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A heightened risk of thrombosis noted early on with the severe acute respiratory syndrome coronavirus 2 infection led to the widespread use of heparin anticoagulation in the coronavirus disease 2019 (COVID-19) pandemic. However, reports soon started appearing in the literature where an apparent failure of heparin to prevent thrombotic events was observed in hospitalized patients with this viral infection. In this review, we explore the likely mechanisms for heparin failure with particular relevance to COVID-19. We also explore the role of anti-Xa assays and global hemostatic tests in this context. The current controversy of dosing heparin in this disease is detailed with some possible mechanistic reasons for anticoagulant failure. We hope that lessons learnt from the use of heparin in COVID-19 could assist us in the appropriate use of this anticoagulant in the future.

Introduction: Predicting the bleeding phenotype is crucial for the management of patients with moderate haemophilia. Global coagulation assays evaluate haemostasis more comprehensively than conventional methods. Aim: To explore global coagulation assays and the bleeding phenotype of patients with moderate haemophilia A (MHA) and B (MHB). Methods: The MoHem study is a cross-sectional, multicentre study covering Nordic patients with MHA and MHB. Thromboelastometry in whole blood and thrombin generation (TG) in platelet-poor plasma (1, 2.5 and 5 pM tissue factor (TF)) were compared with joint health (Haemophilia Joint Health Score (HJHS)) and treatment modality. Results: We report on 61 patients from Oslo and Helsinki: 24 MHA and 37 MHB. By TG (2.5 pM TF), patients who had been without replacement therapy during the previous 12 months depicted higher endogenous thrombin potential (P =.03). In contrast, those who had low ETP (< median) captured higher HJHS (P =.02). Patients who had undergone orthopaedic surgery generated least thrombin (P =.02). By thromboelastometry, those without the need of factor consumption had short clotting times, and quick times to maximum velocity (< median values) (P =.03). Factor VIII/factor IX activity (FVIII/FIX:C) did not align with the bleeding phenotype, but FIX:C ≤ 3 IU/dL was associated with lower peak thrombin (P =.03). Conclusion: TG differentiated patients with moderate haemophilia according to HJHS, annual factor consumption, and whether orthopaedic surgery had been performed. Thromboelastometry differentiated according to factor consumption only. Global coagulation assays may assist predicting the bleeding phenotype in moderate haemophilia. (Less)

Background Severe carotid stenosis is a common cause of stroke. In addition, previous clinical studies revealed that patients symptomatic of carotid stenosis suffer from increased episodes of stroke compared with their asymptomatic counterparts. However, the mechanism underlying these differences in the recurrence of stroke remains unclear. Objective The present study aimed to evaluate the levels of neutrophil extracellular traps (NETs) in the plasma of patients with severe carotid stenosis and investigate whether NETs induced procoagulant activity (PCA) in severe carotid stenosis. The study also sought to investigate the interactions between platelets or endothelial cells (ECs) and NETs. Methods The levels of NETs in plasma were quantified using enzyme‐linked immunosorbent assay (ELISA). In addition, NETting neutrophils and neutrophil‐platelet aggregates were detected through flow cytometry. On the other hand, the morphology of NETs formation and endothelial cells were analyzed through confocal microscopy. Finally, the procoagulant activity (PCA) of NETs and endothelial cells were assessed through ELISA and fibrin formation. Results Patients with symptomatic carotid stenosis patients had significantly higher levels of NETs markers compared with their asymptomatic counterparts and healthy subjects. In addition, increased levels of neutrophil‐platelet aggregates induced the generation of NETs in patients with symptomatic carotid stenosis. Moreover, NETs contributed to PCA through tissue factor (TF), in patients with carotid stenosis. Furthermore, NETs disrupted the endothelial barrier and converted endothelial cells (ECs) into PCA to enhance the PCA in patients with carotid stenosis. Conclusions The current study revealed differences in the levels of NETs in the plasma of symptomatic and asymptomatic patients suffering from carotid stenosis. The study also uncovered the interaction between NETs and thrombogenicity in carotid stenosis. Therefore, inhibiting NETs may be a potential biomarker and therapeutic target for recurring stroke in severe carotid stenosis., The plasma levels of neutrophil extracellular traps (NETs) are increased in samples from severe carotid stenosis patients. • Platelets (PLT) initiate the formation of NETs in severe carotid stenosis patients. • NETs induce hypercoagulation state in severe carotid stenosis patients and convert endothelial cells (ECs) to procoagulant phenotype.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID‐19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow‐fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore‐size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected “free” MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with “free” MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time., Here we developed a plasma‐based coagulation assay that allowed for clot formation potential of mesenchymal stromal cells (MSCs) across a range of exposures. We conclude that immobilization of MSCs in a hollow fiber filter contributes to a reduced clot initiation potential relative to directly injected MSCs. Further removal of cellular byproducts through saline flushing of the bioreactor further reduces clot formation potential.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti‐TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody‐drug conjugates (ADCs) and molecular imaging probes. We prepared an anti‐TF mAb, clone 1084, labeled with astatine‐211 (211At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50‐100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211At‐conjugated clone 1084 (211At‐anti‐TF mAb) was disrupted by an 211At‐induced radiochemical reaction, we demonstrated that astatinated anti‐TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF–expressing gastric cancer xenograft model, 211At‐anti‐TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211At‐anti‐TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211At‐radioimmunotherapy., Although the astatinated anti–tissue factor (TF) monoclonal antibody (mAb) was disrupted by an astatine‐211–induced radiochemical reaction, we demonstrated that the immunoconjugates eluted in 0.6 or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to maintaining their cellular binding and antitumor activities. In gastric cancer xenograft models, astatine‐211–conjugated anti‐TF mAb protected by 1.2% SA exerted a potent antitumor effect dependent on TF expression on the cell membrane.

