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Chronic urticaria (CU) is a chronic inflammatory mast cell-driven disorder. Endothelial cells (ECs) contribute importantly to key features of CU. Several markers of EC (dys)function in CU have been reported, but have not yet been systematically reviewed. In this study, we systematically reviewed and categorized all published markers of EC functions in CU through a comprehensive search in Pubmed, The Cochrane Library, Web of Science, and SCOPUS using the following Mesh terms: CU AND pathogenesis AND (vasculopathy OR microangiopathy OR ECs OR marker). In total, 79 articles were selected and the identified biomarkers were categorized according to EC (dys)function in CU. The most frequent and consistently reported upregulated biomarkers in CU skin were adhesion molecules, TF, and P-selectin. The most frequently reported upregulated and reliable biomarkers in sera of CU patients were F1+2 for coagulation cascade involvement, D-dimers for fibrinolysis, and MMP-9 for vascular permeability. Emerging biomarkers described in the selected articles were endostatin, heat shock proteins, cleaved high molecular weight kininogen, and adipokines. This systematic review contributes to the pool of growing evidence for vascular involvement in CU where EC dysfunction is present in different aspects of cell survival, maintenance of vascular structure, and coagulation/fibrinolysis balance., SCOPUS: re.j, info:eu-repo/semantics/published

Standard pharmacokinetic (PK) assessments are demanding for persons with hemophilia A, requiring a 72-hour washout and 5 to 11 timed blood samples. A no-washout, single-clinic visit, sparse sampling population PK (PPK) protocol is an attractive alternative. Here, we compared PK parameters obtained with a traditional washout, 6-sampling time point PPK protocol with a no-washout, single-clinic visit, reverse 2-sampling time point PPK protocol in persons with severe hemophilia A (SHA) receiving ADVATE. A total of 39 inhibitor-negative males with SHA (factor VIII activity [FVIII:C]

DNA methylation has become increasingly recognized in the etiology of complex diseases, including thrombotic disorders. Blood is often collected in epidemiological studies for genotyping and has recently also been used to examine DNA methylation in epigenome-wide association studies. DNA methylation patterns are often tissue-specific, thus, peripheral blood may not accurately reflect the methylation pattern in the tissue of relevance. Here, we collected paired liver and blood samples concurrently from 27 individuals undergoing liver surgery. We performed targeted bisulfite sequencing for a set of 35 hemostatic genes primarily expressed in liver to analyze DNA methylation levels of >10,000 cytosine-phosphate-guanine (CpG) dinucleotides. We evaluated whether DNA methylation in blood could serve as a proxy for DNA methylation in liver at individual CpGs. Approximately 30% of CpGs were nonvariable and were predominantly hypo- (70%) in both tissues. While blood can serve as a proxy for liver at these CpGs, the low variability renders these unlikely to explain phenotypic differences. We therefore focused on CpG sites with variable methylation levels in liver. The level of blood–liver tissue correlation varied widely across these variable CpGs; moderate correlations (0.5 ≤ r

Background The aim of this meta-analysis is to assess the prevalence of coagulation dysfunction in Chinese COVID-19 patients and to determine the association of coagulopathy with the severity and prognosis of COVID-19. Methods A meta-analysis of the prevalence of different abnormal coagulation indicators in COVID-19 patients in China was performed. The difference of coagulation indicators and the incidence of DIC were compared between severe cases and nonsevere cases as well as nonsurvivors and survivors, respectively. Results A total of 22 Chinese studies involving 4,889 confirmed COVID-19 inpatients were included. The average D-dimer value of COVID-19 patients is 0.67 µg/mL (95% confidence interval [CI]: 0.56–0.78), and 29.3% (95% CI: 20.1–38.5%) of patients showed elevated D-dimer values. Severe patients had significantly higher D-dimer levels and prolonged prothrombin time (PT) compared with nonsevere patients. Nonsurvivors had significantly higher D-dimer levels, prolonged PT, and decreased platelet count compared with survivors. In total, 6.2% (95% CI: 2.6–9.9%) COVID-19 patients were complicated by disseminated intravascular coagulation (DIC), in which the log risk ratio in nonsurvivors was 3.267 (95% CI: 2.191–4.342, Z = 5.95, p Conclusion The prevalence of coagulopathy in Chinese COVID-19 inpatients is high, and both the abnormal coagulation indicators and DIC are closely associated with the severity and poor prognosis of these COVID-19 patients. Therefore, attention should be paid to coagulation dysfunction in COVID-19 patients. Closely monitoring of coagulation indicators and application of appropriate anticoagulation may improve the prognosis of COVID-19 inpatients in China.

