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It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20–42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice. AAV mediated gene editing using CRISPR/Cas9 facilitates integration of a bypass coagulation factor VIIa and rescues bleeding phenotype in hemophilia B mice. Image created using Biorender.com. [Display omitted] • CRISPR/Cas9 mediated targeting of Rosa26 locus was found to have 20-42% efficiency in vitro. • Successful insertion of murine activated factor VII (mFVIIa) into the Rosa26 locus was achieved by CRISPR/Cas9 gene editing. • AAV mediated knock-in of bypass clotting factor mFVIIa using CRISPR/Cas9 mitigated bleeding phenotype in hemophilia B mice. • Increased FVIIa activity and shortened prothrombin time was attained after gene editing therapy in hemophilia B mice. [ABSTRACT FROM AUTHOR]

Use of anabolic-androgenic steroids (AAS) is associated with adverse cardiovascular (CV) effects, including potential prothrombotic effects. This study aimed to assess platelet activation and aggregation, coagulation, and fibrinolysis, in long-term AAS users compared to non-using strength-trained athletes. Thirty-seven strength-trained men using AAS were compared to seventeen non-using professional strength-trained athletes at similar age (median 33 years). AAS use was verified by blood and urine analyses. Platelet Function Analyzer 100 (PFA-100) and whole blood impedance aggregometry with thrombin, arachidonic acid, and ADP as agonists, were performed to evaluate platelet aggregation. ELISA methods were used for markers of platelet activation. Fibrinogen, D-dimer, the coagulation inhibitors protein S and C activity, and antithrombin were measured by routine. Fibrinolysis was evaluated by Plasminogen Activator Inhibitor-1 (PAI-1) activity. There were no significant differences in platelet aggregation between the two groups. Von Willebrand factor was lower among the AAS users (p < 0.01), and P-Selectin was slightly higher (p = 0.05), whereas CD40 Ligand, β-thromboglobulin, and thrombospondin did not differ significantly. No differences were found in the assessed coagulation inhibitors. Higher D-dimer levels (p < 0.01) and lower PAI-1 activity (p < 0.01) were found among the AAS users. The investigated long-term users of AAS did not exhibit elevated platelet activity compared to strength-trained non-using athletes. However, AAS use was associated with higher D-dimer levels and lower PAI-1 activity. These findings suggest that any prothrombotic effect of long-term AAS use may predominantly involve other aspects of the hemostatic system than blood platelets. [Display omitted] • Anabolic steroids are associated with an increased risk of cardiovascular diseases. • It has been hypothesized that anabolic steroids might have a prothrombotic effect. • The prothrombotic effects of anabolic androgenic steroids are still unclear. • We found an increased activation of coagulation and fibrinolysis. • No increase of blood platelet activation was observed. [ABSTRACT FROM AUTHOR]

Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5′-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis. • Recombinant VIII (rFVIII) does not affect thrombus formation on normal intima. • rFVIII enhances platelet-fibrin thrombus on smooth muscle cell-rich neointima. • Enhanced thrombosis under elevated FVIII is neutrophil-independent. [ABSTRACT FROM AUTHOR]

• NXT007 is a bispecific IgG engineered from emicizumab to potentiate the activity. • The natural anticoagulant function of AT is unaffected by the presence of emicizumab. • NXT007 was here found not to affect the AT-function despite its enhanced FX-binding. • The results suggest that NXT007 would not increase the AT-related thrombotic risk. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) in sepsis patients increases patient mortality. Endothelial cells are important players in the pathophysiology of sepsis-associated AKI (SA-AKI), yet knowledge regarding their spatiotemporal involvement in coagulation disbalance and leukocyte recruitment is lacking. This study investigated the identity and kinetics of responses of different microvascular compartments in kidney cortex in response to SA-AKI. Laser microdissected arterioles, glomeruli, peritubular capillaries, and postcapillary venules from kidneys of mice subjected to cecal ligation and puncture (CLP) were analyzed using RNA sequencing. Differential expression and pathway enrichment analyses identified genes involved in coagulation and inflammation. A selection of these genes was evaluated by RT-qPCR in microvascular compartments of renal biopsies from patients with SA-AKI. The role of two identified genes in lipopolysaccharide-induced endothelial coagulation and inflammatory activation were determined in vitro in HUVEC using siRNA-based gene silencing. CLP-sepsis in mice induced altered expression of approximately 400 genes in the renal microvasculature, with microvascular compartments exhibiting unique spatiotemporal responses. In mice, changes in gene expression related to coagulation and inflammation were most extensive in glomeruli at early and intermediate time points, with high induction of Plat , Serpine1 , Thbd , Icam1 , Stat3 , and Ifitm3. In human SA-AKI, PROCR and STAT3 were induced in postcapillary venules, while SERPINE1 expression was diminished. IFITM3 was increased in arterioles and glomeruli. In vitro studies revealed that STAT3 and IFITM3 partly control endothelial coagulation and inflammatory activation. Renal microvascular compartments in mice and humans exhibited heterogeneous changes in coagulation- and inflammation-related gene expression in response to SA-AKI. Additional research should aim at understanding the functional consequences of the here described heterogeneous microvascular responses to establish the usefulness of identified genes as therapeutic targets in SA-AKI. [Display omitted] • Laser microdissection combined with RNA-seq enabled identification of unique renal microvascular transcriptome profiles. • Sepsis induced microvascular compartment-specific spatiotemporal coagulation- & inflammation-related gene expression changes. • Renal microvascular responses to sepsis partially overlapped between mice and patients. • STAT3 and IFITM3 were identified as novel endothelial therapeutic targets for sepsis. [ABSTRACT FROM AUTHOR]

Sepsis is a common and critical condition encountered in clinical practice that can lead to multi-organ dysfunction. Sepsis-induced coagulopathy (SIC) significantly affects patient outcomes. However, the precise mechanisms remain unclear, making the identification of effective prognostic and therapeutic targets imperative. The analysis of transcriptome data from the whole blood of sepsis patients, facilitated the identification of key genes implicated in coagulation. Then we developed a prognostic model and a nomogram to predict patient survival. Consensus clustering classified sepsis patients into three subgroups for comparative analysis of immune function and immune cell infiltration. Single-cell sequencing elucidated alterations in intercellular communication between platelets and immune cells in sepsis, as well as the role of the coagulation-related gene FYN. Real-time quantitative PCR determined the mRNA levels of critical coagulation genes in septic rats' blood. Finally, administration of a FYN agonist to septic rats was observed for its effects on coagulation functions and survival. This study identified four pivotal genes—CFD, FYN, ITGAM, and VSIG4—as significant predictors of survival in patients with sepsis. Among them, CFD, FYN, and ITGAM were underexpressed, while VSIG4 was upregulated in patients with sepsis. Moreover, a nomogram that incorporates the coagulation-related genes (CoRGs) risk score with clinical features of patients accurately predicted survival probabilities. Subgroup analysis of CoRGs expression delineated three molecular sepsis subtypes, each with distinct prognoses and immune profiles. Single-cell sequencing shed light on heightened communication between platelets and monocytes, T cells, and plasmacytoid dendritic cells, alongside reduced interactions with neutrophils in sepsis. The collagen signaling pathway was found to be essential in this dynamic. FYN may affect platelet function by modulating factors such as ELF1, PTCRA, and RASGRP2. The administration of the FYN agonist can effectively improve coagulation dysfunction and survival in septic rats. The research identifies CoRGs as crucial prognostic markers for sepsis, highlighting the FYN gene's central role in coagulation disorders associated with the condition and suggesting novel therapeutic intervention strategies. [Display omitted] • Found four key genes for sepsis prognosis linked to coagulation. • Created sepsis survival nomogram combining gene scores and clinical data. • Unveiled three sepsis subtypes with unique prognoses and immune traits. • Showed potential of FYN agonist in treating sepsis-induced coagulopathy. [ABSTRACT FROM AUTHOR]

Strenuous exercise may occasionally cause coronary thrombosis with myocardial infarction and sudden cardiac death. Patients with stable coronary artery disease (CAD) (n = 164) and healthy individuals (n = 25) performed strenuous exercise on a bicycle ergometer. Blood was drawn at baseline, immediately after exercise and 2 h later. Platelet aggregation was measured with Multiplate® Analyzer. Thrombin generation was determined using a thrombogram and by measuring prothrombin fragment 1 + 2 (F1 + 2). A clot lysis assay was used to investigate fibrinolysis. From baseline to immediately after exercise, thrombin receptor activating peptide (TRAP)-induced platelet aggregation increased in CAD patients (Δ77 AU × min, 95 % confidence interval (CI): 46;107) and in healthy individuals (Δ153 AU × min, 95%CI: 75;232). Endogenous thrombin potential (ETP) was unaffected by exercise, whilst F1 + 2 increased (Δ17%, 95%CI: 11;24) in CAD patients. Fibrin clot lysis time increased by 9 % (95%CI: 1–17) in CAD patients and by 26 % (95%CI: 8;45) in healthy individuals. When comparing baseline to 2 h post-exercise, TRAP-induced platelet aggregation remained slightly elevated in both CAD patients (Δ53 AU × min, 95%CI: 22;84) and healthy individuals (Δ140 AU × min, 95%CI: 62;219). In contrast, ETP and F1 + 2 decreased in CAD patients (Δ-6 %, 95%CI: −10;-1 and Δ-8 %, 95%CI: -14;-2). Moreover, clot lysis time decreased (Δ-19 %, 95%CI: −27;-11) in patients with CAD and returned to baseline in healthy individuals. All p -values were <0.05. Platelet aggregation and F1 + 2 were substantially elevated immediately after exercise in CAD patients, indicating a pro-thrombotic state. After 2 h of recovery, they exhibited a markedly increase in fibrinolysis. Similar results were observed in healthy individuals. • Acute strenuous exercise may occasionally induce coronary thrombosis. • Immediately after strenuous exercise, CAD patients were in a pro-thrombotic state. • Two hours after strenuous exercise, CAD patients were in a pro-fibrinolytic state. [ABSTRACT FROM AUTHOR]

