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Background: Understanding of the hemostatic and complement alterations associated with cardiopulmonary bypass (CPB) in pediatric patients and the impact of these alterations on outcome is limited., Objectives: The present study prospectively characterized these alterations and their association with postoperative outcomes in pediatric CPB., Methods: All patients aged <21 years undergoing CPB at the authors' institution between 2020 and 2021 who weighed >3 kg, were >36 weeks gestational age, and had no known prothrombotic or hemorrhagic disorders were eligible. Blood samples were analyzed for multiple hemostatic and complement biomarkers pre-, intra-, and 24 hours post-CPB. Biomarker levels were compared to clinical outcomes, including chest tube output (CTO)., Results: Fifty consecutive patients were enrolled. CPB resulted in multiple significant alterations in hemostatic and complement components. Lower platelet counts (<80 × 10 9 platelets/L) at CPB termination were associated with increased postoperative CTO (P = .003). Lower factor (F)VIII levels (<60 IU/dL) at the end of CPB were associated with a longer hospital stay (P < .001) and increased postoperative CTO (P < .001). Patients undergoing staged single ventricle reconstruction were more likely to have lower platelet counts at CPB termination (P = .009) and higher CTO postoperatively (P = .001) than patients undergoing other types of surgical repair. These differences were not due to different preoperative platelet counts, increased incidences of circulatory arrest, or longer CPB times., Conclusion: These data suggest that intraoperative alterations in hemostatic system components may predict postoperative outcomes in pediatric CPB. Further study is needed to determine if interventions targeting platelets or FVIII could improve outcomes in pediatric CPB., Competing Interests: Declaration of competing interests K.T., S.J.H., J.S.T., J.A.R., E.M., M.G.B., L.R., B.F., B.K.S., A.L., C.S., M.K., D.L.S.M., and J.S.P. have no disclosures. The authors report that S. J. has performed consultancy for the following companies that is unrelated to the work presented here: Omeros, Sobi, and Alexion., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Thrombin promotes the proliferation and function of CD8+ T cells. To test if thrombin prevents exhaustion and sustains antiviral T cell activity during chronic viral infection, we depleted the thrombin-precursor prothrombin to 10% of normal levels in mice prior to infection with the clone 13 strain of lymphocytic choriomeningitis virus. Unexpectedly, prothrombin insufficiency resulted in 100% mortality after infection that was prevented by depletion of CD8+ T cells, suggesting that reduced availability of prothrombin enhances virus-induced immunopathology. Yet, the number, function, and apparent exhaustion of virus-specific T cells were measurably unaffected by prothrombin depletion. Histological analysis of the lung, heart, liver, kidney, spleen, intestine, and brain did not reveal any evidence of hemorrhage or increased tissue damage in mice with low levels of prothrombin that could explain mortality. Viral loads were also similar in infected mice regardless of prothrombin levels. Instead, infection of prothrombin-depleted mice resulted in a severe, T cell-dependent anemia associated with increased hemolysis. Thus, thrombin plays an unexpected protective role in preventing hemolytic anemia during virus infection, with potential implications for patients who are using direct thrombin inhibitors as an anticoagulant therapy.

Background: Protease-activated receptor-1 (PAR1) has emerged as an important link between coagulation and the complications of obesity including metabolic dysfunction-associated steatotic liver disease (MASLD). PAR1 is expressed by various cells and cleaved by different proteases to generate unique tethered agonists that activate distinct signaling pathways. Mice expressing PAR1 with an R41Q mutation have disabled canonical thrombin-mediated signaling, whereas R46Q mice express PAR1 resistant to noncanonical signaling by activated protein C., Methods: Mice with whole body and hepatocyte-selective PAR1 deficiency as well as PAR1 R41Q and R46Q mice were fed a high-fat diet (HFD) to induce MASLD., Results: HFD-fed R41Q mice displayed reduced hepatic steatosis and liver/body weight ratio. In contrast, HFD-fed R46Q mice displayed increased relative liver weight and hepatic steatosis alongside increased serum alanine aminotransferase activity. Surprisingly, despite the distinct impact of PAR1 mutations on steatosis, selective deletion of PAR1 in hepatocytes had no impact. To evaluate a viable PAR1-targeted approach, mice with HFD-induced obesity were treated with the allosteric PAR1 modulator NRD-21, which inhibits canonical PAR1 inflammatory signaling but promotes PAR1 protective, noncanonical anti-inflammatory signaling. NRD-21 treatment reduced plasma tumor necrosis factor-alpha, serum alanine aminotransferase activity, hepatic steatosis, and insulin resistance (Homeostatic Model Assessment for Insulin Resistance) but increased plasma active glucagon-like peptide-1., Conclusion: The results suggest that nonhepatocellular canonical PAR1 cleavage drives MASLD in obese mice and provide translational proof-of-concept that selective pharmacologic modulation of PAR1 yields multiple metabolic benefits in experimental obesity., Competing Interests: Declaration of competing interests C.D. is Chief Strategy Officer and Chief Executive Officer at Function Therapeutics, which provided NRD-21 for these studies. Function Therapeutics did not provide financial support. Beyond research support noted in acknowledgments, no other authors have relevant conflicts of interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Extracorporeal membrane oxygenation (ECMO) is often associated with disturbances in acid/base status that can be triggered by the underlying pathology or the ECMO circuit itself. Extracorporeal membrane oxygenation is known to cause hypocapnia, but the impact of reduced partial pressure of carbon dioxide (pCO 2 ) on biomarkers of tissue perfusion during veno-arterial (VA)-ECMO has not been evaluated. To study the impact of low pCO 2 on perfusion indices in VA-ECMO, we placed Sprague-Dawley rats on an established VA-ECMO circuit using either an oxygen/carbon dioxide mixture (O 2 95%, CO 2 5%) or 100% O 2 delivered through the oxygenator (n = 5 per cohort). Animals receiving 100% O 2 developed a significant VA CO 2 difference (pCO 2 gap) and rising blood lactate levels that were inversely proportional to the decrease in pCO 2 values. In contrast, pCO 2 gap and lactate levels remained similar to pre-ECMO baseline levels in animals receiving the O 2 /CO 2 mixture. More importantly, there was no significant difference in venous oxygen saturation (SvO 2 ) between the two groups, suggesting that elevated blood lactate levels observed in the rats receiving 100% O 2 were a response to oxygenator induced hypocapnia and alkaline pH rather than reduced perfusion or underlying tissue hypoxia. These findings have implications in clinical and experimental extracorporeal support contexts., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2024.)

