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Abstract: Plasma kallikrein (PKa) is an important activator of factor XII (FXII) of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin-generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway-initiated TG compared with that of wild-type controls and was comparable with that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared with wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway-initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway-initiated TG in the complex milieu of mouse WB, and a component of this contribution occurs in an FXII-independent manner., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis., (© 2024 The Authors.)

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.

Peatlands are globally significant carbon sinks, but when disturbed, have the potential to release carbon back to the atmosphere as greenhouse gases. Feral horse populations in the Australian Alps degrade Sphagnum peatlands, which are highly sensitive to disturbance. However, the link between this degradation and peatland carbon cycling is not understood. Here, we compared the autumn daytime carbon dioxide (CO 2 ) and methane (CH 4 ) fluxes of 12 alpine and subalpine Sphagnum peatlands in Kosciuszko National Park, Australia. The presence of feral horses at these sites was correlated with higher carbon loss: sites with horses were losing carbon to the atmosphere (4.83 and 8.18 g CO 2 -e m -2 d -1 in areas of Sphagnum moss and bare soil, respectively), whereas sites without horses were removing carbon from the atmosphere (-6.39 g CO 2 -e m -2 d -1 ). Sites with feral horses also had higher soil bulk density, temperature, and electrical conductivity (EC), and higher water pH, EC, and turbidity, than sites without horses. Our findings suggest that excluding feral horses from peatland areas could reduce rates of carbon loss to the atmosphere, in addition to improving overall site condition, peat soil condition, and water quality. We discuss potential management applications, further research, and restoration opportunities arising from these results., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Declaration of competing interests There are no competing interests to disclose.

The chemotherapeutic drug doxorubicin is cardiotoxic and can cause irreversible heart failure. In addition to being cardiotoxic, doxorubicin also induces the activation of coagulation. We determined the effect of thrombin-mediated activation of protease-activated receptor 1 (PAR1) on doxorubicin-induced cardiac injury. Administration of doxorubicin to mice resulted in a significant increase in plasma prothrombin fragment 1+2, thrombin-antithrombin complexes, and extracellular vesicle tissue factor activity. Doxorubicin-treated mice expressing low levels of tissue factor, but not factor XII-deficient mice, had reduced plasma thrombin-antithrombin complexes compared to controls. To evaluate the role of thrombin-mediated activation of PAR1, transgenic mice insensitive to thrombin (Par1R41Q) or activated protein C (Par1R46Q) were subjected to acute and chronic models of doxorubicin-induced cardiac injury and compared with Par1 wild-type (Par1+/+) and PAR1 deficient (Par1-/-) mice. Par1R41Q and Par1-/- mice, but not Par1R46Q mice, demonstrated similar reductions in the cardiac injury marker cardiac troponin I, preserved cardiac function, and reduced cardiac fibrosis compared to Par1+/+ controls after administration of doxorubicin. Furthermore, inhibition of Gαq signaling downstream of PAR1 with the small molecule inhibitor Q94 significantly preserved cardiac function in Par1+/+ mice, but not in Par1R41Q mice subjected to the acute model of cardiac injury when compared to vehicle controls. In addition, mice with PAR1 deleted in either cardiomyocytes or cardiac fibroblasts demonstrated reduced cardiac injury compared to controls. Taken together, these data suggest that thrombin-mediated activation of PAR1 contributes to doxorubicin-induced cardiac injury., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

C1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency-associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway-mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway-mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway-mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway-mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE., (© 2023 by The American Society of Hematology.)