Hormone replacement therapy is associated with an increased thromboembolic risk. The effects of testosterone (T) on coagulation markers in people assigned female at birth (AFAB) under gender affirming hormone therapy (GAHT) are not well described. Systematic review and meta-analysis on English-language articles retrieved from PubMed, Scopus and Cochrane Library up to April 2023 investigating T therapy in AFAB people. Coagulation parameters included international normalized ratio (INR), fibrinogen, activated partial thromboplastin clotting time (aPTT), plasminogen activator inhibitor-1 (PAI-1); hematological variables included hemoglobin (Hb) and hematocrit (HCT). We also reported the rate of thromboembolic events. Data were combined as mean differences (MD) with a 95 % confidence interval (CI) of pre- vs post-follow-up values, using random-effects models. We included 7 studies (6 prospective and 1 retrospective) providing information on 312 subjects (mean age: 23 to 30 years) who underwent GAHT with variable T preparation. T therapy was associated with a significant increase in INR values [MD: 0.02, 95 % confidence interval (CI): 0.01–0.03; p = 0.0001], with negligible heterogeneity (I2 = 4 %). T therapy was associated with increased Hb (MD: 1.48 g/dL, 95%CI: 1.17 to 1.78; I2 = 9 %) and HCT (4.39 %, 95%CI: 3.52 to 5.26; I2 = 23 %) values. No effect on fibrinogen, aPTT and PAI-1 was found. None of the study reported thromboembolic events during the follow-up. Therapy with T increased blood viscosity in AFAB men. A slight increase in INR values was also found, but the clinical relevance and mechanism(s) of this finding needs to be clarified. • Testosterone therapy in AFAB people is associated with pro-coagulant effects, such as increased HCT and Hb values. • Studies show lengthening of INR of uncertain clinical value. • No apparent thrombotic risk is present during testosterone-based affirming therapy in AFAB people. [ABSTRACT FROM AUTHOR]

Viscoelastic testing has been used in adult hematologic malignancies in conjunction with conventional coagulation tests (CCTs) to predict coagulopathies and tailor blood product replacement. However, there is a paucity of similar pediatric studies. Analyze and correlate leukemia-associated coagulopathy in newly diagnosed pediatric leukemia patients using CCT's and Rotational Thromboelastometry (ROTEM). Pediatric patients with newly diagnosed acute leukemia underwent testing with ROTEM and CCTs on days 0, 15 and 29 of induction chemotherapy. Sixty-two patients were enrolled. At presentation, 54.8 % of patients had platelets <50 K/μL, 73 % had prolonged PT, 1.6 % had fibrinogen <150 mg/dL. Fifteen patients (24.2 %) had WHO grade 1 bleeding and two patients (3 %) had WHO grade 4 bleeding. EXTEM/INTEM values at presentation (day 0) reflected hypocoagulability, however FIBTEM revealed hypercoagulability. Patients showed a progressive hypocoagulability in all ROTEM assays by day 15 (day 0 vs day 15, p < 0.001), with improvement by day 29 (day 15 vs day 29, p < 0.001). Day 0 ROTEM parameters were comparable to day 29. Fibrinogen strongly correlated with ROTEM at all three time points (p < 0.0001), along with platelet count with moderate correlations (p < 0.001). Fibrinogen and platelets appear to be the drivers of leukemia associated coagulopathy in the pediatric population, suggesting the utility of using CCTs and ROTEM in this population to better evaluate hemostatic function and guide blood product replacement. • Rotational Thromboelastometry (ROTEM) has been used to assess leukemia-associated coagulopathy in adult malignancies, with a paucity of similar studies in pediatric leukemia • Abnormal coagulation test (PT/aPTT) values in leukemia patients do not clearly predict the risk of bleeding • Pediatric leukemia patients had evidence of hypocoagulability during induction chemotherapy using ROTEM assays despite acquired hypofibrinogenemia and thrombocytopenia and a low bleeding risk in our cohort • Strategies adopting ROTEM as a point of care test along with conventional coagulation tests (CCTs) can better evaluate hemostatic function to guide non-erythrocyte hemostatic factor replacement in this patient population • Our study demonstrates the utility of CCTs and ROTEM in newly diagnosed pediatric leukemia patients, with fibrinogen and platelets as the main drivers of leukemia-associated coagulopathy. [ABSTRACT FROM AUTHOR]

Growing evidence supports the importance of factor (F) XI activation for thrombosis and hemostasis as well as inflammation and complement systems. In this study, we evaluated the effect of activated FXI (FXIa) on the detection of factor deficiencies by global hemostasis assays of thrombin generation (TG), plasmin generation (PG), and clot formation and lysis (CFL). An absorbance and fluorescence microplate assay was used to simultaneously observe TG, PG, and CFL in FV-, FVII-, FVIII-, and FIX-deficient plasmas supplemented with purified factors. Coagulation was initiated with tissue factor with or without FXIa in the presence of tissue plasminogen activator. Thrombin and plasmin peak heights (TPH and PPH), maximal clot density (MCD), times to clotting (CT), thrombin and plasmin peaks (TPT and PPT) and clot lysis (LyT) and a new parameter, clot lifetime (LiT), were evaluated. TG/CFL were elevated by the FXIa at low FV (below 0.1 IU/mL), and at FVIII and FIX above 0.01 IU/mL. FXIa affected PG only at low FV and FVII. At high factor concentrations, FXIa reduced MCD. Thrombin and plasmin substrates had effect on CT, LyT, LiT and MCD parameters. FXIa reveals new relationships between TG, PG and CFL parameters in factor deficiencies suggesting potential benefits for discrimination of bleeding phenotypes. • Similar to Tissue factor (TF), factor XIa (FXIa) can mediate thrombosis and hemostasis • Thrombin and plasmin generation (TG and PG), Clot Formation and Lysis (CFL) were studied after activation with TF and FXIa • Addition of FXIa to TF reveals new relationships between TG/PG and CFL parameters in coagulation factors deficient plasmas • CFL parameters differ between TG and PG assays [ABSTRACT FROM AUTHOR]

Thrombotic disease may be an underdiagnosed condition of prolonged exposure to microgravity and yet the underlying factors remain poorly defined. Recently, an internal jugular vein thrombosis was diagnosed in a low-risk female astronaut after an approximately 7-week space mission. Six of the additional 10 crew members demonstrated jugular venous flow risk factors, such as suspicious stagnation or retroversion. Fortunately, all were asymptomatic. Observations in space as well as clinical and in vitro microgravity studies on Earth, where experiments are designed to recapitulate the conditions of space, suggest effects on blood flow stasis, coagulation, and vascular function. In this article, the related literature on thrombotic disease in space is reviewed, with consideration of these elements of Virchow's triad. • Asymptomatic internal jugular vein thrombosis (IJV) was detected in an astronaut. • Exposure to microgravity may cause unforeseen risk during long-term spaceflight. • Studies on thrombosis risk in space and analogous models on Earth are limited. • Microgravity affects the blood flow stasis, coagulation, and endothelial triad. • Here, the pathophysiological and biomolecular evidence are examined. [ABSTRACT FROM AUTHOR]

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions. • Platelets were redundant for clotting in normal plasma under physiological conditions. • Platelets provided robustness of the coagulation system to the changes in initial conditions. • Platelets were different from artificial lipids in effect on the thrombin generation. [ABSTRACT FROM AUTHOR]

Patients with acute myeloid leukemia (AML) are at increased risk of thrombohemorrhagic complications. Overexpressed tissue factor (TF) on AML blasts contributes to systemic coagulation activation. We have recently shown that the heme enzyme myeloperoxidase (MPO) negatively regulates TF procoagulant activity (PCA) on myelomonocytic cells in vitro. We now aimed to further characterize the functional interaction of MPO and TF in AML in vivo. We prospectively recruited 66 patients with newly diagnosed AML. TF PCA of isolated peripheral blood mononuclear cells (PBMC) was assessed by single-stage clotting assay in the presence or absence of inhibitors against MPO catalytic activity (ABAH) or against MPO-binding integrins (anti-CD18). MPO in plasma and in AML blasts was measured by ELISA, and plasma D-dimers and prothrombin fragment F1+2 were quantified by automated immunoturbidimetric and chemiluminescence assays, respectively. Patients with AML had significantly higher MPO plasma levels compared to healthy controls and exhibited increased levels of D-dimers and F1+2. In vivo thrombin generation was mediated by TF PCA on circulating PBMC. Ex vivo incubation of isolated PBMC with ABAH or anti-CD18 antibody resulted in either increased or decreased TF PCA. The strong and robust correlation of F1+2 with TF PCA of circulating PBMC was abrogated at MPO plasma levels higher than 150 ng/mL, indicating a modulatory role for MPO on TF-mediated in vivo thrombin generation above this threshold. Our study indicates that catalytically active MPO released by circulating myeloblasts regulates TF-dependent coagulation in patients with newly diagnosed AML in a CD18-dependent manner. [ABSTRACT FROM AUTHOR]

HIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. In vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl 3 -injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of β3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block β3-integrin outside-in signaling. These results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting β3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs. [Display omitted] • Efficient medications with combination antiretroviral therapies (cARTs) make AIDS no longer an acute life-threaten for HIV-patients, but components of that can differently affect thrombotic cardiovascular outcomes • Potent and safe anti-platelet abilities uncover a pharmacological rationale for less cardiovascular implications in HIV-infected individuals receiving RPV • RPV is identified as a novel c-Src inhibitor to control β3 Integrin outside-in signaling pathway [ABSTRACT FROM AUTHOR]

The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbβ3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11 -mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients. [Display omitted] • The PTPN11 inhibitor SHP099 was used to study effects in platelets. • SHP099 increased collagen-induced thrombus build-up in whole blood microfluidics. • SHP099 enhanced platelet- and shear-dependent coagulation. • SHP099 restored thrombus formation in Noonan syndrome patients with mutated PTPN11. [ABSTRACT FROM AUTHOR]

Sepsis-induced hemostatic disturbances are common and are associated with poor outcomes. Additionally, conventional coagulation tests (CCTs) overdiagnose hypocoagulation and cannot detect hypercoagulation and hyperfibrinolysis. The aim of this study was to describe the coagulation profiles of patients with sepsis/septic shock using rotational thromboelastometry (ROTEM) and to compare coagulation states between sepsis and septic shock groups and between surviving and non-surviving groups. This prospective, observational, single-center study was conducted in the intensive care unit (ICU) of the University Medical Center Ho Chi Minh City, from 6/2020–12/2021. Patients aged ≥18 years with sepsis or septic shock according to the Sepsis-3 criteria were included. ROTEM and CCTs were concurrently performed within the first 24 h of ICU admission. In total, 161 patients were enrolled. Based on ROTEM, 72.7 % of patients with sepsis/septic shock had coagulation disorders, including 25.5 % hypercoagulation, 54.7 % hypocoagulation, 13.6 % mixed hypo-hypercoagulation patterns, and 18.6 % hyperfibrinolysis. A common mixed disorder subtype was characterized by prolonged initial clotting time (CT) with subsequently increased clot firmness. Fibrinogen levels and maximum clot formation (MCF)-fibtem were strongly correlated (rho = 0.73, p < 0.05). Hypocoagulation was observed more in the septic shock group than in the sepsis group. Compared to survivors, non-survivors had more prolonged CT-extem. ROTEM could identify hypocoagulability, hypercoagulability, mixed hypo-hypercoagulability patterns, and hyperfibrinolysis in patients with sepsis/septic shock. Elevated MCF-fibtem and elevated fibrinogen levels were notably common and strongly correlated. The septic shock group had more hypocoagulation than the sepsis group. Lastly, non-survivors had more prolonged CT-extem than survivors. • Rotational thromboelastometry (ROTEM) detected various types of coagulopathy in sepsis. • Typical patterns were hypocoagulability, hypercoagulability, and hyperfibrinolysis. • Mixed hypo-hypercoagulable subtypes were found in 13.7 % of the patients. • Increased fibrinogen levels and MCF-fibtem were common and strongly correlated. • Hypocoagulation was more observed in the septic shock group than in the sepsis group. [ABSTRACT FROM AUTHOR]

Cancer survivors are at an increased risk of thromboembolism compared to the general pediatric population. Anticoagulant therapy decreases the risk of thromboembolism in cancer patients. We hypothesized that pediatric cancer survivors are in a chronically hypercoagulable state compared to healthy controls. Children who survived for more than five years from cancer diagnosis at the UT Health Science Center at San Antonio Cancer Survivorship Clinic were compared to healthy controls. The exclusion criteria were recent NSAID use or a history of coagulopathy. Coagulation analysis included platelet count, thrombin-antithrombin complexes (TAT), plasminogen activator inhibitor (PAI), routine coagulation assays, and thrombin generation with and without thrombomodulin. We enrolled 47 pediatric cancer survivors and 37 healthy controls. Platelet count was significantly lower in cancer survivors at a mean of 254 × 109/L (95%CI: 234–273 × 109/L) compared at 307 × 109/L (283–331 × 109/L) in healthy controls (p < 0.001), although not outside the normal range. Routine coagulation assays showed no differences, except for a significantly lower prothrombin time (PT) in cancer survivors (p < 0.004). Cancer survivors has significantly elevated biomarkers of the procoagulant state, such as TAT and PAI, compared to healthy controls (p < 0.001). A multiple logistic regression model controlling for age, BMI, gender, and race/ethnicity documented that a low platelet count, short prothrombin clot time, and higher procoagulant biomarkers (TAT and PAI) were significantly associated with past cancer therapy. Survivors of childhood cancer have a persistent procoagulant imbalance for more than five years after diagnosis. Further studies are needed to establish whether procoagulant imbalance increases the risk of thromboembolism in childhood cancer survivors. • Anticoagulants decrease thrombotic events in adults receiving cancer therapy. • Survivors of childhood cancer have increased risk of a thromboembolic stroke. • Elevated coagulation protein levels are sensitive biomarkers of thrombosis risk. • Our study identified that childhood survivors have chronically elevated proteins. • These biomarkers could be used to design anticoagulant trials for survivors. [ABSTRACT FROM AUTHOR]

Elderly people without atrial fibrillation (AF) still have a high incidence of cardioembolic stroke, suggesting that thrombus formation within the left atrial appendage (LAA) may also occur in an AF-independent manner. In the present study, we explored the potential mechanisms for aging-induced LAA thrombus formation and stroke in mice. We monitored stroke events in 180 aging male mice (14–24 months) and assessed left atrium (LA) remodeling by echocardiography at different ages. Mice that had stroke were implanted with telemeters to confirm AF. Histological features of LA and LAA thrombi were examined, as well as collagen content, expression of matrix metalloproteinases (MMPs), and leukocyte density in the atria at different ages, in mice with or without stroke. Also, the effects of MMP inhibition on stroke incidence and atrial inflammation were tested. We detected 20 mice (11 %) with stroke, 60 % of which were within 18–19 months of age. Although we did not detect AF in mice with stroke, we detected the presence of LAA thrombi, suggesting that stroke originated from the hearts of these mice. Compared with 18-month-old mice without stroke, 18-month-old stroke mice had enlarged LA with a very thin endocardium, that was associated with less collagen and heightened MMP expression in the atria. During aging, we found that the expression of mRNAs for atrial MMP7, MMP8, and MMP9 peaked at 18 months, which closely correlated with reductions in collagen content and the time-window for cardioembolic stroke in these mice. Treatment of mice with an MMP inhibitor at 17–18 months of age reduced atrial inflammation and remodeling, and stroke incidence. Taken together, our study demonstrates that aging-induced LAA thrombus formation occurs through a mechanism involving upregulation of MMPs and breakdown of collagen, and that treatment with an MMP inhibitor may be effective as a treatment strategy for this heart condition. Possible mechanisms of left atrial appendage (LAA) thrombus formation in aging mice Normal mice (2-month-old) display intact and thick connective tissue in the atrial endocardium. During 18–19 months of age, increased infiltration of leukocytes into the mouse atria induces the up-regulation of matrix metalloproteinases (MMPs), leading to the reduction of collagen. LAA thrombus might start from the breakdown of endocardial fibril collagen by MMPs, which then triggers platelet adhesion and aggregation along the slit. An enlarged left atrium chamber may trigger blood clotting and the formation of an unstable mural thrombus in the early phase. After 19 months, reduced expression of MMPs causes collagen accumulation and atrial fibrosis. The remaining or recurrent thrombi are infiltrated by leukocytes and myofibroblasts at the late phase, and then covered by fibrotic tissue to form a stable, organized thrombus (A). Temporal changes of leukocytes, MMPs, and total collagen in the atria of aging mice (B). [Display omitted] • Cardioembolic strokes that occurred in aged mice are independent of atrial fibrillation. • Stroke mice had more dilated left atrium and heightened atrial inflammatory responses compared with mice of similar age but without stroke. • Elevated levels of MMPs and reductions in collagen content within the atria of mice at 18 months are associated with LAA thrombus formation and cardioembolic stroke. • Treatment with the MMP inhibitor XL-784 between 18 and 19 months of age repressed atrial dilatation as well as the incidence of stroke in aged mice. [ABSTRACT FROM AUTHOR]

Despite cardiovascular diseases and thrombosis being major causes of death in patients with chronic kidney disease (CKD), there remains no effective biomarker to predict thrombotic risk in this population. To evaluate global coagulation assays in patients with CKD and correlate the biomarkers to clinical outcomes. Patients with eGFR<30 mL/min/1.73m2 were recruited (n = 90) in this prospective observational study. Blood samples were collected for global coagulation assays, including thromboelastography, calibrated automated thrombogram (CAT), overall hemostatic potential (OHP) and tissue factor pathway inhibitor (TFPI). Following adjustment for age and gender, CKD subjects (mean age 66 years, 36 % female) had increased maximum amplitude on thromboelastography (70.1 vs 60.2 mm, p < 0.001), higher peak thrombin (233.2 vs 219.7 mm, p = 0.030) and increased OHP (16.1 vs 6.4 units, p < 0.001) compared to healthy controls (n = 153). TFPI was also increased in CKD patients (36.4 vs 14.5 ng/mL, p < 0.001). Compared to hemodialysis patients (n = 43), peritoneal-dialysis patients (n = 25) had more hypercoagulable parameters. Thirty-five CKD patients reported thrombotic complications – key predictors included dialysis, higher fibrinogen, reduced endogenous thrombin potential, elevated D-dimer and increased TFPI. Using the dialysis cohort, the predictive risk model based on the key predictors performed better than Framingham heart score and number of cardiovascular risk factors (Harrell's C-stat 0.862 vs 0.585 vs 0.565). CKD appears to confer a hypercoagulable state compared to healthy controls. Interestingly, reduced thrombin generation and raised TFPI was paradoxically associated with increased thrombotic risks, highlighting possible complex compensatory mechanisms within the coagulation system, which may be important in predicting clinical outcomes. • There is no effective risk assessment model for thrombosis risk for CKD patients. • CKD patients have more hypercoagulable global coagulation assays than controls. • Peritoneal dialysis patients appear to have the most hypercoagulable parameters. • Lower thrombin generation is paradoxically associated with thrombotic outcomes. [ABSTRACT FROM AUTHOR]