Background: Factor XII (FXII) is a multifunctional protease capable of activating thrombotic and inflammatory pathways. FXII has been linked to thrombosis in extracorporeal membrane oxygenation (ECMO), but the role of FXII in ECMO-induced inflammatory complications has not been studied. We used novel gene-targeted FXII- deficient rats to evaluate the role of FXII in ECMO-induced thromboinflammation., Methods: FXII-deficient (FXII -/- ) Sprague-Dawley rats were generated using CRISPR/Cas9. A minimally invasive venoarterial (VA) ECMO model was used to compare wild-type (WT) and FXII -/- rats in 2 separate experimental cohorts: rats placed on ECMO without pharmacologic anticoagulation and rats anticoagulated with argatroban. Rats were maintained on ECMO for 1 hour or until circuit failure occurred. Comparisons were made with unchallenged rats and rats that underwent a sham surgical procedure without ECMO., Results: FXII -/- rats were maintained on ECMO without pharmacologic anticoagulation with low resistance throughout the 1-hour experiment. In contrast, WT rats placed on ECMO without anticoagulation developed thrombotic circuit failure within 10 minutes. Argatroban provided a means to maintain WT and FXII -/- rats on ECMO for the 1-hour time frame without thrombotic complications. Analyses of these rats demonstrated that ECMO resulted in increased neutrophil migration into the liver that was significantly blunted by FXII deficiency. ECMO also resulted in increases in high molecular weight kininogen cleavage and complement activation that were abrogated by genetic deletion of FXII., Conclusions: FXII initiates hemostatic system activation and key inflammatory sequelae in ECMO, suggesting that therapies targeting FXII could limit both thromboembolism and inopportune inflammatory complications in this setting., (Copyright © 2023 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Objectives: The activated partial thromboplastin time (aPTT) is the most frequently used monitoring assay for bivalirudin in children and young adults on mechanical circulatory support including ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO). However, intrinsic variability of the aPTT complicates management and risks bleeding or thrombotic complications. We evaluated the utility and reliability of a bivalirudin-calibrated dilute thrombin time (Bival dTT) assay for bivalirudin monitoring in this population., Design: Retrospective analysis of clinical data (including aPTT, dilute thrombin time [dTT]) and results of residual plasma samples from VAD patients were assessed in two drug-calibrated experimental assays. One assay (Bival dTT) was validated for clinical use in VAD patients, and subsequently used by clinicians in ECMO patients. Pearson correlation and simple linear regression were used to determine R2 correlation coefficients between the different laboratory parameters using Statistical Package for Social Sciences (Armonk, NY)., Setting: ICUs at Cincinnati Children's Hospital Medical Center., Subjects: Children on VAD or ECMO support anticoagulated with bivalirudin., Interventions: None., Measurements and Main Results: One hundred fifteen plasma samples from 11 VAD patients were analyzed. Both drug-calibrated experimental assays (anti-IIa and Bival dTT) showed excellent correlation with each other ( R2 = 0.94) and with the dTT ( R2 = 0.87), but poor correlation with aPTT ( R2 = 0.1). Bival dTT was selected for validation in VAD patients. Subsequently, clinically ordered results (105) from 11 ECMO patients demonstrated excellent correlation between the Bival dTT and the standard dTT ( R2 = 0.86) but very poor correlation with aPTT ( R2 = 0.004)., Conclusions: APTT is unreliable and correlates poorly with bivalirudin's anticoagulant effect in ECMO and VAD patients. A drug-calibrated Bival dTT offers superior reliability and opportunity to standardize results across institutions. Additional studies are needed to determine an appropriate therapeutic range and correlation with clinical outcomes., Competing Interests: Drs. Block, Palumbo, and Luchtman-Jones disclosed the off-label product use of the dilute thrombin time monitoring assay kit. Dr. Lorts’ institution received funding from Abbott, Berlin Heart, Bayer, and Abiomed. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2024 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch., Competing Interests: Declaration of interests I.M.C. serves on the SAB of GSK Pharmaceuticals. Provisional patent application serial no. 63/438,668, of which some co-authors are inventors, was filed based on these findings., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.)