Ovarian cancer is a known risk factor of venous thromboembolism (VTE). Thrombogenic factor expression and lymphocytic infiltrate have been reported in endometriosis and ovarian cancers. We reviewed 30 cases of ovarian carcinomas (high grade serous carcinoma, 10; endometrioid carcinoma, 10; clear cell carcinoma (CCC), 10) and 16 endometriotic lesions. We immunohistochemically investigated the expressions of tissue factor (TF), podoplanin, P-selectin, and number of CD4 and CD8 positive lymphocytes in cancer tissue and endometriotic lesions, along with their relationship with VTE. The expression of TF was higher in CCC. The TF expression and the number of CD8 positive cells were higher in cancer tissues with VTE than in those without VTE. The podoplanin or P-selectin expression did not differ among histological types or between cases with and without VTE. Our results demonstrated a high TF expression and intraepithelial CD8 cells in CCC, which were associated with VTE. The results suggest that infiltrating lymphocytes may affect TF expression that, in turn, influences VTE.

Uncontrolled hemorrhage accounts for a significant proportion of annual mortality worldwide. The development of bioinspired hemostatic composites can effectively reduce hemorrhage and related deaths. This work aims to develop an efficient hemostatic agent by incorporating tissue factor (TF) integrated liposomes and collagen, which are capable of augmenting different inherent hemostatic mechanisms, into hemostasis-stimulating alginate matrix. The composite of TF, collagen and alginate (TCA) was made into hydrogel beads with a diameter range of 2.5-3.5 mm, followed by electron microscopy, infrared spectroscopy, rheological, and swelling characterization to confirm its composition and hydrogel nature. When the TCA beads were introduced into simulated body fluid, a controlled release of the loaded TF-liposomes was observed, which also accelerated with the increase of temperature, obtaining intact free proteoliposomes as demonstrated by fluorescence measurement. It is further seen that TCA beads induced the coagulation of whole rabbit blood in about 4.5 min, as compared to ~14.4 min for the control with only recalcified blood. The lipidated TF, collagen and alginate in TCA beads showed a positive synergistic effect on coagulation, while among them a decreasing procoagulant effect was observed. Finally, we demonstrated by a live/dead cell assay that TCA particles had undetectable cytotoxicity. Thus, the TCA hydrogel macrobeads may offer a potential platform for the development of potent hemostatic agents.