Studies exploring alterations in blood coagulation and platelet activation induced by COVID-19 vaccines are not concordant. We aimed to assess the impact of four COVID-19 vaccines on platelet activation, coagulation, and inflammation considering also the immunization dose and the history of SARS-CoV-2 infection. TREASURE study enrolled 368 consecutive subjects (161 receiving viral vector vaccines -ChAdOx1-S/Vaxzevria or Janssen- and 207 receiving mRNA vaccines -Comirnaty/Pfizer-BioNTech or Spikevax/Moderna). Blood was collected the day before and 8 ± 2 days after the vaccination. Platelet activation markers (P-selectin, aGPIIbIIIa and Tissue Factor expression; number of platelet-monocyte and -granulocyte aggregates) and microvesicle release were analyzed by flow cytometry. Platelet thrombin generation (TG) capacity was measured using the Calibrated Automated Thrombogram. Plasma coagulation and inflammation markers and immune response were evaluated by ELISA. Vaccination did not induce platelet activation and microvesicle release. IL-6 and CRP levels (+30%), D-dimer, fibrinogen and F 1 +2 (+13%, +3.7%, +4.3%, respectively) but not TAT levels significantly increased upon immunization with all four vaccines, with no difference among them and between first and second dose. An overall minor post-vaccination reduction of aPC, TM and TFPI, all possibly related to endothelial function, was observed. No anti-PF4 seroconversion was observed. This study showed that the four COVID-19 vaccines administered to a large population sample induce a transient inflammatory response, with no onset of platelet activation. The minor changes in clotting activation and endothelial function might be potentially involved at a population level in explaining the very rare venous thromboembolic complications of COVID-19 vaccination. [ABSTRACT FROM AUTHOR]

Oxidative stress derived from severe systemic inflammation promotes conversion from high-density lipoprotein HDL to oxidized HDL (oxHDL), which interacts with vascular endothelial cells (ECs). OxHDL acquires procoagulant features playing a role in modulating coagulation, which has been linked with organ failure in ICU patients. However, whether oxHDL elicits a ECs-mediated procoagulant phenotype generating organ failure and death, and the underlying molecular mechanism is not known. Therefore, we studied whether oxHDL-treated rats and high-oxHDL ICU patients exhibit a procoagulant phenotype and its association with kidney injury and mortality and the endothelial underlying molecular mechanism. Human ECs, oxHDL-treated rats and ICU patients were subjected to several cellular and molecular studies, coagulation analyses, kidney injury assessment and mortality determination. OxHDL-treated ECs showed a procoagulant protein expression reprograming characterized by increased E-/P-selectin and vWF mRNA expression through specific signaling pathways. OxHDL-treated rats exhibited a procoagulant phenotype and modified E-/P-selectin, vWF, TF and t-PA mRNA expression correlating with plasma TF, t-PA and D-dimer. Also, showed increased death events and the relative risk of death, and increased creatinine, urea, BUN/creatinine ratio, KIM-1, NGAL, β2M, and decreased eGFR, all concordant with kidney injury, correlated with plasma TF, t-PA and D-dimer. ICU patients showed correlation between plasma oxHDL and increased creatinine, cystatin, BUN, BUN/creatinine ratio, KIM-1, NGAL, β2M, and decreased GFR. Notably, ICU high-oxHDL patients showed decreased survival. Interestingly, altered coagulation factors TF, t-PA and D-dimer correlated with both increased oxHDL levels and kidney injury markers, indicating a connection between these factors. Increased circulating oxHDL generates an endothelial-dependent procoagulant phenotype that associates with acute kidney injury and increased risk of death. [ABSTRACT FROM AUTHOR]

Local coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and thrombin play a role in tumor progression by activating cellular receptors and local formation of fibrin. However, whether venous thromboembolism (VTE) or a hypercoagulable state has a direct impact on cancer progression is unknown. Here we have combined an orthotopic murine breast cancer model, using female Nod-SCID mice, with siRNA-mediated silencing of antithrombin (siAT) leading to the induction of a systemic hypercoagulable state. We show that, compared to control siRNA-treated (not experiencing a hypercoagulable state) tumor-bearing mice, siAT treated tumor-bearing mice do not show enhanced tumor growth nor enhanced metastasis. We conclude that, in this murine model for hypercoagulability, induction of a hypercoagulable state does not contribute to breast cancer progression. • Cancer results in increased risk for venous thrombosis • Effect of venous thrombosis on cancer progression remains unclear • In mice, induction of a hypercoagulable state did not influence breast cancer progression [ABSTRACT FROM AUTHOR]

The balance between thrombosis and hemostasis is a difficult issue during extracorporeal membrane oxygenation (ECMO) support. The pathogenesis leading to thrombotic complications during ECMO support is not well understood. Neutrophil extracellular traps (NETs) were reported to participate in thrombosis and have a key role in inflammation. This study aimed to explore the role of NETs in thrombosis during ECMO support and investigate NETs as a predictive biomarker for thrombotic complications during ECMO assistance. Ten ovine models of ECMO support were established. Animals were then randomly divided into 2 groups (5 sheep/group): venoarterial (VA) ECMO group and venovenous (VV) ECMO group. The venous blood samples were collected at different time points. Markers of NETs were detected in plasma, neutrophils, and thrombi from the vessels and membrane. Moreover, circulating NETs levels in 8 adults treated in the intensive care unit (ICU) who received VA-ECMO and 8 healthy controls were detected; patient survival was also recorded. In vivo study showed that neutrophils and NETs markers (dsDNA and citH3) levels were significantly elevated 6 h after ECMO compared to baseline. Isolated neutrophils from fresh blood at 6 h could release more NETs. dsDNA and citH3 levels were significantly higher in the VA mode than in the VV mode. NETs were found in thrombi from the vessel and membrane. Clinical data further revealed that dsDNA, citH3, and nucleosomes were higher in patients who received ECMO than in healthy controls. These data suggest NETs might be associated with thrombus during ECMO support, especially in the VA mode. These findings provide new insight into preventing thrombotic complications by targeting NETs. Also, NETs may potentially become an early warning biomarker for thrombosis under ECMO assistance. [ABSTRACT FROM AUTHOR]

Coagulation activation on medical devices remains a significant problem as it can lead to dramatic thromboembolic complications. Understanding its poorly described mechanisms and finding optimal pharmacological prevention means is crucial to improve patient safety. We developed an in vitro model to study thrombin generation (TG) initiated by the contact of plasma with the surface of catheters. Interventional cardiology catheters were cut into segments and inserted in the bottom of multi-well plates; TG was then measured with the calibrated automated thrombogram (CAT). Model performance (analytical, intra- and inter-individual variability) was investigated and compared with activation of thrombin generation by tissue factor (TF) or contact pathway activator (ellagic acid), in the presence (PRP) and absence (PPP) of platelets. Model response to unfractionated heparin (UFH) was also assessed. TG was greater when measured in presence of catheter segments, compared to conditions without activators. The analytical variability of the model was good (CV ≤ 5 %), both with PPP and PRP. Intra-individual variability was between 15 and 30 % with PPP and between 10 and 15 % with PRP. Inter-individual variability was between 15 and 30 % with both kinds of plasma samples. The analytical performance of the catheter-initiated TG model was equivalent to that observed when TG was initiated with TF or ellagic acid. Catheter-initiated TG was measurable until 0.1 IU/mL UFH with PPP and until 1.0 IU/mL UFH with PRP, highlighting the crucial requirement of platelets. Our model is suitable for studying TG initiated with catheters. Inhibition of TG by UFH is overestimated in the absence of platelets. • Foreign material, including catheters, strongly activates hemostasis. • Platelets are essential to assess the effect of heparin on thrombin generation (TG). • We developed a model to study TG at the surface of catheters in presence of platelets. • The model showed low analytical, intra- and inter-individual variability. • This model will enable to study pharmacological inhibition of TG at foreign surfaces. [ABSTRACT FROM AUTHOR]

Heparin-binding protein (HBP) levels have been linked to organ failure and may represent an inflammatory biomarker of sepsis. We found disseminated intravascular coagulation (DIC) is associated with higher HBP levels in patients and in in vivo and in vitro models. This prospective, single-center observational study investigated the effects and underlying mechanisms of HBP on the coagulation cascade in sepsis. 538 patients with sepsis from June 2016 to December 2019 were enrolled. Mechanisms underlying HBP and the coagulation system were investigated in human umbilical vein endothelial cells (HUVEC) and C57 mice. Increased HBP was associated with sepsis-induced DIC. The optimal cutoff value was 37.5 ng/mL (sensitivity: 56 %, specificity: 65 %). Antithrombin-III (AT-III) activity, plasmin-a2 plasmin inhibitor complex (PIC), procalcitonin (PCT), hemoglobin, and HBP ≥37.5 ng/mL were associated with of DIC occurrence. In HUVECs &C57 mice models, Western blotting, qPCR, and immunohistochemistry analysis showed that the binding between HBP and TGF-β receptor 2 (TGFBR2) caused elevation of plasminogen activator inhibitor-1 (PAI-1) levels. Furthermore, we found that mice stimulated with HBP had higher levels of fibrinogen and D-dimer in the blood. HBP treatment caused the accumulation of fibrinogen in mice lung tissue. Treatment with TGFBR2-small interfering RNAs inhibited the effects. Patients with sepsis having HBP ≥37.5 ng/mL at admission were more likely to develop DIC. HBP upregulates the expression of fibrinogen and PAI-1 via TGFBR2 and the TGF-β signalling pathway. [Display omitted] • Septic patients with high heparin-binding protein (HBP) levels were likely to develop disseminated intravascular coagulation. • HBP activates TGF-β-PAI-1 signalling pathway in DIC model. • HBP as an inflammatory factor activates coagulation system. • The Early Warning Effect of HBP on the Diagnosis of DIC. [ABSTRACT FROM AUTHOR]