Aim Atypical hemolytic uremic syndrome (aHUS), characterized by thrombotic microangiopathy (TMA), is a genetic, life-threatening disease which needs many differential diagnoses. This study aimed to reveal coagulation and fibrinolysis profiles in aHUS and secondary TMA patients. Furthermore, we investigated whether aHUS patients progress to, and meet, disseminated intravascular coagulation (DIC) criteria. Methods The acute phase samples were available in 15 aHUS and 20 secondary TMA patients. We measured PT-ratio, activated partial thromboplastin time (APTT), fibrinogen, fibrin degradation product (FDP), fibrin monomer complex (FMC), antithrombin (AT), plasmin-α2 plasmin inhibitor complex (PIC), and von Willebrand factor antigen (VWF:Ag). We examined and compared these tests among aHUS, secondary TMA patients, and healthy volunteer (HV), and evaluated whether patients with aHUS and secondary TMA met DIC criteria. Results PT-ratio, APTT, FDP, FMC and PIC in patients with aHUS and secondary TMA were higher than those in HV. Fibrinogen and AT showed no significant difference among three groups. VWF:Ag was higher in only aHUS patients. No tests showed significant difference between aHUS and secondary TMA patients. Three aHUS patients out of 15 met DIC criteria. Conclusion We revealed the profiles and distributions of coagulation and fibrinolysis tests of aHUS and secondary TMA patients. All tests were enhanced compared to HV; however, our results showed the no specificities in distinguishing aHUS from secondary TMA patients. We also clarified that some aHUS patients fulfilled DIC diagnostic criteria, indicating that DIC itself cannot be an exclusion criterion of aHUS.

Hypofibrinolysis is a key abnormality in diabetes but the role of impaired clot lysis in predicting vascular events and mortality in this population is yet to be determined. We aimed to investigate the relationship between fibrin clot properties and clinical outcomes in patients with diabetes and recent acute coronary syndrome (ACS). Plasma samples were collected at hospital discharge from 974 ACS patients with diabetes randomised to clopidogrel or ticagrelor in the PLATO trial. A validated turbidimetric assay was employed to study fibrin clot lysis and maximum turbidity. One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were determined using Cox proportional analysis. After adjusting for CV risk factors, each 50% increase in lysis time was associated with increased risk of CV death/MI (HR 1.21; 95% confidence interval [CI] 1.02–1.44; p = 0.026) and CV death alone (HR 1.38; 1.08–1.76; p = 0.01). Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death/MI (HR 1.25; 1.02–1.53; p = 0.031) and CV death alone (HR 1.49; 1.08–2.04; p = 0.014). The relationship between lysis time and the combined outcome of CV death and MI remained significant after adjusting for multiple prognostic vascular biomarkers (p = 0.034). Neither lysis time nor maximum turbidity was associated with major bleeding events. Impaired fibrin clot lysis predicts 1-year CV death and MI in diabetes patients following ACS. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier NCT00391872.

Prudent administration of fluids helps restore or maintain hemodynamic stability in the setting of perioperative blood loss. However, fluids may arguably exacerbate the existing coagulopathy. We sought to investigate the influence of balanced crystalloid and synthetic gelatine infusions on coagulation and fibrinolysis in healthy volunteers. This prospective randomized crossover study included 25 males aged 18–30 years. Infusions performed included 20 mL/kg of a balanced crystalloid solution (Optilyte®) or 20 mL/kg of gelatine 26.500 Da (Geloplasma®) in a random order over a period of 2 weeks. Laboratory analysis included conventional coagulation parameters and rotational thromboelastometry (ROTEM) assays. We confirmed a decrease in fibrinogen concentration and the number of platelets, and prolongation of PT after infusions. Compared to baseline values, differences in the ROTEM assays’ results after infusions signified the decrease in coagulation factors and fibrinogen concentration, causing impaired fibrin polymerization and clot structure. The ROTEM indicator of clot lysis remained unaffected. In the case of both Optilyte® and Geloplasma®, the results suggested relevant dilution. Gelatine disrupted the process of clot formation more than balanced crystalloid. Infusions of both crystalloid and saline-free colloid solutions causing up to 30% blood dilution cause significant dilution of the coagulation factors, platelets, and fibrinogen. However, balanced crystalloid infusion provides less infusion-induced coagulopathy compared to gelatine.