Previous studies suggested that contact pathway factors drive thrombosis in mechanical circulation. We used a rabbit model of veno-arterial extracorporeal circulation (VA-ECMO) to evaluate the role of factors XI and XII in ECMO-associated thrombosis and organ damage. Factors XI and XII (FXI, FXII) were depleted using established antisense oligonucleotides before placement on a blood-primed VA-ECMO circuit. Decreasing FXII or FXI to < 5% of baseline activity significantly prolonged ECMO circuit lifespan, limited the development of coagulopathy, and prevented fibrinogen consumption. Histological analysis suggested that FXII depletion mitigated interstitial pulmonary edema and hemorrhage whereas heparin and FXI depletion did not. Neither FXI nor FXII depletion was associated with significant hemorrhage in other organs. In vitro analysis showed that membrane oxygenator fibers (MOFs) alone are capable of driving significant thrombin generation in a FXII- and FXI-dependent manner. MOFs also augment thrombin generation triggered by low (1 pM) or high (5 pM) tissue factor concentrations. However, only FXI elimination completely prevented the increase in thrombin generation driven by MOFs, suggesting MOFs augment thrombin-mediated FXI activation. Together, these results suggest that therapies targeting FXII or FXI limit thromboembolic complications associated with ECMO. Further studies are needed to determine the contexts wherein targeting FXI and FXII, either alone or in combination, would be most beneficial in ECMO. Moreover, studies are also needed to determine the potential mechanisms coupling FXII to end-organ damage in ECMO., (© 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.)

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival., (© 2023. The Author(s).)

The mechanisms driving the pathologic state created by extracorporeal membrane oxygenation (ECMO) remain poorly defined. We developed the first complete blood-primed murine model of veno-arterial ECMO capable of maintaining oxygenation and perfusion, allowing molecular studies that are unavailable in larger animal models. Fifteen C57BL/6 mice underwent ECMO by cannulating the left common carotid artery and the right external jugular vein. The mean arterial pressure was measured through cannulation of the femoral artery. The blood-primed circuit functioned well. Hemodynamic parameters remained stable and blood gas analyses showed adequate oxygenation of the animals during ECMO over a 1-hour timeframe. A significant increase in plasma-free hemoglobin was observed following ECMO, likely secondary to hemolysis within the miniaturized circuit components. Paralleling clinical data, ECMO resulted in a significant increase in plasma levels of multiple proinflammatory cytokines as well as evidence of early signs of kidney and liver dysfunction. These results demonstrate that this novel, miniature blood-primed ECMO circuit represents a functional murine model of ECMO that will provide unique opportunities for further studies to expand our knowledge of ECMO-related pathologies using the wealth of available genetic, pharmacological, and biochemical murine reagents not available for other species., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2022.)

Anticoagulant treatment of pediatric cancer-associated venous thromboembolism (VTE) has not been prospectively evaluated. Management of anticoagulation for cancer-associated VTE is often challenged by drug interactions and treatment interruptions. A total of 56 of the 500 children (11.2%) with VTE who participated in the recent EINSTEIN-Jr randomized study had cancer (hematologic malignancy, 64.3%, solid malignant tumor, 35.7%). Children were allocated to either therapeutic-dose bodyweight-adjusted oral rivaroxaban (n=40) or standard anticoagulation with heparins, with or without vitamin K antagonists (n=16) and received a median of 30 concomitant medications. Based on sparse blood sampling at steady-state, pharmacokinetic (PK) parameters of rivaroxaban were derived using population PK modeling. During the 3 months of treatment, no recurrent VTE or major bleeding occurred (95% confidence interval, 0.0%-6.4%), and 3-month repeat imaging showed complete or partial vein recanalization in 20 and 24 of 52 evaluable children (38.5% and 46.2%, respectively). Anticoagulant treatment was interrupted 70 times in 26 (46.4%) children because of thrombocytopenia, invasive procedures, or adverse events, for a mean individual period of 5.8 days. Anticoagulant therapy was resumed in therapeutic doses and was not associated with thrombotic or bleeding complications. Rivaroxaban exposures were within the adult exposure range and similar to those observed in children with VTE who did not have cancer-associated VTE. Rivaroxaban and standard anticoagulants appeared safe and efficacious and were associated with reduced clot burden in most children with cancer-associated VTE, including those who had anticoagulant treatment interruptions. Rivaroxaban exposures were within the adult exposure range despite significant polypharmacy use. This trial was registered at www.clinicaltrials.gov as #NCT02234843., (© 2022 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.)

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by immunoglobulin G (IgG)-mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with thrombopoietin mimetics. However, there are no universally safe and effective treatments for patients presenting with severe life-threatening bleeding. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia, and IdeS is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes, would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of nontargeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from an FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from patients with ITP and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding., (© 2022 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.)