• Preanalytical activation of the contact system may interfere with coagulation assays • Different sample tubes may activate the contact system differently • Activation can be quantified by Cleaved H-Kininogen and Fast form α 2 − Macroglobulin • Activation was much higher in Vacutainer tubes than in Monovette tubes • Tubes with a low contact activation are preferable for some coagulation assays [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase (PDI) contributes to tissue factor (TF) regulation in monocytes. While bacitracin and quercetin-3-rutinoside mitigate myeloid TF production, the effect of PACMA-31, a more specific PDI inhibitor with distinct pharmacologic properties, remains unclear. Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMCs) or citrate-anticoagulated whole blood was carried out in the presence of PACMA-31 or DMSO vehicle before monocytes were analyzed for TF expression, including antigen, procoagulant activity (PCA) and mRNA, release of IL-6 and TNFα, and LPS-induced signaling pathways. While PACMA-31 alone had no effect, coincubation with LPS and PACMA-31 (25 μM) enhanced LPS-induced monocyte TF production in whole blood. The effect was at least partially regulated on the transcriptional level and could not be explained by increased phosphatidylserine membrane exposure. In contrast, the same PACMA-31 concentrations were cytotoxic in isolated PBMCs. A lower dose of PACMA-31, however, restored the stimulating effect by enhancing IκB-NFκB signaling that also increased the release of IL-6 and TNFα. The protease-activated receptor 2 (PAR2) inhibitor ENMD 547 but not TF antibody 10H10 or factor Xa inhibitor rivaroxaban prevented the stimulatory effect of PACMA-31 on inflammatory monocytes. In sharp contrast, short time incubation of LPS-stimulated PBMCs with 25 μM PACMA-31 was non-cytotoxic and significantly inhibited cellular TF PCA but not surface antigen expression. PACMA-31 regulates monocyte TF in a concentration-dependent manner by opposing transcriptional and posttranscriptional mechanisms. While low concentrations of PACMA-31 augment monocyte TF production by amplifying LPS-dependent PAR2 signaling, high concentrations convert monocyte TF into its non-coagulant state. Proposed mechanisms of PACMA - 31 - mediated regulation of LPS - induced monocyte TF production LPS stimulation induces TF production by monocytes in an NFκB- and PAR2-dependent manner (left upper panel). Co-stimulation with LPS and PACMA-31 at low, non-cytotoxic concentrations amplifies LPS-induced monocyte TF production through a PAR2-dependent signaling pathway (right upper panel). In contrast, high concentrations of PACMA-31 convert surface-located TF into its cryptic, non-coagulant form and downregulate LPS-induced TF production through cytotoxic effects (left lower panel). [Display omitted] • Protein disulfide isomerase (PDI) contributes to TF regulation in monocytes. • Propynoic acid carbamoyl methyl-amide-31 (PACMA) is a potent PDI inhibitor. • PACMA regulates monocyte TF by transcriptional and posttranscriptional mechanisms. • PACMA can convert preformed procoagulant TF into its cryptic state. • PACMA amplifies LPS-induced monocyte TF production in a PAR2-dependent manner. [ABSTRACT FROM AUTHOR]

Untreated menopause may have serious health implications, but treatments can have dangerous side effects. We evaluate menopausal symptoms as well as available treatments -the routes of administration and their effect on blood coagulation. Menopausal females may experience hot flushes, vulva- and vaginal atrophy and osteoporosis. Many treatments are available to relieve these symptoms such as Conjugated Equine Estrogen and bioidentical hormones. The routes of administration include oral and transdermal. Hormones that are administered orally undergo a hepatic first pass metabolism. The by-products have a lower efficacy and possibly enhanced side effects. Furthermore, hormone treatments influence the coagulation cascade through coagulation factors or their regulators. Increased coagulation poses a risk for venous thromboembolism. Currently a definite conclusion on whether the side effects from hormone treatments exceed the risk of untreated menopause cannot be made. However, a more individualised approach to hormone treatments may be the most feasible solution to this dilemma. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) in pregnant women is characterized by immune activation and inflammation despite suppressive antiretroviral therapy (ART). The extent to which ongoing inflammation contributes to activation of coagulation and fibrinolysis is unknown. This cross-sectional study included pregnant women in the following three groups: HIV negative (n = 109), HIV infected virologically suppressed (n = 109) and HIV infected with HIV viral load (VL) of >50 copies/mL (n = 80). Fibrinolytic activity was evaluated by measuring d-dimer and plasminogen activator inhibitor-1 (PAI-1) as well as thrombin-antithrombin (TAT) complex concentrations, as an index of coagulation, in the first, second and third trimesters. In this population, with a mean age of 33 ± 6 years, pregnancy outcomes were recorded for 277 (93.0 %) participants with live births. HIV infected participants with virological suppression and VL of >50 copies/mL showed significantly increasing levels of d-dimer and PAI-1 in the first, second and third trimesters, as compared to HIV negative participants. No significant differences were observed between HIV infected participants with virological suppression and HIV infected participants with VL > 50 copies/mL for levels of first and third trimester d-dimer and PAI-1 in each trimester. In addition, TAT complex levels in the first trimester were significantly increased in HIV infected virologically suppressed participants as compared to HIV negative participants. HIV infected virologically suppressed pregnant women show evidence of persistently impaired markers of fibrinolysis. Future research should explore the risk of adverse pregnancy complications among HIV infected pregnant women in the modern era of ART. [ABSTRACT FROM AUTHOR]

Acute bleeding leads to significant morbidity and mortality. Recombinant wildtype Factor VIIa (WT FVIIa) had been reported to have some therapeutic effects in some clinical trials, however, its use was associated with thromboembolic events. We sought to develop a novel FVIIa molecule (CT-001) with enhanced activity and lowered thrombogenicity risk. CT-001 has 4 N-glycans (T106N/N145/V253N/N322) with terminal sialic acid residues removed to promote active clearance via the asialoglycoprotein receptor, and P10Q/K32E substitutions introduced to its gamma-carboxyglutamic acid (Gla) domain for enhanced phospholipid affinity and activity. In mice, CT-001 had a half-life of 5 min and a clearance of 467 mL/h/kg at 3 mg/kg, significantly faster than WT FVIIa (t 1/2 = 1.8 h, Cl = 39 mL/h/kg). Interestingly, CT-001 was efficacious in reducing blood loss even with its rapid clearance. In a severe hemorrhage mouse model with tail amputated 5 cm from the tip, 1 mg/kg CT-001 provided efficacy comparable to 3 mg/kg WT FVIIa. The fast clearance of CT-001 resulted in significantly reduced thrombogenicity in comparison to WT FVIIa in a FeCl 3 -induced carotid artery thrombosis mouse model, and further confirmed in a soluble tissue factor-induced thrombosis model. The data on CT-001 demonstrate that a short duration of highly active FVIIa procoagulant activity has the potential to be an optimal paradigm for the treatment of acute bleeds. [ABSTRACT FROM AUTHOR]

Hutchinson-Gilford Progeria Syndrome (HGPS) is an ultra-rare premature aging genetic disorder caused by a point mutation in the lamin A gene, LMNA. Children with HGPS display short lifespans and typically die due to myocardial infarction or ischemic stroke, both acute cardiovascular events that are tightly linked to arterial thrombosis. Despite this fact, the effect of the classic HGPS LMNA gene mutation on arterial thrombosis remains unknown. Heterozygous Lmna G609G knock-in (Lmna G609G/+ ) mice, yielding an equivalent classic mutation observed in HGPS patients (c.1824C>T; pG608G mutation in the human LMNA gene) and corresponding wild-type (WT) control littermates underwent photochemically laser-induced carotid injury to trigger thrombosis. Coagulation and fibrinolytic factors were measured. Furthermore, platelet activation and reactivity were investigated. Lmna G609G/+ mice displayed accelerated arterial thrombus formation, as underlined by shortened time to occlusion compared to WT littermates. Levels of factors involved in the coagulation and fibrinolytic system were comparable between groups, while Lmna G609G/+ animals showed higher plasma levels of thrombin-antithrombin complex and lower levels of antithrombin. Bone marrow analysis showed larger megakaryocytes in progeric mice. Lastly, enhanced platelet activation upon adenosine diphosphate, collagen-related peptide, and thrombin stimulation was observed in Lmna G609G/+ animals compared to the WT group, indicating a higher platelet reactivity in progeric animals. LMNA mutation in HGPS mice accelerates arterial thrombus formation, which is mediated, at least in part, by enhanced platelet reactivity, which consequently augments thrombin generation. Given the wide spectrum of antiplatelet agents available clinically, further investigation is warranted to consider the most suitable antiplatelet regimen for children with HGPS to mitigate disease mortality and morbidity. • Hutchinson-Gilford progeria syndrome (HGPS) mouse model displays accelerated arterial thrombus formation. • Extrinsic coagulation and fibrinolytic pathways are not affected by the HGPS classic LMNA mutation. • Platelets isolated from HGPS animals show intensified reactivity upon stimulation. • HGPS animals display increased levels of circulating thrombin-antithrombin complex. [ABSTRACT FROM AUTHOR]