Beyond the absolute and indisputable relevance and efficacy of anti-SARS-CoV-2 vaccines, the rapid transmission, the severity of infection, the absence of the protection on immunocompromised patients, the propagation of variants, the onset of infection and/or disease in vaccinated subjects and the lack of availability of worldwide vaccination require additional antiviral treatments. Since 1987, lactoferrin (Lf) is well-known to possess an antiviral activity related to its physico-chemical properties and to its ability to bind to both heparan sulfate proteoglycans (HSPGs) of host cells and/or surface components of viral particles. In the present review, we summarize in vitro and in vivo studies concerning the efficacy of Lf against DNA, RNA, enveloped and non-enveloped viruses. Recent studies have revealed that the in vitro antiviral activity of Lf is also extendable to SARS-CoV-2. In vivo, Lf oral administration in early stage of SARS-CoV-2 infection counteracts COVID-19 pathogenesis. In particular, the effect of Lf on SARS-CoV-2 entry, inflammatory homeostasis, iron dysregulation, iron-proteins synthesis, reactive oxygen formation, oxidative stress, gut-lung axis regulation as well as on RNA negativization, and coagulation/fibrinolysis balance will be critically reviewed. Moreover, the molecular mechanisms underneath, including the Lf binding to HSPGs and spike glycoprotein, will be disclosed and discussed. Taken together, present data not only support the application of the oral administration of Lf alone in asymptomatic COVID-19 patients or as adjuvant of standard of care practice in symptomatic ones but also constitute the basis for enriching the limited literature on Lf effectiveness for COVID-19 treatment.

Functional point-of-care tests (POCTs) have evolved into useful tools for diagnosing disorders of blood coagulation and fibrinolysis. We aimed to describe the in vivo association between standard and functional parameters of coagulation and fibrinolysis in the setting of acute hemodilution induced by an infusion of balanced crystalloid or synthetic gelatine solutions. This prospective randomized crossover in vivo study included healthy male volunteers aged 18–30 years. Enrolled participants were randomly assigned to receive either the Optilyte® or Geloplasma® infusion. Laboratory analysis included conventional coagulation parameters and rotational thromboelastometry (ROTEM) assays. A total of 25 healthy Caucasian males were included. ROTEM viscoelastic assays presented moderate to strong correlations with conventional coagulation tests, regardless of the fluid type utilized. Irrespectively of the extent of hemodilution, significant correlations remained unaffected. The strongest associations were found between the ROTEM clot formation and clot strength and the fibrinogen concentration and platelet count, and between the ROTEM clotting time and the APTT and PT. This in vivo experimental study in healthy male volunteers demonstrated that ROTEM may be used as a credible alternative to standard laboratory tests to assess blood coagulation and fibrinolysis in the setting of fluid resuscitation with both crystalloid and colloid solutions. The study was registered online in the ClinicalTrials.gov database (NCT05148650).

Introduction FVIII inhibitor development is the most serious contemporary treatment complication in haemophilia A, particularly in previously untreated patients (PUPs). No inhibitors developed in clinical trials in previously treated patients treated with simoctocog alfa (Nuwiq), a fourth-generation recombinant FVIII produced in a human cell line. Methods The NuProtect study investigated the immunogenicity of simoctocog alfa in PUPs. NuProtect was a prospective, multinational, open-label, non-controlled, phase III study. PUPs with severe haemophilia A (FVIII:C Results A total of 108 PUPs with a median age at first treatment of 12.0 months (interquartile range: 8.0–23.5) were treated with simoctocog alfa. F8 mutation type was known for 102 patients (94.4%) of whom 90 (88.2%) had null F8 mutations and 12 (11.8%) had non-null mutations. Of 105 PUPs evaluable for inhibitor development, 28 (26.7%) developed inhibitors; 17 high titre (16.2%) and 11 low titre (10.5%). No PUPs with non-null F8 mutations developed inhibitors. Conclusion In the NuProtect study, the rate of inhibitor development in PUPs with severe haemophilia A treated with simoctocog alfa was lower than the rate reported for hamster-cell-derived recombinant factor VIII products in other recent clinical trials. No inhibitors were reported in PUPs with non-null F8 mutations.

BAY 94-9027 (damoctocog alfa pegol, Jivi) is an extended-half-life recombinant factor VIII (rFVIII) shown to be well-tolerated and efficacious in bleeding prevention in previously treated patients with severe hemophilia A. During the PROTECT VIII study, prophylaxis patients received BAY 94-9027 at intervals determined based on their bleeding phenotype, observed during a 10-week run-in treatment period with twice-weekly dosing. Those with ≤ 1 spontaneous joint or muscle bleed were randomized to either 45 to 60 IU/kg every 5 days or 60 IU/kg every 7 days; patients could increase dosing frequency to every 5 days or twice weekly in the case of bleeds. Those enrolled after the randomization arms were full, and those with ≥ 2 bleeds in the run-in period, received 30 to 40 IU/kg twice weekly. Patients completing the main study could receive open-label BAY 94-9027 in the extension phase. Dosing regimen, total, and joint annualized bleeding rates were analyzed over three periods: prestudy, main study, and extension. A total of 80 patients who were on prophylaxis treatment prior to and during the study and had prior bleed data available were evaluated in this post hoc analysis of PROTECT VIII. Most patients (> 80%) required fewer infusions with BAY 94-9027 prophylaxis versus their previous standard-half-life (SHL) rFVIII product. Lower bleeding and joint bleeding rates were observed over time from the prestudy to the extension study period in all treatment regimens. Compared with SHL FVIII, BAY 94-9027 prophylaxis allows patients to reduce infusion frequency with maintained or improved protection from bleeds.