Introduction: Extracorporeal membrane oxygenation (ECMO) profoundly impacts inflammatory and coagulation pathways, and strict monitoring is essential to guide therapeutic anticoagulation. Thromboelastography (TEG) offers a global evaluation of whole blood hemostatic system components and may be a valuable measurement of hemostatic function in these patients. There is a paucity of data correlating TEG parameters with standard measures of coagulation in heparinized pediatric patients., Methods: Children on ECMO during a 10-year period were retrospectively reviewed. Standard measures of coagulation were matched to TEGs drawn within 30 min of each other., Results: Out of 296 unique patients with 331 ECMO runs, 74.3% (n = 246) had at least one set of matched laboratory samples for a total of 2502 matched samples. The aPTT correlated with R-time (p<0.001). Platelets and fibrinogen correlated with α-angle (p<0.001). Fibrinogen (p<0.001) and platelets (p<0.001) were each associated with maximum amplitude (MA). 158 (47.7%) patients had at least one bleeding complication, and 100 (30.2%) had at least one thrombotic complication. Interestingly, a decreasing MA was associated with increased thrombotic complications (p<0.001)., Discussion: TEG correlated well with traditional measures of hemostasis in pediatric ECMO patients. However, there was not a clear benefit of the TEG over these other measures LEVEL OF EVIDENCE: III., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Significant data have accumulated demonstrating a reciprocal relationship between cancer and the hemostatic system whereby cancer promotes life-threatening hemostatic system dysregulation (e.g., thromboembolism, consumptive coagulopathy), and hemostatic system components directly contribute to cancer pathogenesis. The mechanistic underpinnings of this relationship continue to be defined, but it is becoming increasingly clear that many of these mechanisms involve crosstalk between the hemostatic and immune systems. This is perhaps not surprising given that there is ample evidence for bidirectional crosstalk between the hemostatic and immune systems at multiple levels that likely evolved to coordinate the response to injury, host defense, and tissue repair. Much of the data linking hemostasis and immunity in cancer biology focus on innate immune system components. However, the advent of adaptive immunity-based cancer therapies such as immune checkpoint inhibitors has revealed that the relationship of hemostasis and immunity in cancer extends to the adaptive immune system. Adaptive immunity-based cancer therapies appear to be associated with an increased risk of thromboembolic complications, and hemostatic system components appear to regulate adaptive immune functions through diverse mechanisms to affect tumor progression. In this review, the evidence for crosstalk between hemostatic and adaptive immune system components is discussed, and the implications of this relationship in the context of cancer therapy are reviewed. A better understanding of these relationships will likely lead to strategies to make existing adaptive immune based therapies safer by decreasing thromboembolic risk and may also lead to novel targets to improve adaptive immune-based cancer treatments., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Studies spanning the last 3 decades have fundamentally altered our understanding of the interplay between the hemostatic and immune systems. A plethora of studies have revealed that there is bidirectional crosstalk between these two systems at multiple levels that likely evolved as a means to coordinate the host response to numerous challenges, including trauma, infection, and thermal or chemical injury. Such challenges require reestablishment of vascular integrity, the clearance of pathogens, and removal of cellular and external debris. Clearly, bidirectional coordination of hemostasis and immunity would be beneficial in such contexts. Many types of malignancies take advantage of the interplay between hemostasis and immunity, co-opting these mechanisms to promote tumorigenesis, the formation of a supportive stroma, and metastasis to distant organs. Three important "bridges" that mechanistically link the hemostatic system to immune functions that have been shown to play a key role in cancer biology include the platelet/fibrinogen axis, protease activated receptor-1 (PAR-1) and protease activated receptor-2 (PAR-2). These hemostatic system components have been shown to regulate a variety of immune functions that support tumorigenesis in the context of inflammation-driven malignancy, metastasis, and escape from adaptive antitumor immunity. Understanding the mechanisms coupling these bridges between hemostasis and immunity, as well as others, could provide novel targets for the prevention and treatment of a variety of cancers., (Copyright © 2021. Published by Elsevier Ltd.)

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models., (© 2022 by The American Society of Hematology.)

Background: We previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC)., Objective: Our aim was to define the mechanisms coupling fibrin(ogen) to CRC growth., Results: CRC tumors transplanted into the dorsal subcutis of Fib - mice were less proliferative and demonstrated increased senescence relative to those grown in Fib + mice. RNA-seq analyses of Fib + and Fib - tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib - mice was stratifin, encoding 14-3-3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14-3-3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib - mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed three-dimensional environment compared with controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib - tumors. Furthermore, nuclear magnetic resonance-based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib - relative to Fib + mice. Together, these findings suggest that fibrin(ogen)-mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14-3-3σ and p21, thereby promoting cellular proliferation and preventing senescence., Conclusions: These studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target., (© 2021 International Society on Thrombosis and Haemostasis.)

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication., (© 2021. The Author(s).)

Background: Protease-activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll-like receptor 3-dependent interferon- β expression in cardiac fibroblasts., Objectives: We investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection., Methods: We used mice with either a global deficiency of PAR1, cell type-specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites., Results: PAR1-deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type-specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin-8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression., Conclusions: These results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Fanconi anemia (FA) is a complex genetic disorder associated with progressive marrow failure and a strong predisposition to malignancy. FA is associated with metabolic disturbances such as short stature, insulin resistance, thyroid dysfunction, abnormal body mass index (BMI), and dyslipidemia. We studied tryptophan metabolism in FA by examining tryptophan and its metabolites before and during the stress of hematopoietic stem cell transplant (HSCT). Tryptophan is an essential amino acid that can be converted to serotonin and kynurenine. We report here that serotonin levels are markedly elevated 14 days after HSCT in individuals with FA, in contrast to individuals without FA. Kynurenine levels are significantly reduced in individuals with FA compared with individuals without FA, before and after HSCT. Most peripheral serotonin is made in the bowel. However, serotonin levels in stool decreased in individuals with FA after transplant, similar to individuals without FA. Instead, we detected serotonin production in the skin in individuals with FA, whereas none was seen in individuals without FA. As expected, serotonin and transforming growth factor β (TGF-β) levels were closely correlated with platelet count before and after HSCT in persons without FA. In FA, neither baseline serotonin nor TGF-B correlated with baseline platelet count (host-derived platelets), only TGF-B correlated 14 days after transplant (blood bank-derived platelets). BMI was negatively correlated with serotonin in individuals with FA, suggesting that hyperserotonemia may contribute to growth failure in FA. Serotonin is a potential therapeutic target, and currently available drugs might be beneficial in restoring metabolic balance in individuals with FA., (© 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.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity., (© 2020 by The American Society of Hematology.)