Plasma proteins involved in coagulation and fibrinolysis are essential to hemostasis. Consequently, their circulating levels and functionality are critical in bleeding and thrombosis development. Well-established laboratory tests to assess these are available; however, said tests do not allow high multiplicity, require large volumes of plasma and are often costly. A novel technology to quantify plasma proteins is quantitative protein mass spectrometry (QPMS). Aided by stable isotope-labeled internal standards a large number of proteins can be quantified in one single analytical run requiring <30 μL of plasma. This provides an opportunity to improve insight in the etiology and prognosis of bleeding and thrombotic disorders, in which the balance between different proteins plays a crucial role. This manuscript aims to give an overview of the QPMS potential applications in thrombosis and hemostasis research (quantifying the 38 proteins assigned to coagulation and fibrinolysis by the KEGG database), but also to explore the potential and hurdles if designed for clinical practice. Advantages and limitations of QPMS are described and strategies for improved analysis are proposed, using as an example the test requirements for antithrombin. Application of this technology in the future could represent a step towards individualized patient care. • Well-established laboratory tests are available for coagulation and fibrinolysis. • Unmet clinical needs for test development exist for undiagnosed bleeding disorders and venous thrombosis risk stratification. • Quantitative targeted proteomics (QPMS) might address these needs through multiplex proteins quantitation. • Application of QPMS in the future could represent a step towards individualized patient care. [ABSTRACT FROM AUTHOR]

Dysregulated host response to infection causes life-threatening organ dysfunction. Excessive inflammation and abnormal blood coagulation can lead to disseminated intravascular coagulation (DIC) and multiple-organ failure in the late sepsis stages. Platelet function impairment in sepsis contributes to bleeding, secondary infection, and tissue injury. Platelet transfusion is considered in patients with sepsis with DIC and bleeding; however, its benefits are limited and of low quality. Fibrinogen plays a crucial role in platelet function, and establishing a fibrin network binds to activated integrin αIIbβ3 and promotes outside-in signaling that amplifies platelet functions. However, the role of fibrinogen in sepsis-induced platelet dysfunction remains unclear. We evaluated the effects of fibrinogen on platelet hyporeactivity during septic shock in adult male Wistar rats using lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP) surgery. Changes in the hemodynamic, biochemical, and coagulation parameters were examined. Platelet activation and aggregation were measured using whole-blood assay, 96-well plate-based aggregometry, and light-transmission aggregometry. Additionally, platelet adhesion, spreading, and fibrin clot retraction were evaluated. Rats with LPS- and CLP-induced sepsis displayed considerable decreases in plasma fibrinogen levels and platelet aggregation, adhesion, spreading, and clot retraction. The aggregation of platelets obtained from rats with sepsis was markedly augmented by fibrinogen supplementation. Additionally, fibrinogen administration improved platelet adhesion, spreading, and clot retraction in rats with sepsis. Fibrinogen supplementation could serve as a potential therapeutic intervention for alleviating platelet hyporeactivity in patients with sepsis and bleeding. [Display omitted] • Septic rats revealed significant decreases in plasma fibrinogen levels and platelet reactivity to endogenous agonists. • Fibrinogen improved impairment of platelet aggregation, adhesion, spreading, and clot retraction in rats with sepsis. • Fibrinogen supplementation could be a potential therapeutic intervention for alleviating platelet hyporeactivity in sepsis. [ABSTRACT FROM AUTHOR]

Protein C (PC), a vitamin K–dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE. • The hereditary deficency of PC caused by mutations in PROC gene is mainly associated with an increased risk of venous thrombosis. • This study provides insight into the function of Protein C mutation *462Qext*17 and its contribution to thrombosis development in patients. • *462Qext*17 variant significantly impairs PC activation and its anticoagulant function, constituting a genetic risk factor for venous thrombosis. [ABSTRACT FROM AUTHOR]

Excessive activation of the coagulation cascades leads to life-threatening disseminated intravascular coagulation (DIC) in sepsis. Two recent studies by our group and others have both demonstrated the noncanonical inflammasome is pivotal for the endotoxin or gram-negative bacterial-induced coagulation. Based on this, we further evaluated the function of the NLRP3 inflammasome, the most studied inflammasome, in endotoxin-induced coagulation. We established an endotoxin-induced coagulation model by intraperitoneal injection of sublethal doses of LPS in mice. Mice were sacrificed 8 h after injection and blood was collected for thrombin-antithrombin (TAT), plasminogen activator inhibitor-1 (PAI-1), prothrombin time (PT), D-dimer, IL-1β and tissue factor (TF) measurements by commercial ELISA. Lungs and livers were examined via HE staining images to determine injury scores and immunohistochemistry for TF expression and fibrin deposits. The role of NLRP3 activation was evaluated in wild-type (WT), Nlrp3−/−, Asc−/− (apoptosis-associated speck-like protein containing a CARD), Caspase-11−/− mice and 30 min after treatment with MCC950, a potent inhibitor of NLRP3. Western blotting and Q-PCR were performed to assess TF expression in the lungs and livers. To uncover the different effects of NLRP3 and Caspase-11, we also compared the time-dependent IL-1β release in LPS-treated Nlrp3−/− and Caspase-11−/− mice. Correlation analysis of TAT, PAI-1 were estimated the relationship of coagulation and release of IL-1β, as well as IL-1β and TF. Inhibition of NLRP3 by MCC950 as well as NLRP3 or ASC deficiency decreased TAT, PAI-1, PT, D-dimer, and TF levels in blood and impaired the thrombus formation and fibrin deposition, as well as declined expression of TF in the liver and lung in endotoxin-induced coagulation but not caspase-11 deficiency. Impressively, IL-1β release is increased in LPS-treated Caspase-11−/− mice, but not in Nlrp3−/− mice. Moreover, the correlation analysis is indicated that downstream of the NLRP3 inflammasome, IL-1β expression, is positively correlated with TAT, PAI-1 and TF in blood circulation. The NLRP3 inflammasome contributes to endotoxin-induced coagulation by promoting TF expression at least in part through the induction of IL-1β release. These findings broadened our understanding of the mechanism of coagulation and implicated a possible therapeutic strategy for preventing coagulation in sepsis. [ABSTRACT FROM AUTHOR]

Extracorporeal membrane oxygenation (ECMO) can be life-saving, but suffers from thrombus formation in the circuit with associated risks of oxygenator occlusion, hemolysis and arterial embolism. The formation of thrombin is the key step to thrombus formation and two factors are needed for sustained thrombin generation, a coagulation activator to initiate the process and a procoagulant phospholipid surface for the coagulation system to assemble on. The purpose of this study was to use thrombin generation potential (TGP) and other assays to determine the specific coagulation activators and sources of procoagulant phospholipid that are present in ECMO patient plasma. Samples were collected from 60 patients on ECMO (age 1d-19y) followed evaluation of native and stimulated TGP, measurement of factor II levels and determination of procoagulant extracellular vesicle levels by flow cytometry. During ECMO, native (unstimulated) TGP was increased, followed by a decrease back towards normal after ECMO ended. The main activator of TGP in ECMO plasma was increased FXIa (100% of samples tested), while increased tissue factor activity was present in 7%. Procoagulant phospholipids were present in plasma from ECMO patients in the form of circulating platelet and red cell extracellular vesicles, which were increased 2 to 7-fold compared to normal. Procoagulant extracellular vesicle levels correlated with increased plasma native TGP activity. ECMO activates coagulation in plasma primarily through activation of the contact system and formation of activated factor XIa and generation of circulating procoagulant extracellular vesicles through platelet and red cell activation. • In pediatric patients, ECMO stimulates increased thrombin generation potential. • ECMO activates the contact system generating activated factor IXa. • ECMO increases procoagulant phospholipids through extracellular vesicle release. • Maquet circuits showed higher TGP and platelet activation than Cardiohelp circuits. [ABSTRACT FROM AUTHOR]

No reference values are currently available for coagulation assays performed for thrombophilia screening prescribed according to guidelines, after a first venous thromboembolic (VTE) event, and we have no idea of the intra-patient associations between results. We performed a retrospective study of consecutive prescriptions fulfilling guidelines in a French university hospital from 2010 to 2019 (n = 3842) from the Glims® laboratory information system. We collected results of 12 parameters: aPTT, PT, fibrinogen (Fg), one-stage clotting methods for factors VIII, IX, XI and II (FVIII, FIX, FXI, FII), antithrombin (using an amidolytic assay: AT), protein C and S (using clotting assays: PC and PS) and mixing tests of a lupus-anticoagulant sensitive aPTT and of DRVVT. We show the results of the 12 parameters from 3603 individual files with less than 6 missing values, then describe these distributions and correlations between results from 2930 files with no missing value. We give the frequency of results described as indicating a risk of first VTE or of VTE recurrence. We propose 2 quantitative scores linking the 12 parameters at the individual level and reflecting their degree of dispersion with respect to their mean, describe the values of these scores and their associations with thrombophilic results. These normal values should help laboratory workers to validate process results and to assess their degree of originality. Our 2 scores should help to determine the intra-patient plausibility of associations of results. The usefulness of these laboratory scores for predicting clinically-relevant outcomes deserves to be investigated. [ABSTRACT FROM AUTHOR]