The prospective observational cohort study COMPASS-COVID-19 aimed to develop a risk assessment model for early identification of hospitalized COVID-19 patients at risk for worsening disease. Patients with confirmed COVID-19 (n = 430) hospitalized between March 18 and April 21, 2020 were divided in derivation (n = 310) and validation (n = 120) cohorts. Two groups became evident: (1) good prognosis group (G-group) with patients hospitalized at the conventional COVID-19 ward and (2) Worsening disease group (W-group) with patients admitted to the intensive care unit (ICU) from the emergency departments. The study end point was disease worsening (acute respiratory failure, shock, myocardial dysfunction, bacterial or viral coinfections, and acute kidney injury) requiring ICU admission. All patients were routinely evaluated for full blood count, prothrombin time, fibrinogen, D-dimers, antithrombin (AT), and protein C activity. Data from the first hospitalization day at the conventional ward or the ICU were analyzed. Cardiovascular risk factors and comorbidities were routinely registered. Obesity, hypertension, diabetes and male gender, increased fibrinogen and D-dimers, thrombocytopenia, AT deficiency, lymphopenia, and an International Society on Thrombosis and Haemostasis (ISTH) score for compensated disseminated intravascular coagulation score (cDIC-ISTH) ≥5 were significant risk factors for worsening disease. The COMPASS-COVID-19 score was derived from multivariate analyses and includes obesity, gender, hemoglobin, lymphocyte, and the cDIC-ISTH score (including platelet count, prothrombin time, D-dimers, AT, and protein C levels). The score has a very good discriminating capacity to stratify patients at high and low risk for worsening disease, with an area under the receiver operating characteristic curve value of 0.77, a sensitivity of 81%, and a specificity of 60%. Application of the COMPASS-COVID-19 score at the validation cohort showed 96% sensitivity. The COMPASS-COVID-19 score is an accurate clinical decision-making tool for an easy identification of COVID-19 patients being at high risk for disease worsening.

Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a central role in haemostasis, and plasma TAFI concentrations are heritable. Candidate gene studies have identified several variants within the gene encoding TAFI, CPB2, that explain part of the estimated heritability. Here, we describe an exploratory genome-wide association study to identify novel variants within and outside of the CPB2 locus that influence plasma concentrations of intact TAFI and/or the extent of TAFI activation (measured by released TAFI activation peptide, TAFI-AP) amongst 3,260 subjects from Southern Sweden. We also explored the role of rare variants on the HumanExome BeadChip. We confirmed the association with previously reported common variants in CPB2 for both intact TAFI and TAFI-AP, and discovered novel associations with variants in putative CPB2 enhancers. We identified a gene-based association with intact TAFI at CPB2 (PSKAT-O = 2.8 × 10-8), driven by two novel rare nonsynonymous single nucleotide polymorphisms (SNPs; I420N and D177G). Carriers of the rare variant of D177G (rs140446990; MAF 0.2%) had lower intact TAFI and TAFI-AP concentrations compared with non-carriers (intact TAFI, geometric mean 53 vs. 78%, PT-test = 5 × 10-7; TAFI-AP 63 vs. 99%, PT-test = 7.2 × 10-4). For TAFI-AP, we identified a genome-wide significant association at an intergenic region of chromosome 3p14.1 and five gene-based associations (all PSKAT-O

SummaryEmicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab–antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (K D = 1.58, 1.52, 1.85, and 0.978 μM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens’ epidermal growth factor (EGF)-like domains. We then performed K D-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX–emicizumab–FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab’s cofactor activity. The simulation also predicted that at 10.0–100 μg/ml of emicizumab–levels shown in a previous study to be clinically effective–the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The K D-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab’s cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd.Supplementary Material to this article is available online at www.thrombosis-online.com.