Anticoagulant treatment of pediatric central venous catheter-related venous thromboembolism (CVC-VTE) has not been specifically evaluated. In EINSTEIN-Jr, 500 children with any VTE received rivaroxaban or standard anticoagulants. A predefined analysis of the CVC-VTE cohort was performed. Children with CVC-VTE (age, birth to 17 years) were administered rivaroxaban or standard anticoagulants during the 1-month (children <2 years) or 3-month (all other children) study period. Predefined outcomes were recurrent VTE, change in thrombotic burden on repeat imaging, and bleeding. Predictors for continuation of anticoagulant therapy beyond the study period were evaluated. One hundred twenty-six children with symptomatic (n = 76, 60%) or asymptomatic (n = 50, 40%) CVC-VTE received either rivaroxaban (n = 90) or standard anticoagulants (n = 36). There was no recurrent VTE (0%; 95% confidence interval [CI], 0.0%-2.8%). Three children had the principal safety outcome: none had major bleeding and 3 children had clinically relevant nonmajor bleeding (2.4%; 95% CI, 0.7%-6.5%), all in the rivaroxaban arm. Complete or partial vein recanalization occurred in 57 (55%) and 38 (37%) of 103 evaluable children, respectively. Results were similar for symptomatic and asymptomatic CVC-VTE. Continuation of anticoagulant therapy beyond the study period occurred in 61 (48%) of children and was associated with residual VTE but only in children <2 years (odds ratio [OR], 20.9; P = .003) and continued CVC use (OR, 6.7; P = .002). Anticoagulant therapy appeared safe and efficacious and was associated with reduced clot burden in most children with symptomatic or asymptomatic CVC-VTE. Residual VTE and continued CVC use were associated with extended anticoagulation. This trial was registered at www.clinicaltrials.gov as #NCT02234843., (© 2020 by The American Society of Hematology.)

Background: Hematologic trends after pancreatectomy with islet autotransplantation (IAT), which involves splenectomy, have been rarely studied. Reactive thrombocytosis (RT, platelets ≥500 K/μL) often occurs postoperatively, similar to other postsplenectomy states, but the degree of similarities and true incidence are unknown., Study Design: A single-site, retrospective, observational cohort study of patients who underwent total splenectomy between 2010 and 2018 was performed. Thrombocytosis incidence and pharmacologic management strategies were evaluated, including cohort-based analyses for IAT versus other splenectomy indications., Results: Analyses included 112 patients overall, 42 of whom underwent IAT. RT occurred frequently (93.8%) despite most patients having normal preoperative platelet counts. IAT patients had significantly higher peak platelet counts compared to non-IAT patients and the rate of platelet rise for IAT patients was significantly faster. IAT was uniquely predictive of developing extreme thrombocytosis (ExT, platelets ≥1000 K/μL, 90% vs. 15.7%, risk ratio 4.11, P < 0.0001) despite standardized hydroxyurea use. Thrombotic events were infrequent and did not differ between groups., Conclusions: RT was common regardless of splenectomy indication but ExT was uniquely associated with IAT despite cytoreductive pharmacotherapy. These results strongly suggest that splenectomy is unlikely to be the sole contributor to post-IAT RT but further investigations into this phenomenon are needed., Level-Of-Evidence Rating: Treatment study, Level III (retrospective comparative study)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Recently, the randomized EINSTEIN-Jr study showed similar efficacy and safety for rivaroxaban and standard anticoagulation for treatment of pediatric venous thromboembolism (VTE). The rivaroxaban dosing strategy was established based on phase 1 and 2 data in children and through pharmacokinetic (PK) modeling., Methods: Rivaroxaban treatment with tablets or the newly developed granules-for-oral suspension formulation was bodyweight-adjusted and administered once-daily, twice-daily, or thrice-daily for children with bodyweights of ≥30, ≥12 to <30, and <12 kg, respectively. Previously, these regimens were confirmed for children weighing ≥20 kg but only predicted in those <20 kg. Based on sparse blood sampling, the daily area under the plasma concentration-time curve [AUC (0-24)ss ] and trough [C trough,ss ] and maximum [C max,ss ] steady-state plasma concentrations were derived using population PK modeling. Exposure-response graphs were generated to evaluate the potential relationship of individual PK parameters with recurrent VTE, repeat imaging outcomes, and bleeding or adverse events. A taste-and-texture questionnaire was collected for suspension-recipients., Results: Of the 335 children (aged 0-17 years) allocated to rivaroxaban, 316 (94.3%) were evaluable for PK analyses. Rivaroxaban exposures were within the adult exposure range. No clustering was observed for any of the PK parameters with efficacy, bleeding, or adverse event outcomes. Results were similar for the tablet and suspension formulation. Acceptability and palatability of the suspension were favorable., Discussion: Based on this analysis and the recently documented similar efficacy and safety of rivaroxaban compared with standard anticoagulation, we conclude that bodyweight-adjusted pediatric rivaroxaban regimens with either tablets or suspension are validated and provide for appropriate treatment of children with VTE., (© 2020 International Society on Thrombosis and Haemostasis.)

The last half century of cancer research has seen an explosion in our understanding of the complex interplay between cancer cells and host-derived factors critical for cancer progression. Two important host-derived arms that are part of this complex interplay are the inflammatory immune compartment and the hemostatic system. Chronic pathological inflammation is a major factor in the development of multiple common malignancies, including adenocarcinomas of the colon, pancreas, prostate and breast. Hemostatic system components have also been shown to promote cancer progression in multiple contexts. What is only recently been recognized is the link between inflammation and hemostasis in cancer progression. The hemostatic and inflammatory innate immune systems co-evolved to deal with many of the same challenges, including trauma, infections, and thermal/chemical injuries. Their co-evolution necessarily led to bidirectional cross-talk whereby inflammatory cells can activate and alter hemostasis, and hemostatic system components serve as important regulators of inflammatory processes. This cross-talk is critical for the maintenance of vascular integrity, host defense, and wound healing. However, in the context of malignancy, the interplay of these integrated host systems has the capacity to promote multiple stages of malignancy, including tumorigenesis, tumor growth and metastatic dissemination. This review focuses on the interplay of inflammatory cells with the thrombin-fibrinogen axis and protease-activated receptor-1 in cancer pathobiology. Dissecting the mechanisms by which the inflammatory and hemostatic systems cooperatively promote cancer progression will fill in critical knowledge gaps in our understanding of malignancy, and also likely reveal novel therapeutic targets to treat and/or prevent cancer., Competing Interests: Conflict of interest statement J.S.P. receives research funding from Ionis Pharmaceuticals., (© 2020 Elsevier Ltd. All rights reserved.)

