (VHH) targeting.

BACKGROUND: Factor XII (FXII) triggers contact activation by binding to foreign surfaces, with the EGF-1 domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis. OBJECTIVES: To unravel and prevent EGF-1 mediated FXII surface-binding with VHH. METHODS: FXII variants with glutamine substitutions of two positively-charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain specific VHHs were raised to inhibit EGF-1 mediated FXII contact activation while preserving quiescence. RESULTS: Two unique, positively-charged patches in the EGF1 domain were identified (upstream: 73K74K76K78H81K82H; downstream: 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in auto-activation of 94% and prolongation of clot formation in aPTT assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1 specific VHHs were developed, that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V HH 'F2' binds the positively-charged patches and thereby dose-dependently extends aPTT clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. CONCLUSION: The two unique, positively-charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding making both patches crucial for contact activation. Targeting these with FXII EGF-1 specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable target in preventing medical device-induced thrombosis.

Viscoelastic blood coagulation testing system enabled by a non-contact triboelectric angle sensor.

Thromboelastography (TEG) remains a convenient and effective viscoelastic blood coagulation testing device for guiding blood component transfusion and assessing the risk of thrombosis. Here, a TEG enabled by a non-contact triboelectric angle sensor (NTAS) with a small size (∼7 cm3) is developed for assessing the blood coagulation system. With the assistance of a superelastic torsion wire structure, the NTAS-TEG realizes the detection of blood viscoelasticity. Benefiting from a grating and convex design, the NTAS holds a collection of compelling features, including accurate detection of rotation angles from -2.5° to 2.5°, high linearity (R 2 = 0.999), and a resolution of 0.01°. Besides, the NTAS exhibits merits of low cost and simplified fabrication. Based on the NTAS-TEG, a viscoelastic blood coagulation detection and analysis system is successfully constructed, which can provide a graph and parameters associated with clot initiation, formation, and stability for clinicians by using 0.36 mL of whole blood. The system not only validates the feasibility of the triboelectric coagulation testing sensor, but also further expands the application of triboelectric sensors in healthcare.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Factor XIII in major burns coagulation.

BACKGROUND: In the days following a burn injury, major burn patients (MBP) present a multifactorial coagulation disorder known as acute burn-induced coagulopathy. Several studies have investigated coagulation in MBPs; however, Factor XIII (FXIII), which converts fibrin monomers into a stable clot and promotes wound healing, has not yet been studied. OBJECTIVE: To determine the kinetics of FXIII and other coagulation factors and cofactors in MBPs in order to clarify coagulopathy in these patients and its potential relationship with surgical bleeding. METHODS: Prospective observational pilot study of the kinetics of FXIII and other coagulation factors and cofactors in MBPs during the first 30 days of burn injury. RESULTS: FXIII levels show a significant decline of 75.10% in the interval between the burn injury and surgery, and a decline of 87.70% in the 24 h following surgery. Patients undergo surgery with a median antigenic FXIII of 32%. Plasma levels of most factors decrease significantly 24 h after the burn injury. CONCLUSION: MBPs experience a significant decrease in plasma levels of FXIII from the time of admission up to 24 h after surgery. Abnormally low levels were observed at the time of surgery that could not be detected by other coagulation tests. The decrease in most factors at 24 h seems to be associated with dilution due to intensive fluid resuscitation.

Minimal interference of concizumab with standard clinical coagulation laboratory assays - An in vitro study.

INTRODUCTION: Non-factor replacement therapies are emerging as prophylactic treatment options in haemophilia A or B (HA/HB) with and without inhibitors. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody preventing factor (F)Xa inhibition and enhancing thrombin generation. Based on experience with other non-factor therapies and extended half-life products, there is a focus on potential interference with common clinical coagulation assays used to monitor patients treated with concizumab. AIM: To evaluate the impact of concizumab on standard clinical coagulation assays. METHODS: Plasma samples (normal, HA/HB with/without inhibitors) in the presence/absence of added concizumab (250-16,000 ng/mL) were analysed in clinical assays including activated partial thromboplastin time (aPTT), prothrombin time (PT), FVIII and FIX one-stage clot and chromogenic substrate assay, assays for detecting FVIII or FIX inhibitors and other assays for coagulation factors. RESULTS: Concizumab did not impact PT assays, but resulted in a small shortening of aPTT (up to 5 s in haemophilia plasma and 0.4 s in normal plasma). Concizumab had no, or only a minor impact on FVIII and FIX activity assays or Bethesda inhibitor assays. FXI and FXII activity in normal plasma, as measured by single factor aPTT-based assay, was significantly increased in the presence of concizumab (+11% each). This was also the case for FVII and FX measured by PT-based assays using plasma with 25% of FVII or FX (+64% and +22%, respectively). CONCLUSION: The presence of concizumab did not, or only slightly, influence the outcome of standard clinical coagulation assays relevant for HA and HB.

Identifying hemophilia B carriers: Utility of aPTT, factor IX levels and ratios of factor IX to other Vitamin K dependent factors.