SummaryThe ARISTOTLE trial showed a risk reduction of stroke/systemic embolism (SE) and major bleeding in non-valvular atrial fibrillation (NVAF) patients treated with apixaban compared to warfarin. This retrospective study used four large US claims databases (MarketScan, PharMetrics, Optum, and Humana) of NVAF patients newly initiating apixaban or warfarin from January 1, 2013 to September 30, 2015. After 1:1 warfarin-apixaban propensity score matching (PSM) within each database, the resulting patient records were pooled. Kaplan-Meier curves and Cox proportional hazards models were used to estimate the cumulative incidence and hazard ratios (HRs) of stroke/SE and major bleeding (identified using the first listed diagnosis of inpatient claims) within one year of therapy initiation. The study included a total of 76,940 (38,470 warfarin and 38,470 apixaban) patients. Among the 38,470 matched pairs, 14,563 were from MarketScan, 7,683 were from PharMetrics, 7,894 were from Optum, and 8,330 were from Humana. Baseline characteristics were balanced between the two cohorts with a mean (standard deviation [SD]) age of 71 (12) years and a mean (SD) CHA2DS2-VASc score of 3.2 (1.7). Apixaban initiators had a significantly lower risk of stroke/SE (HR: 0.67, 95 % CI: 0.59–0.76) and major bleeding (HR: 0.60, 95 % CI: 0.54–0.65) than warfarin initiators. Different types of stroke/SE and major bleeding – including ischaemic stroke, haemorrhagic stroke, SE, intracranial haemorrhage, gastrointestinal bleeding, and other major bleeding – were all significantly lower for apixaban compared to warfarin treatment. Subgroup analyses (apixaban dosage, age strata, CHA2DS2-VASc or HAS-BLED score strata, or dataset source) all show consistently lower risks of stroke/SE and major bleeding associated with apixaban as compared to warfarin treatment. This is the largest “real-world” study on apixaban effectiveness and safety to date, showing that apixaban initiation was associated with significant risk reductions in stroke/SE and major bleeding compared to warfarin initiation after PSM. These benefits were consistent across various high-risk subgroups and both the standard-and low-dose apixaban dose regimens.Note: The review process for this manuscript was fully handled by Christian Weber, Editor in Chief.Supplementary Material to this article is available online at www.thrombosis-online.com.

SummaryOf the new direct oral anticoagulants, direct factor Xa inhibitors are limited by the absence of a proven reversal agent. We assessed the safety, tolerability and impact on anticoagulation reversal of ciraparantag (PER977) alone and following a 60 mg dose of the FXa inhibitor edoxaban. Escalating, single IV doses of ciraparantag were administered alone and following a 60 mg oral dose of edoxaban in a double-blind, placebo-controlled fashion to healthy subjects. Serial assessments of the pharmacokinetics and pharmacodynamic effects of ciraparantag were performed. Eighty male subjects completed the study. Following edoxaban (60 mg), a single IV dose of ciraparantag (100 to 300 mg) demonstrated full reversal of anticoagulation within 10 minutes and sustained for 24 hours. Fibrin diameter within clots was restored to normal 30 minutes after a single dose of 100 to 300 mg ciraparantag as determined by scanning electron microscopy and change in fibrin diameter quantified by automated image analysis. Potentially related adverse events were periorbital and facial flushing and cool sensation following IV injection of ciraparantag. Renal excretion of ciraparantag metabolite was the main elimination route. There was no evidence of procoagulant activity following ciraparantag as assessed by D-dimer, prothrombin fragments 1.2, and tissue factor pathway inhibitor levels. In conclusion, ciraparantag in healthy subjects is safe and well tolerated with minor, non-dose limiting adverse events. Baseline haemostasis was restored from the anticoagulated state with doses of 100 to 300 mg ciraparantag within 10–30 minutes of administration and sustained for at least 24 hours.Institution where the work was performed: Duke University Clinical Research Unit, Duke University Medical Center, Durham, NC USA

Mouse models of thromboembolism are frequently used to investigate platelet function in vivo and, according to European Union (EU) legislation, must be conducted in the context of replacement, refinement and reduction. We have previously developed a refined real-time mouse model of thromboembolism as an alternative to models of thromboembolic mortality which inflict considerable pain and suffering. Real-time monitoring involves infusion of radiolabelled platelets into the circulation of anaesthetized mice, and platelet aggregation is measured as increases in platelet-associated counts in the pulmonary vasculature following injection of platelet agonists. This gives a definitive data set on the tissue localization and extent of platelet activation. We developed an additional, more simplistic alternative to mortality models based on blood microsampling which entails the measurement of circulating platelet counts following agonist stimulation. Blood microsamples were collected from the tail vein of anaesthetized mice at three different time points leading to a reduction in animal numbers. Platelet counts significantly dropped 1 minute after stimulation with collagen or thrombin and were restored over 10 minutes. These results correlate with those obtained via real-time monitoring and were confirmed by immunohistochemistry. Pre-treatment of mice with aspirin significantly inhibited the decrease in platelet counts following collagen. These data suggest that blood microsampling may be implemented as a simplistic refined alternative to mortality models of thromboembolism when specialized monitoring equipment, or use of radioactive isotopes for real-time monitoring, which remains the ‘gold standard’, is not feasible. Microsampling refines and reduces animal procedures in compliance with current EU legislation.