Background: Blood coagulation protease activity is proposed to drive hepatic fibrosis through activation of protease-activated receptors (PARs). Whole-body PAR-1 deficiency reduces experimental hepatic fibrosis, and in vitro studies suggest a potential contribution by PAR-1 expressed by hepatic stellate cells. However, owing to a lack of specific tools, the cell-specific role of PAR-1 in experimental hepatic fibrosis has never been formally investigated. Using a novel mouse expressing a conditional PAR-1 allele, we tested the hypothesis that PAR-1 expressed by hepatic stellate cells contributes to hepatic fibrosis., Methods: PAR-1 flox/flox mice were crossed with mice expressing Cre recombinase controlled by the lecithin retinol acyltransferase (LRAT) promoter, which induces recombination in hepatic stellate cells. Male PAR-1 flox/flox /LRATCre and PAR-1 flox/flox mice were challenged twice weekly with carbon tetrachloride (CCl 4 , 1 mL/kg i.p.) for 6 weeks to induce liver fibrosis., Results: PAR-1 mRNA levels were reduced (>95%) in hepatic stellate cells isolated from PAR-1 flox/flox /LRATCre mice. Hepatic stellate cell activation was evident in CCl 4 -challenged PAR-1 flox/flox mice, indicated by increased α-smooth muscle actin labeling and induction of several profibrogenic genes. CCl 4 -challenged PAR-1 flox/flox mice displayed robust hepatic collagen deposition, indicated by picrosirius red staining and type I collagen immunolabeling. Notably, stellate cell activation and collagen deposition were significantly reduced (>30%) in PAR-1 flox/flox /LRATCre mice. Importantly, the reduction in liver fibrosis was not a consequence of reduced acute CCl 4 hepatotoxicity in PAR-1 flox/flox /LRATCre mice., Conclusions: The results constitute the first direct experimental evidence that PAR-1 expressed by stellate cells directly promotes their profibrogenic phenotype and hepatic fibrosis in vivo ., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Background: Thromboembolism (TE) and Fontan-associated liver disease (FALD) are common and lead to significant morbidity in Fontan circulations. Risk factors for TE and the potential link between TE and FALD are not well understood. The objective of this study was to evaluate the association between TE and the severity of FALD based on radiologic liver stiffness. Methods: Using a retrospective cohort study design, 85 Fontan patients (aged 27.7 ± 8.2 years) who had liver stiffness measurement were included. Multivariable logistic regression was used to determine independent associations with TE. Results: Sixteen patients (19%) had a history of TE after the Fontan procedure at a mean age of 21.4 ± 15.0 years. Patients with TE were significantly older at the time of the last evaluation (33.8 ± 11.7 vs. 26.3 ± 6.5 years, p = 0.03). Liver stiffness by MRI and ultrasound was higher in the TE group (5.1 ± 1.4 vs. 4.3 ± 1.2 kPa, p = 0.04 and 2.8 ± 0.4 vs. 2.4 ± 0.5 m/s, p = 0.04, respectively). On multivariable analysis, higher liver stiffness (odds ratio (OR): 2.12, p = 0.03) and older age (OR: 1.11, p = 0.03) were associated with TE. Conclusions: This study found an association between TE, age, and radiologic liver stiffness.