INTRODUCTION: Diagnosing hemophilia B (HB) carrier status is important to manage bleeding in carriers and to prevent bleeding in potential offspring. Without a family history of hemophilia, diagnosing HB carrier status is challenging. Genetic testing is the gold-standard, however it is reserved for individuals with a high suspicion of carrier status. AIMS: To describe the distribution of activated partial thromboplastin time (aPTT) and factor IX coagulant (FIX:C) levels in HB carriers and assess the ratio of FIX:C to other Vitamin K dependent factors (FII:C, FVII:C, FX:C) as an indicator of HB carrier status. METHODS: In this retrospective, single-centre cohort study, subjects were included if they were obligate or genetically proven HB carriers. Distributions of aPTT and FIX:C were described and the relationship between FIX:C levels in carriers and severity of familial HB was analysed. Ratios of FIX:C to FII:C, FVII:C, FX:C were calculated. RESULTS: Seventy-two female HB carriers (median age: 34 years; IQR 24-43) were included. Median aPTT and FIX:C levels were 33.0 s [IQR 30.0-37.0] and 57 IU/dL [IQR 43-74]. Fifteen carriers (21%) had mild HB (FIX:C levels of 10-40 IU/dL). FIX:C levels trended higher in carriers of mild HB versus carriers of moderate/severe HB. In six carriers, the median ratio of FIX:C to other Vitamin K dependent factors was 0.44, with 92% of ratios being ≤ 0.75. CONCLUSION: aPTT and FIX:C levels were unreliable in diagnosing HB carrier status. A low ratio of FIX:C to other Vitamin K dependent factors may be a useful marker of HB carrier status.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

[Pedigree Analysis of Hereditary Coagulation Factor XII Deficiency Caused by Compound Heterozygous Mutation p.Gly175Cys and p.Gly542Ser of F12 Gene].

OBJECTIVE: To analyze the clinical phenotype and gene mutation of a genetic coagulation factor XII (FXII) deficiency pedigree and explore the molecular pathogenesis. METHODS: The activated partial thromboplastin time (APTT) and FXII activity (FXII:C) were detected by clotting method. The FXII antigen (FXII:Ag) was tested with ELISA. All exons and flanks of F12 gene were determined by Sanger sequencing. ClustalX-2.1-win, PROVEAN and Swiss-Pdb Viewer software were used to analyze the conservatism of amino acids at the mutant site, forecast whether the mutant amino acids were harmful and confirm the influence of the mutation on protein structure. RESULTS: The APTT of the proband prolonged to 71.3 s. The FXII:C and FXII:Ag were decreased to 5% and 6%, respectively. There were two heterozygous missense mutations c.580G>T and c.1681G>A detected in exon 7 and exon 14 of F12 gene, resulting in p.Gly175Cys and p.Gly542Ser, severally. Proband's father carried the p.Gly175Cys heterozygous mutation, while mother, brother and daughter had the p.Gly542Ser heterozygous mutation. Software analysis showed that both Gly175 and Gly542 were conserved, the two mutations were harmful and when mutations had occurred, the corresponding sites affected the protein local structure. CONCLUSION: The p.Gly175Cys and p.Gly542Ser compound heterozygous mutations are the molecular pathogenesis of the hereditary coagulation FXII deficiency pedigree. The p.Gly175Cys mutation has been detected for the first time in the world.

[Intervention Effect and Mechanism of Regulating MiR-155 on Young Rats with Dysfunction of Blood Coagulation].

OBJECTIVE: To investigate the intervention effect and mechanism of regulating miR-155 on young rats with dysfunction of blood coagulation. METHODS: Twenty-six healthy and clean SD male rats were selected to establish the coagulopathy models. Twenty-four rats successfully established models and were randomly divided into three groups: model group, up-regulated miR-155 group and down-regulated miR-155 group, with 8 rats in each group. The expression of miR-155 was detected by real-time fluorescence quantitative polymerase chain reaction. The changes of coagulation factors and coagulation indicators were observed. Liver pathological tissues were observed by HE staining. The expressions of HMGB1-RAGE/TLRs-NF-κB signaling pathway related proteins were detected by Western blot. RESULTS: Compared with model group, the expressions of HMGB1, RAGE, TLR2, TLR4 and NF-κB were significantly increased in up-regulated miR-155 group (all P < 0.05), while decreased in down-regulated miR-155 group (all P < 0.05). Compared with model group, the expressions of coagulation factor Ⅱ, Ⅶ, Ⅸ, and Ⅹ were significantly decreased in up-regulated miR-155 group (all P < 0.05), while increased in down-regulated miR-155 group (P < 0.05). There was no significant difference in the expression of coagulation factor Ⅺ among the three groups (P >0.05). Compared with model group, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) were lower and fibrinogen (FIB) was higher in up-regulated miR-155 group (all P < 0.05), while in the down-regulated miR-155 group they were opposite. CONCLUSION: Down-regulation of miR-155 can effectively improve coagulation factors and coagulation indexes and inhibit inflammation in young rats with dysfunction of blood coagulopathy, and the mechanism may be related to HMGB1-RAGE/TLRs-NF-κB signaling pathway.