SummaryRecombinant human soluble thrombomodulin (ART-123) is a novel anticoagulant for patients with disseminated intravascular coagulation (DIC). It is widely used in clinical settings throughout Japan. Furthermore, a global Phase 3 study is currently being conducted. In healthy subjects, ART-123 is excreted mainly via the kidneys. Therefore, ART-123 dose decrease was recommended in DIC patients with severe renal dysfunction. However, the pharmacokinetics of ART-123 in DIC patients with severe acute renal dysfunction has not been elucidated. In an open-label, multicentre, prospective, clinical pharmacological study, we investigated the pharmacokinetics and safety of ART-123 upon repeated administration to DIC patients. ART-123 was administered to patients at a dose of 130 or 380 U/kg/day for six consecutive days. Plasma concentrations of ART-123 were measured at 21 time points until eight days after the final administration. Urinary excretion rates during the first 24 hours (h) were calculated. Patient renal functions were evaluated by measuring 24-h creatinine clearance (Ccr). Forty-three patients were enrolled in the present study. The urinary excretion rates of ART-123 correlated closely with 24-h Ccr. Total body clearance of ART-123 was also weakly related with 24-h Ccr. However, the plasma concentrations of ART-123 were not considerably different among patients with different renal function. Two patients had subcutaneous haemorrhage as an adverse event related to ART-123. In conclusion, plasma concentrations of ART-123 may not be different among patients with different renal functions. ART-123 was well tolerated in these patients.

Background: CO2 artificial pneumothorax creates a sufficient operative field for thoracoscopic esophagectomy. However, it has potential complications and continuous CO2 insufflation may impede coagulation and fibrinolysis. We sought to compare the effects of CO2 artificial pneumothorax on perioperative coagulation and fibrinolysis during thoracoscopic esophagectomy. Methods: We investigated patients who underwent thoracoscopic esophagectomy with (group P, n = 24) or without CO2 artificial pneumothorax (group N, n = 24). The following parameters of coagulation–fibrinolysis function: intraoperative bleeding volume; serum levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), thromboelastogram (TEG), D-Dimer; and arterial blood gas levels were compared with two groups. Results: Group P showed higher levels of PaCO2, reaction time (R) value and kinetics (K) value, but significantly lower pH value, alpha (α) angle and Maximum Amplitude (MA) value at 60 minutes after the initiation of CO2 artificial pneumothorax than group N ((P .05). There was no significant difference in D-dimer. Conclusion: CO2 artificial pneumothorax during thoracoscopic esophagectomy had a substantial impact on coagulation and fibrinolysis, inducing significant derangements in pH and PaCO2. Trial registration: The study was registered at the Chinese clinical trial registry (ChiCTR1800019004)

Summary A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the

Purpose Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach. Methods We utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue. Results Clot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls. Conclusions Our data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.

Recombinant factor XIII-A2 (rFXIII-A2) was developed for prophylaxis and treatment of bleeds in patients with congenital FXIII A-subunit deficiency. mentor™2 (NCT00978380), a multinational, open-label, single-arm, multiple-dosing extension to the pivotal mentor™1 trial, assessed long-term safety and efficacy of rFXIII-A2 prophylaxis in eligible patients (patients with severe [

Summary Colistin electrostatically interacts with lipopolysaccharides (LPS). Preclinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-1 levels decreased numerically by 63 %. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ∼50 % and the two-fold increase in vWF antigen levels by ∼70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected. Note: This work was conducted at the Medical University of Vienna. EudraCT-Nr.: 2014–00285720 Supplementary Material to this article is available online at http://www.thrombosis-online.com

SummaryCarbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

Summary Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of ‘knots’ interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin ‘knots’. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin’s viscoelastic properties by rheometry. PolyP reduced the stiffness (G’) and ability of the fibrin network to deform plastically G”, but to different extents. Consequently, the relative plastic component (loss tangent (G”/G’)) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing ‘knotted’ regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically. Supplementary Material to this article is available online at www.thrombosis-online.com.