Background & Aim: Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury., Methods: Wild-type mice and VWF-deficient (Vwf -/- ) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence., Results: In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf -/- mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf -/- mice, blocking VWF or the platelet integrin α IIb β 3 during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice., Conclusion: These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver., Lay Summary: Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Treatment of venous thromboembolism in children is based on data obtained in adults with little direct documentation of its efficacy and safety in children. The aim of our study was to compare the efficacy and safety of rivaroxaban versus standard anticoagulants in children with venous thromboembolism., Methods: In a multicentre, parallel-group, open-label, randomised study, children (aged 0-17 years) attending 107 paediatric hospitals in 28 countries with documented acute venous thromboembolism who had started heparinisation were assigned (2:1) to bodyweight-adjusted rivaroxaban (tablets or suspension) in a 20-mg equivalent dose or standard anticoagulants (heparin or switched to vitamin K antagonist). Randomisation was stratified by age and venous thromboembolism site. The main treatment period was 3 months (1 month in children <2 years of age with catheter-related venous thromboembolism). The primary efficacy outcome, symptomatic recurrent venous thromboembolism (assessed by intention-to-treat), and the principal safety outcome, major or clinically relevant non-major bleeding (assessed in participants who received ≥1 dose), were centrally assessed by investigators who were unaware of treatment assignment. Repeat imaging was obtained at the end of the main treatment period and compared with baseline imaging tests. This trial is registered with ClinicalTrials.gov, number NCT02234843 and has been completed., Findings: From Nov 14, 2014, to Sept 28, 2018, 500 (96%) of the 520 children screened for eligibility were enrolled. After a median follow-up of 91 days (IQR 87-95) in children who had a study treatment period of 3 months (n=463) and 31 days (IQR 29-35) in children who had a study treatment period of 1 month (n=37), symptomatic recurrent venous thromboembolism occurred in four (1%) of 335 children receiving rivaroxaban and five (3%) of 165 receiving standard anticoagulants (hazard ratio [HR] 0·40, 95% CI 0·11-1·41). Repeat imaging showed an improved effect of rivaroxaban on thrombotic burden as compared with standard anticoagulants (p=0·012). Major or clinically relevant non-major bleeding in participants who received ≥1 dose occurred in ten (3%) of 329 children (all non-major) receiving rivaroxaban and in three (2%) of 162 children (two major and one non-major) receiving standard anticoagulants (HR 1·58, 95% CI 0·51-6·27). Absolute and relative efficacy and safety estimates of rivaroxaban versus standard anticoagulation estimates were similar to those in rivaroxaban studies in adults. There were no treatment-related deaths., Interpretation: In children with acute venous thromboembolism, treatment with rivaroxaban resulted in a similarly low recurrence risk and reduced thrombotic burden without increased bleeding, as compared with standard anticoagulants., Funding: Bayer AG and Janssen Research & Development., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Protease-activated receptor-1 (PAR-1) plays a major role in multiple disease processes, including colitis. Understanding the mechanisms coupling PAR-1 to disease pathogenesis is complicated by the fact that PAR-1 is broadly expressed across multiple cell types., Objective: Determine the specific contributions of PAR-1 expressed by macrophages and colonic enterocytes to infectious colitis., Methods: Mice carrying a conditional PAR-1 allele were generated and bred to mice expressing Cre recombinase in a myeloid- (PAR-1 ΔM ) or enterocyte-specific (PAR-1 ΔEPI ) fashion. Citrobacter rodentium colitis pathogenesis was analyzed in mice with global PAR-1 deletion (PAR-1 -/- ) and cell type-specific deletions., Results: Constitutive deletion of PAR-1 had no significant impact on weight loss, crypt hypertrophy, crypt abscess formation, or leukocyte infiltration in Citrobacter colitis. However, colonic shortening was significantly blunted in infected PAR-1 -/- mice, and these animals exhibited decreased local levels of IL-1β, IL-22, IL-6, and IL-17A. In contrast, infected PAR-1 ΔM mice lost less weight and had fewer crypt abscesses relative to controls. PAR-1 ΔM mice had diminished CD3+ T cell infiltration into colonic tissue, but macrophage and CD4+ T cell infiltration were similar to controls. Also contrasting results in global knockouts, PAR-1 ΔM mice exhibited lower levels of IL-1β, but not Th17-related cytokines (ie, IL-22, IL-6, IL-17A). Infected PAR-1 ΔEPI mice exhibited increased crypt hypertrophy and crypt abscess formation, but local cytokine elaboration was similar to controls., Conclusions: These studies reveal complex, cell type-specific roles for PAR-1 in modulating the immune response to Citrobacter colitis that are not readily apparent in analyses limited to mice with global PAR-1 deficiency., (© 2019 International Society on Thrombosis and Haemostasis.)

Pathological activation of the coagulation system occurs with virtually all forms of cancer, particularly epithelial malignancies. Accordingly, thrombosis is one of the most common comorbidities associated with cancer. Indeed, cancer-associated thromboembolism is the second leading cause of death for cancer patients, second only to the cancer itself. The identification of specific molecular mechanisms whereby tumor cells activate the coagulation system and drive thrombosis has been an active area of investigation for several decades. Studies in animal models and human trials have revealed that there is a bidirectional relationship between coagulation factor activity and cancer, whereby the pathological hemostatic system activation associated with cancer not only promotes thromboembolism but also drives progression of the malignancy. Numerous studies indicate that factors up and down the clotting cascade can contribute to various stages of cancer, including tumorigenesis, primary tumor growth, and metastasis. Although there are some mechanistic points of commonality, there are also clearly context-dependent contributions of coagulation components to cancer progression dependent on the type of cancer and stage of disease. It is also notable that in some instances, coagulation factors appear to contribute to cancer progression independently of their traditional roles in hemostasis and thrombosis. Here, the authors review the current state of the field with regard to hemostatic factor-driven cancer pathogenesis., Competing Interests: J.S.P. reports grants from Ionis Pharmaceuticals, during the conduct of the study; grants from National Institutes of Health, outside the submitted work. The other authors have no conflict to disclose., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Purpose of Review: The purpose of this review is to summarize the diagnosis and management of the antiphospholipid syndrome (APS) in children., Recent Findings: APS is a rare, acquired autoimmune systemic disease that can result in significant morbidity in children related to vascular thrombosis. The diagnosis and management of APS in children can be challenging due to a lack of validated diagnostic criteria and the rarity of the disease. In addition, many healthy children have transient circulating antiphospholipid antibodies without thrombotic complications. Nevertheless, epidemiological studies suggest that APS represents a greater relative proportion of thrombotic disease in children than it does in adults. Management of pediatric APS is largely inferred from adult data despite unique characteristics of pediatric APS. The current recommendations include long-term anticoagulation, which can be problematic in young, active individuals. There is little data on potential benefits of nonantithrombotic therapy in the management of pediatric APS., Summary: Data on pediatric APS are limited, but evidence suggests that using current available diagnostic testing is valuable and, until further evidence is available, treating thrombotic complications with heparins or warfarin should be standard of care.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC ( KRas G12D , TRP53 R172H ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of TF or Par-1 in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1 KO ) failed to generate sizable tumors, a phenotype completely rescued by restoration of Par-1 expression. Expression profiling of KPC and KPC-Par-1 KO cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1 KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised NSG mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1 KO tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment., (©2019 American Association for Cancer Research.)