Acute lung injury (ALI) caused by gas explosion is common, and warrants research on the underlying mechanisms. Specifically, the role of abnormalities of coagulation and fibrinolysis in this process has not been defined. It was hypothesized that the abnormal coagulation and fibrinolysis promoted ALI caused by gas explosion. Based on the presence of ALI, 74 cases of gas explosion injury were divided into the ALI and non‐ALI groups. The results of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and platelet count (PLT) were collected within 24 hours and compared between the groups. ALI models caused by gas explosion were established in Sprague Dawley rats, and injuries were evaluated using hematoxylin and eosin (HE) staining and histopathological scoring. Moreover, the bronchoalveolar lavage fluid (BALF) was collected to examine thrombin‐antithrombin complex (TAT), tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibitor‐1 (PAI‐1) levels by enzyme‐linked immunosorbent assay (ELISA). The patients in ALI group had shorter PT and longer APTT, raised concentration of FIB and decreased number of PLT, as compared to the non‐ALI group. In ALI rats, the HE staining revealed red blood cells in alveoli and interstitial thickening within 2 hours which peaked at 72 hours. The levels of TAT/TF in the BALF increased continually until the seventh day, while the PAI‐1 was raised after 24 hours and 7 days. The TFPI was elevated after 2 hours and 24 hours, and then decreased after 72 hours. Abnormalities in coagulation and fibrinolysis in lung tissues play a role in ALI caused by gas explosion.

Background and Aims: Chemotherapeutic enteritis is a major dose-limiting adverse reaction to chemotherapy, and few effective drugs are currently available in clinic. Recently the mechanism of intestinal ischemic injury has emerged in the research spotlight due to its notable influence on chemotherapeutic enteritis, but the pathological mechanism of ischemic injury remains unclear. Methods: Irinotecan (CPT-11) was used to establish chemotherapeutic enteritis animal model. Western blotting, gelatin zymography, immunohistochemistry (IHC), intestinal blood flow measurement were used to detect the pathogenesis of ischemia-hypoxia injury. In clinic, the effects of ozone treatment and coagulation examination in patients with chemotherapeutic enteritis were recorded. Results: CPT-11 increased levels of tissue factor (TF) both in the blood and in intestines in mice. Interestingly, the elevation of TF in the blood displayed "double-peak", which was consistent with the intestinal mucosal "double-strike" injury trend. CPT-11 decreased the intestinal blood flow. Moreover, we found that ozone therapy could relieve chemotherapeutic enteritis in mice. We also found that ozone autotransfusion therapy could effectively attenuate chemotherapeutic enteritis and the blood hypercoagulability in cancer patients. Ozone therapy could inhibit TF expression induced by CPT-11 and effectively ameliorate the intestinal mucosal injury in mice. Further research showed that ozone could activate AMPK/SOCS3 to inhibit TLR-4/p38/TF signaling pathway. Conclusions: For the first time, we proposed that TF-induced thrombotic intestinal ischemic injury is a core trigger pathological mechanism of chemotherapeutic enteritis, and provided a new treatment strategy, ozone therapy, to suppress TF expression and treat chemotherapeutic enteritis. Funding Statement: This work was supported by grants from Major Science and Technology Innovation Project of Shandong Province (2018CXGC1220), Major Project of "Science and Technology Innovation Fund" of Nanjing Medical University (2017NJMUCX004), the Natural Science Foundation of Jiangsu province (BK20161033), the Natural Science Foundation of Jiangsu province (BK20181363). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All participants provided written informed consent for participation in the study. This study was approved (2017S053) by the internal review board of the Ethics Committee of Shandong Provincial Qianfoshan Hospital. Animal care and husbandry were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and the Nanjing Medical University Animal Care and Use Committee (NMJU-ACUC). Our study protocol was approved by the NMJU-ACUC, and efforts were made to minimize animal suffering and to reduce the number of mice used.