Thrombin generation (TG) assays serve as a valuable tool to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insights into the increased bleeding observed in individuals deficient in factors (F) XI, IX, or VIII. Mice are used extensively in hemostasis research owing to the availability of coagulation factor-deficient mice. However, phenotypic differences between mouse and human TG have become apparent. In this study, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI, or FIX. Expectedly, silica-triggered WB-TG and platelet-poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI, or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet-rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared with an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX-dependent amplification of coagulation suggests an important role of blood cells in this process., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Peatlands account for 15 to 30% of the world's soil carbon (C) stock and are important controls over global nitrogen (N) cycles. However, C and N concentrations are known to vary among peatlands contributing to the uncertainty of global C inventories, but there are few global studies that relate peatland classification to peat chemistry. We analyzed 436 peat cores sampled in 24 countries across six continents and measured C, N, and organic matter (OM) content at three depths down to 70 cm. Sites were distinguished between northern (387) and tropical (49) peatlands and assigned to one of six distinct broadly recognized peatland categories that vary primarily along a pH gradient. Peat C and N concentrations, OM content, and C:N ratios differed significantly among peatland categories, but few differences in chemistry with depth were found within each category. Across all peatlands C and N concentrations in the 10-20 cm layer, were 440 ± 85.1 g kg-1 and 13.9 ± 7.4 g kg-1, with an average C:N ratio of 30.1 ± 20.8. Among peatland categories, median C concentrations were highest in bogs, poor fens and tropical swamps (446-532 g kg-1) and lowest in intermediate and extremely rich fens (375-414 g kg-1). The C:OM ratio in peat was similar across most peatland categories, except in deeper samples from ombrotrophic tropical peat swamps that were higher than other peatlands categories. Peat N concentrations and C:N ratios varied approximately two-fold among peatland categories and N concentrations tended to be higher (and C:N lower) in intermediate fens compared with other peatland types. This study reports on a unique data set and demonstrates that differences in peat C and OM concentrations among broadly classified peatland categories are predictable, which can aid future studies that use land cover assessments to refine global peatland C and N stocks., Competing Interests: There are no competing interests, (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies., Competing Interests: SG has been a consultant for CSL Behring and has received research support from CSL Behring. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grover and Mackman.)

Background: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis., Objective: We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4 fl/fl ;PF4 Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice., Methods: Platelets from PAR4 fl/fl ;PF4 Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors., Results: PAR4 fl/fl ;PF4 Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4 -/- and PAR4 fl/fl ;PF4 Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4 -/- mice and PAR4 fl/fl ;PF4 Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability., Conclusion: We generated a novel PAR4 fl/fl ;PF4 Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability., (© 2021 International Society on Thrombosis and Haemostasis.)

Tissue factor (TF) is induced in a variety of cell types during viral infection, which likely contributes to disseminated intravascular coagulation and thrombosis. TF-expressing cells also release TF-positive extracellular vesicles (EVs) into the circulation that can be measured using an EVTF activity assay. This review summarizes studies that analyze TF expression, TF-positive EVs, activation of coagulation, and thrombosis after infection with influenza A virus (IAV) and coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). The current pandemic of coronavirus disease 2019 (COVID-19) is caused by infection with SARS-CoV-2. Infection of mice with IAV increased TF expression in lung epithelial cells as well as increased EVTF activity and activation of coagulation in the bronchoalveolar lavage fluid (BALF). Infection of mice with MERS-CoV, SARS-CoV, and SARS-CoV-2 also increased lung TF expression. Single-cell RNA sequencing analysis on the BALF from severe COVID-19 patients revealed increased TF mRNA expression in epithelial cells. TF expression was observed in peripheral blood mononuclear cells infected with SARS-CoV. TF was also expressed by peripheral blood mononuclear cells, monocytes in platelet-monocyte aggregates, and neutrophils isolated from COVID-19 patients. Elevated circulating EVTF activity was observed in severe IAV and COVID-19 patients. Importantly, EVTF activity was associated with mortality in severe IAV patients and with plasma D-dimer, severity, thrombosis, and mortality in COVID-19 patients. These studies strongly suggest that increased TF expression in patients infected with IAV and pathogenic CoVs contributes to thrombosis., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: Coronavirus disease 2019 (COVID-19) is associated with activation of coagulation that mainly presents as thrombosis. Sepsis is also associated with activation of coagulation that mainly presents as disseminated intravascular coagulation. Many studies have reported increased levels of plasma d-dimer in patients with COVID-19 that is associated with severity, thrombosis, and mortality., Objectives: The aim of this study was to compare levels of circulating extracellular vesicle tissue factor (EVTF) activity and active plasminogen activator inhibitor 1 (PAI-1) in plasma from patients with COVID-19 or sepsis., Methods: We measured levels of d-dimer, EVTF activity, and active PAI-1 in plasma samples from patients with COVID-19 (intensive care unit [ICU], N = 15; and non-ICU, N = 20) and patients with sepsis (N = 35)., Results: Patients with COVID-19 had significantly higher levels of d-dimer, EVTF activity, and active PAI-1 compared with healthy controls. Patients with sepsis had significantly higher levels of d-dimer and EVTF activity compared with healthy controls. Levels of d-dimer were significantly lower in patients with COVID-19 compared with patients with sepsis. Levels of EVTF activity were significantly higher in ICU patients with COVID-19 compared with patients with sepsis. Levels of active PAI-1 were significantly higher in patients with COVID-19 compared with patients with sepsis., Conclusions: High levels of both EVTF activity and active PAI-1 may promote thrombosis in patients with COVID-19 due to simultaneous activation of coagulation and inhibition of fibrinolysis. The high levels of active PAI-1 in patients with COVID-19 may limit plasmin degradation of crosslinked fibrin and the release of d-dimer. This may explain the lower levels of D-dimer in patients with COVID-19 compared with patients with sepsis., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