Fertility treatment with frozen thawed embryo transfer (FET) is widely used. Women treated in artificial cycles (AC-FET) receive high doses of estrogen in contrast to natural cycles (NC-FET), where no estrogen is administered. Estrogen substitution may be associated with increased risk of thromboembolism. Our aim is therefore to characterize changes in blood coagulation parameters defined as surrogate thrombotic risk markers in women undergoing estrogen substitution during AC-FET. In our prospective cohort study, we enrolled 34 women in either: AC-FET (n = 19) or NC-FET (n = 15). Women were recruited at the Department of Obstetrics and Gynaecology, Horsens Fertility Clinic, Denmark, from August 2019 – November 2020. Blood samples were obtained at four timepoints. Thrombin generation, platelet aggregation and fibrinolysis were evaluated as thrombotic risk markers. Within the AC-FET group, we found a significantly shorter lagtime (p < 0.05) and time to peak (TTP) (p < 0.001) after hormone substitution compared to baseline. Furthermore, a significantly higher mean peak (p < 0.0001) and larger endogenous thrombin potential (ETP) (p < 0.0001) was observed. When compared to the NC-FET group, women receiving AC-FET had a significantly shorter mean TTP (p < 0.005), higher mean peak (p < 0.0001) and larger ETP (p < 0.05). Additionally, we demonstrated a significantly prolonged lysis time within the AC-FET group (p < 0.001). Our results indicate that women receiving AC-FET have a significantly increased thrombin generation which may increase the thromboembolic risk in women being estrogen substituted. • Frozen embryo transfer is often proceeded by high doses of estrogen. • Estrogen stimulation in frozen embryo transfers activate thrombotic risk markers. • Special attention should be given to women with an increased thromboembolic risk. • A restriction in the use of unnecessary hormone exposure is important. [ABSTRACT FROM AUTHOR]

Endothelial damage and thrombosis caused by COVID-19 may imperil cardiovascular health. More than a year since the WHO declared COVID-19 pandemic, information on its effects beyond the acute phase is lacking. We investigate endothelial dysfunction, coagulation and inflammation, 3 months post-COVID-19. A cohort study was conducted including 203 patients with prior COVID-19. Macrovascular dysfunction was assessed by measuring the carotid artery diameter in response to hand immersion in ice-water. A historic cohort of 312 subjects served as controls. Propensity score matching corrected for baseline differences. Plasma concentrations of endothelin-1 were measured in patients post-COVID-19, during the acute phase, and in matched controls. Coagulation enzyme:inhibitor complexes and inflammatory cytokines were studied. The prevalence of macrovascular dysfunction did not differ between the COVID-19 (18.6%) and the historic cohort (22.5%, RD −4%, 95%CI: −15–7, p = 0.49). Endothelin-1 levels were significantly higher in acute COVID-19 (1.67 ± 0.64 pg/mL) as compared to controls (1.24 ± 0.37, p < 0.001), and further elevated 3 months post-COVID-19 (2.74 ± 1.81, p < 0.001). Thrombin:antithrombin(AT) was high in 48.3%. Markers of contact activation were increased in 16–30%. FVIIa:AT (35%) and Von Willebrand Factor:antigen (80.8%) were elevated. Inflammatory cytokine levels were high in a majority: interleukin(IL)-18 (73.9%), IL-6 (47.7%), and IL-1ra (48.9%). At 3 months after acute COVID-19 there was no indication of macrovascular dysfunction; there was evidence, however, of sustained endothelial cell involvement, coagulation activity and inflammation. Our data highlight the importance of further studies on SARS-CoV-2 related vascular inflammation and thrombosis, as well as longer follow-up in recovered patients. • No indication of macrovascular dysfunction 3 months after acute COVID-19 • Elevated ET-1 levels during acute COVID-19, and further elevated after 3 months • Increased coagulation activity & high inflammatory cytokines 3 months post-COVID-19 [ABSTRACT FROM AUTHOR]

Background: Intracerebral hemorrhage (ICH) of undetermined etiology occurs infrequently in young and middle-aged adults. We hypothesized that slight decreases in coagulation factors and formation of less compact fibrin clots prone to faster lysis predispose to this type of ICH., Methods: We recruited 44 consecutive patients aged <50 years following ICH of unknown cause at least 3 months since the event. Subjects free of ICH (n = 47) matched for age, sex, BMI, and hypertension served as the control group. We assessed plasma fibrin clot permeability, turbidity and fibrinolytic capacity, along with thrombin generation, coagulation factors (F) II, FV, FVII, FVIII, FIX, FX, FXI, antithrombin, and fibrinolysis proteins., Results: ICH patients (median age 41 years, 45.5 % women) had 8.4 % lower FII (p = 0.0001) and 10.1 % lower FVII activity (p = 0.0003), 9.4 % higher antithrombin activity (p = 0.0004) and 13.5 % lower platelet count (p = 0.02). Other factors and thrombin generation did not differ between the two groups. The ICH survivors were characterized by impaired fibrin polymerization reflected by 10.1 % longer lag phase of the turbidimetry curve (p = 0.0002), decreased fiber density indicated by 11.8 % lower maximum absorbance (p = 0.004), as well as 11.1 % shorter clot lysis time (p = 0.014) and 10.0 % faster increase of maximal D-Dimer levels (p = 0.000001)., Conclusions: We demonstrated a prohemorrhagic fibrin clot phenotype, along with lower FII, FVII and higher antithrombin activity in adults below 50 years of age who suffered from ICH of unknown cause, which might indicate novel mechanisms contributing to ICH in younger individuals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Human body functions exhibit a circadian rhythm generated in peripheral cells and synchronized by the suprachiasmatic nucleus (SCN), which mostly is entrained by the daily light/dark cycles. Activity, meals and posture are capable of interfering with the endogenous circadian rhythm of coagulation parameters. An increasing number of human disorders show a circadian component, and epidemiological studies find cardiovascular events to peak in the morning hours. The aim was to review the circadian rhythms impact on fibrinolysis and coagulation in healthy individuals and cardiovascular patients. A total number of 25 studies were identified where 8 enrolled cardiovascular patients with or without healthy individuals. Using a MeSH-search in MEDLINE PubMed. Only original peer-reviewed papers were included. Results showed substantial variance with respect to exhibition of circadian rhythms and/or peak/trough times. Circadian rhythms of fibrinolysis were less pronounced in cardiovascular patients than in healthy individuals with decreased levels in the morning hours compared to healthy inducing higher risk of blood clotting. Because of small studied group sizes and failure to control for entraining factors, larger studies are needed to fully establish the effects of the circadian rhythm on especially coagulation. The findings of chronobiologic rhythms in coagulation and fibrinolysis could suggest a need for a chrono-pharmacological approach when treating/preventing cardiovascular diseases. • Risk of cardiovascular events peak in the morning pointing to a circadian factor • Diurnal thrombosis appears to be disturbed in cardiovascular patients. • Trend toward lower fibrinolysis in the morning hours in cardiovascular patients • Antithrombotic medication in the evening may be the best protective therapy [ABSTRACT FROM AUTHOR]

Chronic liver disease (CLD) is characterized by changes in haemostasis, embracing both hypo- and hypercoagulability. Global hemostatic tests such as thrombin generation assays evaluate the hemostatic balance, to better assess bleeding and thrombotic risks. In addition, procoagulant state in patients with CLD has been demonstrated using modified thrombin generation assays with thrombomodulin, a cofactor for protein C activation. In this study, we prospectively determined thrombin generation and thrombomodulin resistance in patients with CLD staged with liver stiffness measurement (LSM), using both the fully automated analyzer ST Genesia® Thrombin Generation System (STG) and the calibrated automated thrombogram assay (CAT). Demographic, clinical and laboratory characteristics, and blood samples were collected from 65 patients with CLD. Liver stiffness was measured by transient elastography, and thrombin generation and thrombomodulin resistance, by STG and CAT. Patients were separated based on LSM of <21 and ≥21 kilopascals (kPa). The propagation rate of thrombin generation was higher in patients with LSM ≥21 kPa and the thrombin generation rate increased as LSM increased. In addition, thrombomodulin resistance assessed by STG and CAT was higher in patients with LSM ≥21 kPa. However, ETP inhibition by activated protein C was comparable in patients with LSM <21 and ≥21 kPa. Finally, LSM correlated with most thrombin generation parameters. The STG automated system may have value in the assessment of patients with chronic liver disease in the routine coagulation laboratory. LSM ≥21 kPa identify a procoagulant phenotype in these patients, including thrombomodulin resistance. • ST Genesia® is a new automated instrument that may be a suitable routine coagulation laboratory instrument for the measurement of thrombin generation parameters in patients with chronic liver disease. • We showed that high liver stiffness measurements identify a procoagulant phenotype in these patients, including thombomodulin resistance. • Liver stiffness showed correlation with both ST Genesia® and calibrated automated thrombogram assays. • ST Genesia® assay may be better than calibrated automated thrombogram assay as more of its parameters correlate with liver stiffness in patients with chronic liver disease. • Liver stiffness measurements ≥21 kPa identify a procoagulant phenotype in the studied cohort, including thombomodulin resistance. [ABSTRACT FROM AUTHOR]