Summary Established primary prevention strategies of cardiovascular diseases are based on understanding of risk factors, but whether the same risk factors are associated with atrial fibrillation (AF) remains unclear. We conducted a systematic review and field synopsis of the associations of 23 cardiovascular risk factors and incident AF, which included 84 reports based on 28 consented and four electronic health record cohorts of 20,420,175 participants and 576,602 AF events. We identified 3-19 reports per risk factor and heterogeneity in AF definition, quality of reporting, and adjustment. We extracted relative risks (RR) and 95 % confidence intervals [CI] and visualised the number of reports with inverse (RR [CI]1.00) associations. For hypertension (13/17 reports) and obesity (19/19 reports), there were direct associations with incident AF, as there are for coronary heart disease (CHD). There were inverse associations for non-White ethnicity (5/5 reports, with RR from 0.35 to 0.84 [0.82–0.85]), total cholesterol (4/13 reports from 0.76 [0.59–0.98] to 0.94 [0.90–0.97]; 8/13 reports with non-significant inverse associations), and diastolic blood pressure (2/11 reports from 0.87 [0.78–0.96] to 0.92 [0.85–0.99]; 5/11 reports with non-significant inverse associations), and direct associations for taller height (7/10 reports from 1.03 [1.02–1.05] to 1.92 [1.38–2.67]), which are in the opposite direction of known associations with CHD. A systematic evaluation of the available evidence suggests similarities as well as important differences in the risk factors for incidence of AF as compared with other cardiovascular diseases, which has implications for the primary prevention strategies for atrial fibrillation.

SummaryElevated platelet turnover contributes to high platelet reactivity. High platelet reactivity after percutaneous coronary intervention (PCI) is associated with major adverse cardiovascular events (MACE). The purpose of this study was to determine the prognostic value of platelet turnover and function with regard to MACE after PCI with stent implantation. In this prospective observational study, 486 consecutive patients after PCI on aspirin and clopidogrel were included to determine platelet turnover (mean platelet volume (MPV), reticulated platelet fraction (RPF)) and platelet function (multiple electrode aggregometry (MEA), vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) assay). At six-months follow-up, MACE occurred in 10.7 % of patients. RPF (odds ratio [OR]=1.173 (95% confidence interval [CI 95 %] 1.040–1.324), p=0.009) and MPV (OR=1.459 (CI 95 % 1.059–2.008), p=0.021) were univariable predictors of MACE, whereas VASP-P (OR=1.016 (CI 95 % 1.000–1.032), p=0.052) and MEA (OR=0.999 (CI 95 % 0.980–1.017), p=0.895) failed to predict MACE. RPF remained the only platelet variable independently associated with MACE. The best model to predict MACE included: troponin I (OR=1.007 (CI 95 % 1.002–1.012), p=0.009), RPF (OR=1.136 (CI 95 % 1.001–1.288), p=0.048), CRP (OR=1.008 (CI 95 % 1.001–1.014), p=0.023) and history of myocardial infarction (OR=2.039 (CI 95 % 1.093–3.806), p=0.025). RPF (OR=1.211 (CI 95 % 1.042–1.406), p=0.012) was also independently associated with in-hospital bleedings. In conclusion, RPF as index of platelet turnover is an independent predictor of MACE and bleeding events in PCI patients on dual antiplatelet therapy. Since RPF can reliably be quantified along with routine haemograms, RPF might easily be applied in the setting of cardiovascular risk prediction.

SummaryVenous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0% were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5%). The diagnosis was deep-vein thrombosis (DVT) in 59.5% and pulmonary embolism (PE) in 40.5%. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5%), hypertension (42.3%) and dyslipidaemia (21.1%). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2%), almost half received a vitamin K antagonist (48.7%) and nearly a quarter received a DOAC (24.5%). Almost a quarter of all presentations were for recurrent VTE, with >80% of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes.

Interaction between hypoxia and coagulation is important given the increased risk of thrombotic diseases in chronically hypoxic patients who reside at sea level and in residents at high altitude. Hypoxia alters the proteome of platelets favouring a prothrombotic phenotype, but studies of activation and consumption of specific coagulation factors in hypoxic humans have yielded conflicting results. We tested blood from 63 healthy lowland volunteers acclimatizing to high altitude (5,200 m) using thromboelastometry and assays of platelet function to examine the effects of hypoxia on haemostasis. Using data from two separate cohorts of patients following identical ascent profiles, we detected a significant delay in clot formation, but increased clot strength by day 7 at 5,200 m. The latter finding may be accounted for by the significant rise in platelet count and fibrinogen concentration that occurred during acclimatization. Platelet function assays revealed evidence of platelet hyper-reactivity, with shortened PFA-100 closure times and increased platelet aggregation in response to adenosine diphosphate. Post-expedition results were consistent with the normalization of coagulation following descent to sea level. These robust findings indicate that hypoxia increases platelet reactivity and, with the exception of the paradoxical delay in thromboelastometry clotting time, suggest a prothrombotic phenotype at altitude. Further work to elucidate the mechanism of platelet activation in hypoxia will be important and could impact upon the management of patients with acute or chronic hypoxic respiratory diseases who are at risk of thrombotic events.