[Figure: see text].

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention., (© 2021 by The American Society of Hematology.)

Objective: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 infection leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19., Conclusions: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality.

Pancreatic cancer patients have a high risk of venous thromboembolism (VTE). Plasminogen activator inhibitor 1 (PAI-1) inhibits plasminogen activators and increases the risk of thrombosis. PAI-1 is expressed by pancreatic tumors and human pancreatic cell lines. However, to date, there are no studies analyzing the association of active PAI-1 and VTE in pancreatic cancer patients. We investigated the association of active PAI-1 in plasma and VTE in pancreatic cancer patients. In addition, we determined if the presence of human pancreatic tumors expressing PAI-1 impairs venous thrombus resolution in mice. Plasma levels of active PAI-1 in patients with pancreatic cancer and mice bearing human tumors were determined by enzyme-linked immunosorbent assay. We measured PAI-1 expression in 5 different human pancreatic cancer cell lines and found that PANC-1 cells expressed the highest level. PANC-1 tumors were grown in nude mice. Venous thrombosis was induced by complete ligation of the inferior vena cava (IVC). Levels of active PAI-1 were independently associated with increased risk of VTE in patients with pancreatic cancer (subdistribution hazard ratio per doubling of levels: 1.39 [95% confidence interval, 1.09-1.78], P = .007). Mice bearing PANC-1 tumors had increased levels of both active human and active mouse PAI-1 and decreased levels of plasmin activity. Importantly, mice bearing PANC-1 tumors exhibited impaired venous thrombus resolution 8 days after IVC stasis compared with nontumor controls. Our results suggest that PAI-1 contributes to VTE in pancreatic cancer., (© 2021 by The American Society of Hematology.)

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp , but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections., Competing Interests: The authors declare no competing interest.