The antiphospholipid syndrome (APS) is diagnosed by the presence of lupus anticoagulant and/or antibodies against cardiolipin or β 2 -glycoprotein-1 and the occurrence of thrombosis or pregnancy morbidity. The assessment of overall coagulation is known to differ in APS patients compared to normal subjects. The accelerated production of key factor thrombin causes a prothrombotic state in APS patients, and the reduced efficacy of the activated protein C pathway promotes this effect. Even though significant differences exist in the coagulation profile between normal controls and APS patients, it is not possible to rely on a single test result to diagnose APS. A neural network is a computing system inspired by the human brain that can be trained to distinguish between healthy subjects and patients based on subject specific data. In a first cohort of patients, we developed a neural networking that diagnoses APS. We clinically validated this neural network in a separate cohort consisting of APS patients, normal controls, controls visiting the hospital for other indications and two diseased control groups (thrombosis patients and auto-immune disease patients). The positive predictive value ranged from 62% in the hospital controls to 91% in normal controls and the negative predictive value of the neural network ranged from 86% in the thrombosis control group to 95% in the hospital controls. The sensitivity of the neural network was higher than 90% in all control groups. In conclusion, we developed a neural network that accurately diagnoses APS in the validation cohort. After further clinical validation in newly diagnosed patients, this neural network could possibly be clinically implemented to diagnose APS based on thrombin generation data. • The antiphospholipid syndrome is an auto-immune disease associated with thrombosis. • The thrombin generation (TG) method can detect a thrombotic risk. • A neural network (NN) is a method to classify subjects into categories. • The TG-based NN accurately diagnoses APS patients from normal controls. • The NN distinguishes APS patients from thrombosis and auto-immune disease patients. [ABSTRACT FROM AUTHOR]

Venothrombolism (VTE) prophylaxis is increasingly utilized in pediatric intensive care units (PICUs). Enoxaparin, a low-molecular weight heparin, is frequently used for this purpose. Enoxaparin can also be used for therapeutic anticoagulation in cases of known thrombus. In such cases, monitoring involves obtaining serum anti- Xa levels with a target value of 0.5–1 units/mL. No monitoring recommendations currently exist for enoxaparin when intended for pediatric VTE prophylaxis. We hypothesize that a clinically important number of patients on VTE prophylaxis with enoxaparin have serum anti-Xa levels consistent with values targeted for therapeutic anticoagulation. We found that over 20% of patients on VTE prophylaxis with enoxaparin had serum anti-Xa levels consistent with true therapeutic anticoagulation (anti-Xa level 0.5–1 units/mL) during their enoxaparin course and 5% achieved values of supratherapeutic anticoagulation (anti-Xa level >1 units/mL). Serum anti-Xa level did not correlate with once versus twice daily dosing, body mass index (BMI), or creatinine level. Blood urea nitrogen (BUN) was found to have a positive odds ratio for an anti-Xa level ≥ 0.5 units/mL. We believe that this incidence of unintended therapeutic anticoagulation indicates a clinically significant number and therefore routine anti-Xa evaluation while on VTE prophylaxis is warranted within our population. [ABSTRACT FROM AUTHOR]

Blood coagulation and innate immunity are closely interrelated. At sites of inflammation, DNA and myeloperoxidase (MPO) are released from polymorphonuclear leukocytes (PMNs) as an integral component of neutrophil extracellular traps (NETs). NETs exert pleiotropic thrombogenic effects, with DNA-mediated contact activation of factor XII (FXII) likely playing a role. We have previously shown that MPO, a highly cationic protein, regulates coagulation through heteromolecular interactions with various negatively charged structures, including membrane phospholipids and low-molecular-weight heparin. The aims of our current study were to confirm that DNA activates coagulation and to investigate whether its procoagulant activity (PCA) is regulated by PMN-derived MPO. To this end, we used thrombin generation and FXIIa amidolytic activity assays to analyze the PCA of cell-free DNA isolated with silica membrane-based (cfDNA) or silica-free procedures (PaxDNA). cfDNA potently activated FXII and promoted thrombin generation in a concentration-dependent manner, but its PCA was largely attributable to contaminating silica particles. In contrast, pure, i.e. silica-free, PaxDNA was markedly less procoagulant. Although PaxDNA amplified thrombin generation in plasma, it was devoid of any direct FXII activating activity. MPO supershifted both cfDNA and PaxDNA in gel electrophoresis, but only silica-associated PCA of cfDNA was neutralized by MPO independently of its catalytic properties. Moreover, pretreatment with DNase I abolished silica-induced thrombin generation. In summary, we show that pure DNA has rather weak PCA, which is not further inhibited by heteromolecular complex formation with exogenous MPO. Our study thus provides novel mechanistic insights into the regulation of coagulation by extracellular DNA under inflammatory conditions. • Extracellular MPO regulates coagulation and is released in close proximity to DNA. • Pure genomic DNA is a weak initiator of contact-dependent coagulation. • Contaminating silica particles can contribute to DNA procoagulant activity (PCA). • DNase I may inhibit silica-induced coagulation. • MPO neutralizes the PCA of silica particles, but not of DNA. [ABSTRACT FROM AUTHOR]

Venous thrombosis is the leading cause of pregnancy-related maternal morbidity and mortality. The thrombosis risk is increased by caesarean section and blood loss, though underlying mechanisms of these prothrombotic changes remain unknown. This prospective study recruited 50 pregnant women at term undergoing elective caesarean section at University Hospital Magdeburg, Germany. Blood loss during surgery was correlated with the changes in total protein S, full length TFPI (TFPI fl), prothrombin, the endogenous thrombin potential (ETP) and resistance to activated protein C (APCsr) determined via calibrated automated thrombography. Mean blood loss was 506 ml (95%CI: 456 to 557 ml). Total protein S was 0.63 (95%CI: 0.60 to 0.67) U/ml preoperatively, decreased by 14.8% after caesarean section and almost normalised five days later. TFPI fl was 0.47 (95%CI: 0.41 to 0.53) U/ml before, remained unchanged immediately after and increased by 11.5% five days after surgery. Prothormbin was 1.10 (95%CI: 1.03 to 1.16) U/ml preoperatively, reduced by 10.4% immediately after and increased again five days after caesarean section, exceeding the preoperative values by 4.4% (−0.7 to 9.6). The ETP decreased by 3.9%, whereas the APCsr increased by 37.0% immediately after caesarean section. The changes in total protein S, prothrombin, thrombin generation and APC resistance showed a trend to be more pronounced in the subgroups with higher blood loss. Moderate blood loss during caesarean section hardly reduces thrombin generation but aggravates pregnancy-induced APC resistance and combined deficiency of TFPI and protein S, which can account for the increased thrombosis risk in early puerperium. • Peripartal blood loss results in a simultaneous deficiency of anticoagulant proteins and increases APC resistance. • The ETP marginally decreases after a moderate blood loss during caesarean section. • Normalisation of various coagulation proteins postpartum follows different patterns. • Combined deficiency of anticoagulants and APC resistance can contribute the thrombosis risk after postpartum haemorrhage. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection is associated with a clear prothrombotic phenotype. Although the exact pathophysiological mechanisms are not yet fully understood, thrombosis is clearly a highly important in the prognosis and outcome of COVID-19. As such, there is a need for diagnostic analysis and quantification of the coagulation potential in these patients, both at diagnosis and follow-up. Global coagulation assays like thrombin generation (TG) and rotational thromboelastometry (ROTEM) might be suitable in estimating COVID-19 associated coagulopathy and thrombosis risk. Therefore, we aimed at validating both assays for samples with high levels of fibrinogen and in the presence of anticoagulant heparins, such as commonly observed for COVID-19 ICU patients. Calibrated Automated Thrombography (CAT) was optimized to assess plasma thrombin generation in the presence of heparins. The final conditions with either 10 μg/mL Ellagic acid (EA) or PPP Reagent HIGH (high tissue factor; HPPH) were validated according to the EP5 protocol for within-run and between-run variability. Overall variability was well below 10%. To estimate the influences of heparins and high fibrinogen levels, CAT was performed on spiked plasma aliquots from 13 healthy volunteers. Comparable to the CAT method, tPA-ROTEM was used to validate the effect of high fibrinogen and heparins on clotting time, clot firmness and clot lysis parameters. Our adjusted COVID-19 assay showed a heparin dose dependent decrease in peak height and endogenous thrombin potential (ETP) for both EA and HPPH triggered variants. High fibrinogen did not alter the inhibitory effect of either LMWH or UFH, nor did it influence the peak height or ETP in any of the conditions. The tPA-ROTEM showed a significant prolongation in clotting time with the additions of heparin, which normalized with the addition of high fibrinogen. MCF was markedly increased in all hyperfibrinogenemic conditions. A trend towards increased lysis time and, thus, decreased fibrinolysis was observed. Thrombin generation and tPA-ROTEM protocols for measurements in the COVID-19 populations were adjusted and validated. The adjusted thrombin generation assay shows good sensitivity for measurements in heparin spiked plasma. High levels of fibrinogen did not alter the assay or the effectiveness of heparins as measured in this assay. t-PA ROTEM was effective in measurement of both high fibrinogen and heparins spiked samples and was sensitive to the expected relevant coagulant changes by these conditions. No clear fibrinolytic effect was observed in different conditions. • Thrombin Generation triggered with: • PPP Reagent High or ~20 pM Tissue Factor • 10 μg/mL Ellagic Acid (both with 4 μM phospholipids) • ROTEM • EXTEM Reagent with 125 ng/mL tPA or ~35 pM Tissue Factor with 125 ng/mL tPA • These modified global coagulation assays are a sensitive tool to measure coagulation potential within the COVID-19 hospital population. [ABSTRACT FROM AUTHOR]

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma. • PT, APTT and CAT parameters for mouse, rat, rabbit, porcine, and goat plasma • TF-initiated CAT is enhanced upon dilution of rabbit, rat, and mouse plasma. • Description of a novel method to establish plasma anti-FXa potential • Enhanced anti-human FXa activity identified in rabbit, rat and mouse plasma • Rat plasma is identified as a relevant model for the assessment of human FX(a). [ABSTRACT FROM AUTHOR]