Mesothelial cells are fundamental to the maintenance of serosal integrity and homeostasis and play a critical role in normal serosal repair following injury. However, when normal repair mechanisms breakdown, mesothelial cells take on a profibrotic role, secreting inflammatory, and profibrotic mediators, differentiating and migrating into the injured tissues where they contribute to fibrogenesis. The development of new molecular and cell tracking techniques has made it possible to examine the origin of fibrotic cells within damaged tissues and to elucidate the roles they play in inflammation and fibrosis. In addition to secreting proinflammatory mediators and contributing to both coagulation and fibrinolysis, mesothelial cells undergo mesothelial-to-mesenchymal transition, a process analogous to epithelial-to-mesenchymal transition, and become fibrogenic cells. Fibrogenic mesothelial cells have now been identified in tissues where they have not previously been thought to occur, such as within the parenchyma of the fibrotic lung. These findings show a direct role for mesothelial cells in fibrogenesis and open therapeutic strategies to prevent or reverse the fibrotic process.

Complex diseases are influenced by variation at multiple genetic loci and environmental factors. Different risks for such diseases can be seen in different ethnic and racial groups. Gene polymorphisms of factors involved in coagulation and fibrinolysis are potentially important in development of arterial and venous thrombosis. In our study we investigated the incidence of mutated alleles for Factor V Leiden, Prothrombin G20210A, Fibrinogen -455 G/A, -148 C/T and PAI-1 4G/5G in healthy population. Sequence variation in Factor V causes the resistance to activated protein C and variations in Prothrombin, Fibrinogen and PAI-1 are associated with increased gene transcription and may lead to their increased plasma levels and hypercoagulability. A 120 subjects were studied (aged between 20-75 years). Genotypes were determined by PCR-RFLP (Prothrombin G20210A and Fibrinogen 455 G/A, -148 C/T) and PCR-SSCP (Factor V Leiden and PAI-1 4G/5G) analysis. The observed genotype frequencies were: Factor V 92.7% wild type, 7.3% heterozygotes ; Prothrombin 98% wild type, 2% heterozygous ; Fibrinogen 46.7% wild type, 38.5% heterozygotes and 4.4% homozygotes for mutated allele ; the same distribution was observed for the Fibrinogen -148 C/T polymorphism ; PAI-1 29.2% 5G homozygotes, 45.2% 4G/5G heterozygotes and 25.6% 4G homozygotes. Two polymorphisms of fibrinogen gene showed to be in complete linkage disequilibrium. The observed distribution of genotypes was consistent with that predicted by the Hardy-Weinberg equilibrium. The results were consistent with reported genotype and allele frequencies for other white populations.

Background Early intramedullary nailing (IMN) of long bone fractures in severely injured patients has been evaluated as beneficial, but has also been associated with increased inflammation, multi organ failure (MOF) and morbidity. This study was initiated to evaluate the impact of primary femoral IMN on coagulation-, fibrinolysis-, inflammatory- and cardiopulmonary responses in polytraumatized patients. Methods Twelve adult polytraumatized patients with femoral shaft fractures were included. Serial blood samples were collected to evaluate coagulation-, fibrinolytic-, and cytokine activation in arterial blood. A flow-directed pulmonary artery (PA) catheter was inserted prior to IMN. Cardiopulmonary function parameters were recorded peri- and postoperatively. The clinical course of the patients and complications were monitored and recorded daily. Results Mean Injury Severity Score (ISS) was 31 ± 2.6. No procedure-related effect of the primary IMN on coagulation- and fibrinolysis activation was evident. Tumor necrosis factor alpha (TNF-α) increased significantly from 6 hours post procedure to peak levels on the third postoperative day. Interleukin-6 (IL-6) increased from the first to the third postoperative day. Interleukin-10 (IL-10) peaked on the first postoperative day. A procedure-related transient hemodynamic response was observed on indexed pulmonary vascular resistance (PVRI) two hours post procedure. 11/12 patients developed systemic inflammatory response syndrome (SIRS), 7/12 pneumonia, 3/12 acute lung injury (ALI), 3/12 adult respiratory distress syndrome (ARDS), 3/12 sepsis, 0/12 wound infection. Conclusion In the polytraumatized patients with femoral shaft fractures operated with primary IMN we observed a substantial response related to the initial trauma. We could not demonstrate any major additional IMN-related impact on the inflammatory responses or on the cardiopulmonary function parameters. These results have to be interpreted carefully due to the relatively few patients included. Trial Registration ClinicalTrials.gov: NCT00981877