Background: The cremaster arteriole laser-induced injury model is a powerful technique with which to investigate the molecular mechanisms that drive thrombus formation. This model is capable of direct visualization and quantification of accumulation of thrombus constituents, including both platelets and fibrin. However, a large degree of variability in platelet accumulation and fibrin formation is observed between thrombi. Strategies to understand this variability will enhance performance and standardization of the model. We determined whether ablation injury size contributes to variation in platelet accumulation and fibrin formation and, if so, whether incorporating ablation injury size into measurements reduces variation., Methods: Thrombus formation was initiated by laser-induced injury of cremaster arterioles of mice (n=59 injuries). Ablation injuries within the vessel wall were consistently identified and quantified by measuring the length of vessel wall injury observed immediately following laser-induced disruption. Platelet accumulation and fibrin formation as detected by fluorescently-labeled antibodies were captured by digital intra-vital microscopy., Results: Laser-induced disruption of the vessel wall resulted in ablation injuries of variable length (18-95 μm) enabling interrogation of the relationship between injury severity and thrombus dynamics. Strong positive correlations were observed between vessel injury length and both platelet and fibrin when the data are transformed as area under the curve (Spearman r = 0.80 and 0.76 respectively). Normalization of area under the curve measurements by injury length reduced intraclass coefficients of variation among thrombi and improved hypothesis testing when comparing different data sets., Conclusions: Measurement of vessel wall injury length provides a reliable and robust marker of injury severity. Injury length can effectively normalize measurements of platelet accumulation and fibrin formation improving data interpretation and standardization., Competing Interests: Conflict of interest RF is a founder and consultant for Platelet Diagnostics. The other authors have no conflicts of interest to disclose.

Background: The intrinsic pathway factors (F) XII and FXI have been shown to contribute to thrombosis in animal models. We assessed the role of FXII and FXI in venous thrombosis in three distinct mouse models., Methods: Venous thrombosis was assessed in mice genetically deficient for either FXII or FXI. Three models were used: the inferior vena cava (IVC) stasis, IVC stenosis, and femoral vein electrolytic injury models., Results: In the IVC stasis model, FXII and FXI deficiency did not affect the size of thrombi but their absence was associated with decreased levels of fibrin(ogen) and an increased level of the neutrophil extracellular trap marker citrullinated histone H3. In contrast, a deficiency of either FXII or FXI resulted in a significant and equivalent reduction in thrombus weight and incidence of thrombus formation in the IVC stenosis model. Thrombi formed in the IVC stenosis model contained significantly higher levels of citrullinated histone H3 compared with the thrombi formed in the IVC stasis model. Deletion of either FXII or FXI also resulted in a significant and equivalent reduction in both fibrin and platelet accumulation in the femoral vein electrolytic injury model., Conclusions: Collectively, these data indicate that FXII and FXI contribute to the size of venous thrombosis in models with blood flow and thrombus composition in a stasis model. This study also demonstrates the importance of using multiple mouse models to assess the role of a given protein in venous thrombosis., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Objective: Quantitative relationships between the extent of injury and thrombus formation in vivo are not well understood. Moreover, it has not been investigated how increased injury severity translates to blood-flow modulation. Here, we investigated interconnections between injury length, clot growth, and blood flow in a mouse model of laser-induced thrombosis. Approach and Results: Using intravital microscopy, we analyzed 59 clotting events collected from the cremaster arteriole of 14 adult mice. We regarded injury length as a measure of injury severity. The injury caused transient constriction upstream and downstream of the injury site resulting in a 50% reduction in arteriole diameter. The amount of platelet accumulation and fibrin formation did not depend on arteriole diameter or deformation but displayed an exponentially increasing dependence on injury length. The height of the platelet clot depended linearly on injury length and the arteriole diameter. Upstream arteriolar constriction correlated with delayed upstream velocity increase, which, in turn, determined downstream velocity. Before clot formation, flow velocity positively correlated with the arteriole diameter. After the onset of thrombus growth, flow velocity at the injury site negatively correlated with the arteriole diameter and with the size of the above-clot lumen., Conclusions: Injury severity increased platelet accumulation and fibrin formation in a persistently steep fashion and, together with arteriole diameter, defined clot height. Arterial constriction and clot formation were characterized by a dynamic change in the blood flow, associated with increased flow velocity.

The intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor (TF)-FVIIa-initiated thrombin generation. In this study, we determined the contribution of murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild-type and FXI-/- mice. Genetic studies were used to determine the effect of complete deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF;FIX-/y male mice were born at the expected frequency, but none survived to wean. In contrast, low-TF;FXI-/- mice were generated at the expected frequency at wean and had a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa-mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF., (© 2020 by The American Society of Hematology.)