Objective: This study aims to explore the role of thrombopoietin (TPO) production in extreme thrombocytosis that is often observed after pancreatectomy with islet autotransplantation (IAT) and the effectiveness of hydroxyurea in thrombocytosis management., Methods: Retrospective chart review was performed for all patients who underwent pancreatectomy with IAT at our institution between April 1, 2015, and December 31, 2016. Data evaluated included demographics, platelet counts, TPO levels, and thrombocytosis management strategies., Results: Twelve total and 1 subtotal pancreatectomy with IAT cases were reviewed. All operations included splenectomy. No major surgical or thrombotic complications occurred. Thrombopoietin levels, normal preoperatively, rose significantly (median, 219 pg/mL) soon after surgery, peaking on median postoperative day 3. Platelet counts, also normal preoperatively, increased within a week of surgery, with 92% over 1000 K/μL (median peak platelet count, 1403 K/μL). Platelet counts and TPO levels dropped after hydroxyurea initiation in most patients., Conclusions: After pancreatectomy with IAT, patients experienced marked TPO rise and subsequent thrombocytosis, and both decreased significantly after hydroxyurea initiation. These data suggest that TPO elevation and associated increased platelet production may be one driver of early extreme post-total pancreatectomy with islet autotransplantation thrombocytosis, and this process may be modulated by hydroxyurea.

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APC M in/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APC M in/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts., (© 2018 International Society on Thrombosis and Haemostasis.)

The association of malignancy and thrombophilia is bidirectional, as evidenced by four decades of studies in animal models showing that hemostatic system components support cancer progression. Consistent with this view, clinical studies have suggested that anticoagulants not only limit thromboembolic complications associated with cancer, but also improve survival by impeding cancer progression, and may even prevent the development of cancer. In order to fully capitalize on this association, a detailed understanding of the mechanisms coupling hemostatic factors to cancer pathogenesis is required. Multiple studies have shown that thrombin-mediated procoagulant functions strongly promote metastatic potential. In particular, the platelet/fibrin(ogen) axis has been shown to protect newly formed micrometastases from innate immune surveillance, contribute to creation of a metastatic niche by recruitment of prometastatic inflammatory cells, and promote the epithelial to mesenchymal transition of metastatic cells. Thrombin-mediated functions have also been shown to support tumor growth in some contexts, and have even been linked to tumorigenesis in the setting of inflammation-driven colon cancer. Here, local thrombin-mediated extravascular fibrin deposition, and specifically fibrin-α M β 2 integrin interaction, push intestinal inflammatory cells toward a pro-tumorigenic phenotype, resulting in the elaboration of key cytokines and growth factors that support the proliferation and survival of transformed intestinal epithelial cells. These studies reveal that hemostatic factors can serve as a bridge between pathological inflammation and the development of cancer. As a large proportion of cancers are caused by pathological inflammation, these studies suggest that therapies targeting the nexus between hemostasis and inflammation could be used to prevent cancer development., (Copyright © 2017. Published by Elsevier Ltd.)

Purpose: Thrombosis in the healthy pediatric population is a rare occurrence. Little is known about the optimal treatment or outcomes of children with unprovoked acute lower extremity (LE) deep vein thrombosis (DVT) associated with atresia of the inferior vena cava (IVC)., Methods: We retrospectively analyzed the records of patients with acute LE DVT subsequently found to have IVC atresia who presented to two tertiary pediatric institutions between 2008 and 2016. Data were reviewed for thrombophilia risk factors, treatment, and outcomes., Results: Eighteen patients, aged 13-18 years (median: 16 years), presenting with acute LE DVT were found to have IVC atresia. Three patients also presented with pulmonary embolism. Fourteen patients underwent site-directed thrombolysis in addition to anticoagulation. Five patients (28%) had confirmed or suspected recurrent thrombosis. Thirteen patients (72%) had no identified provocation for DVT. Ten patients (56%) had post-thrombotic syndrome, and 17 of 18 patients remain on indefinite anticoagulation., Conclusion: This study suggests that IVC atresia is a risk factor for LE DVT and pulmonary embolism in otherwise healthy children and highlights the importance of dedicated imaging of the IVC in young patients with unprovoked LE DVT. Indefinite anticoagulation may be considered in pediatric patients presenting with unprovoked thrombosis secondary to an atretic IVC., (© 2017 Wiley Periodicals, Inc.)

Despite the common occurrence of thrombosis in Fontan circulations, the mid-term thrombotic risk beyond the first two postoperative years is poorly defined especially in total cavo pulmonary Fontan. This study examines the thrombotic incidence and risk beyond the first 2 years after contemporary Fontan surgery. Using a retrospective cohort study design, 89 Fontan patients, 50 male, were included and evaluated with a median of 8.3 years (IQR 6.8-11.4) follow-up. Hospital records were reviewed for known risk factors of thrombosis, thrombotic events, antiplatelet and anticoagulation management, and basic characteristics. Forty seven patients (52%) had a dominant left ventricle, and 28 (32%) had hypoplastic left heart syndrome. Eight patients had thrombotic events post Fontan surgery at a median age of 9 years (IQR 5.6-13), 5.7 (IQR 2.0-9.7) years following surgery, not including events that occurred immediately peri-operatively. Four thrombotic events were intracardiac whereas the remainder were extra-cardiac. There was no significant univariate correlation between thrombosis and the presence of ventricular morphology, pulmonary arterial reconstruction, or type of cavopulmonary anastomosis (lateral tunnel vs. extracardiac conduit). Thrombosis continues to be an important intermediate-term risk even for patients with contemporary Fontan circulations. These results strongly suggest that thrombophilic risk is not dictated purely by vascular pathway and hemodynamic variables. Further investigation into the pathophysiology, individualized risk, and effectiveness of anticoagulation strategies are required in this high risk population.

Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390-396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390-396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.