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer., (Copyright© 2020 Ferrata Storti Foundation.)

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood "macrophage disappearance reaction." Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity., (© 2019 Zhang et al.)

Murine models are widely used valuable tools to study deep vein thrombosis (VT). Leading experts in VT research came together through the American Venous Forum to develop a consensus on maximizing the utility and application of available mouse models of VT. In this work, we provide an algorithm for model selection, with discussion of the advantages, disadvantages, and applications of the main mouse models of VT. Additionally, we provide a detailed surgical description of the models with guidelines to validate surgical technique., (© 2019 International Society on Thrombosis and Haemostasis.)

Murine models are widely used valuable tools to study deep vein thrombosis. Leading experts in venous thrombosis research came together through the American Venous Forum to develop a consensus on maximizing the utility and application of available mouse models of venous thrombosis. In this work, we provide an algorithm for model selection, with discussion of the advantages, disadvantages, and applications of the main mouse models of venous thrombosis. Additionally, we provide a detailed surgical description of the models with guidelines to validate surgical technique.

Activation of the intrinsic pathway of coagulation contributes to the pathogenesis of arterial and venous thrombosis. Critical insights into the involvement of intrinsic pathway factors have been derived from the study of gene-specific knockout animals and targeted inhibitors. Importantly, preclinical studies have indicated that targeting components of this pathway, including FXI (factor XI), FXII, and PKK (prekallikrein), reduces thrombosis with no significant effect on protective hemostatic pathways. This review highlights the advances made from studying the intrinsic pathway using gene-specific knockout animals and inhibitors in models of arterial and venous thrombosis. Development of inhibitors of activated FXI and FXII may reduce thrombosis with minimal increases in bleeding compared with current anticoagulant drugs.

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors. This review will focus on the roles of TF in protective hemostasis and pathological thrombosis., (© 2018 American Heart Association, Inc.)

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored ( e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro-computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition., (© 2015 American Heart Association, Inc.)

Objective: Deep venous thrombosis is a major health problem. Thrombolytic therapies are effective in recanalizing the veins and preventing post-thrombotic complications, but there is no consensus on selection criteria. The aim of this study was to investigate a fibrin-specific MRI contrast agent (EP-2104R) for the accurate quantification of thrombus' fibrin content in vivo and for the identification of thrombus suitable for thrombolysis., Approach and Results: Venous thrombosis was induced in the inferior vena cava of 8- to 10-week-old male BALB/C mice and MRI performed 2, 4, 7, 10, 14, and 21 days later. Eighteen mice were scanned at each time point pre and 2 hours post injection of EP-2104R (8.0 μmol/kg) with 12 mice at each time point used to correlate fibrin contrast uptake with thrombus' histological stage and fibrin content. Six mice at each time point were immediately subjected to intravascular thrombolytic therapy (10 mg/kg of tissue-type plasminogen activator). Mice were imaged to assess response to lytic therapy 24 hours after thrombolytic treatment. Two mice at each time point were scanned post injection of 0.2 mmol/kg of Gd-DTPA (gadolinium with diethylenetriaminepentacetate, Magnevist, Schering AG, Berlin, Germany) for control purpose. Contrast uptake was correlated positively with the fibrin content of the thrombus measured by Western blotting (R(2)=0.889; P<0.001). Thrombus relaxation rate (R1) post contrast and the change in visualized thrombus size on late gadolinium enhancement inversion recovery MRI pre-EP-2104R and post-EP-2104R injection were the best predictors for successful thrombolysis (area under the curve, 0.989 [95% confidence interval, 0.97-1.00] and 0.994 [95% confidence interval, 0.98-1.00] respectively)., Conclusions: MRI with a fibrin-specific contrast agent accurately estimates thrombus fibrin content in vivo and identifies thrombi that are amenable for thrombolysis., (© 2014 American Heart Association, Inc.)

Objective: Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution., Approach and Results: Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib., Conclusions: Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.