The pathogenesis of chronic spontaneous urticaria (CSU), also called chronic idiopathic urticaria, has been considered to be associated with the activation of the extrinsic blood coagulation cascade. However, the trigger for the extrinsic coagulation cascade in patients with CSU remains unclear. We previously reported that histamine and lipopolysaccharide (LPS) synergistically induced the expression of tissue factor (TF), a trigger for the extrinsic coagulation cascade, in human umbilical vein endothelial cells (HUVEC). Because the elevation of tumor necrosis factor (TNF)-α, interleukin (IL)-33, IL-1β and vascular endothelial growth factor (VEGF) in serum has also been observed in patients with CSU, we examined the effects of LPS, TNF-α, IL-33, IL-1β, VEGF and histamine on TF expression in HUVEC by reverse transcription polymerase chain reaction and flow cytometry, as well as examining the activity that triggers the extrinsic coagulation cascade and induces intercellular gap formation of HUVEC in the presence of plasma by Actochrome® TF activity assay and impedance sensor, respectively. The expression of TF mRNA and surface protein of TF on HUVEC in response to histamine or VEGF were synergistically enhanced by the treatment with LPS, TNF-α, IL-33 or IL-1β. Moreover, the activation of the extrinsic coagulation pathway and intercellular gap formation of HUVEC in response to histamine or VEGF were also synergistically increased in the presence of TNF-α and LPS. Thus, TF expression on vascular endothelial cells was strongly enhanced by co-stimulation with CSU-related molecules in blood. Blocking a common pathway of LPS, TNF-α, IL-33 and IL-1β, and/or that of VEGF and histamine may be an effective therapeutic measure for patients with severe and refractory CSU.

Background Western blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. Objectives We assessed several commercial anti-human and anti-mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. Material and methods We used human pancreatic cancer cell lines expressing different levels of TF and a mouse pancreatic cancer cell line expressing TF with a matched knockout derivative. Results Human and mouse TF protein detected by western blotting correlated with levels of TF mRNA in these cell lines. The apparent molecular weight of TF is increased by N-linked glycosylation and, as expected, deglycosylation decreased the size of TF based on western blotting. We found that four commercial anti-human TF antibodies detected TF in a TF-positive cell line HPAF-II whereas no signal was observed in a TF-negative cell line MIA PaCa-2. More variability was observed in detecting mouse TF. Two anti-mouse TF antibodies detected mouse TF in a TF-positive cell line and no signal was observed in a TF knockout cell line. However, a third anti-mouse TF antibody detected a nonspecific protein in both the mouse TF-positive and TF-negative cell lines. Two anti-human TF antibodies that are claimed to cross react with mouse TF either recognized a nonspecific band or did not detect mouse TF. Discussion Our results indicate that there is a range in quality of commercial anti-TF antibodies. Conclusion We recommend that all commercial antibodies should be validated to ensure that they detect TF.

Exposure of rats to 2-chloroethyl ethyl sulfide (CEES), an analog of sulfur mustard, can cause acute lung injury (ALI), resulting in increased inflammation and coagulation and altered levels of plasma microRNAs (miRNAs). Rats were exposed to aerosolized CEES and euthanized 12 h later for collection of tissue and plasma. Profiling of miRNAs in plasma, using a TaqMan-based RT-PCR array, revealed 14 differentially expressed miRNAs. Target gene prediction and pathway analysis revealed miRNA-mediated regulation of organismal injury, inflammation, and respiratory diseases. miR-140-5p, a marker of ALI, was downregulated in the plasma, lung, liver, and kidney of CEES-exposed rats, with a concomitant increase in the expression of the inflammation markers IL-6 and IL-1α and the coagulation marker tissue factor (F3). Exposure of rat airway epithelial cells (RL-65) to CEES (0.5 mM) caused cell death and a decrease in miR-140-5p both in cells and media supernatant. This was accompanied by an increase in cellular mRNA levels of IL-6, IL-1α, and F3, as well as FGF9 and EGR2, putative targets of miR-140. Knockdown of miR-140 by specific oligos in RL-65 cells mimicked the in vivo CEES-mediated effects, leading to significantly increased mRNA levels of IL-6, IL-1α, F3, FGF9, and EGR2. Our study identifies miR-140-5p as a mediator of CEES-induced ALI, which could potentially be targeted for therapy.

Atherothrombosis is a leading cause of cardiovascular mortality and morbidity worldwide. The underlying mechanisms of atherothrombosis comprise plaque disruption and subsequent thrombus formation. Arterial thrombi are thought to mainly comprise aggregated platelets as a result of high blood velocity. However, thrombi that develop on disrupted plaques comprise not only aggregated platelets, but also large amounts of fibrin, because plaques contain large amount of tissue factor that activate the coagulation cascade. Since not all thrombi grow large enough to occlude the vascular lumen, the propagation of thrombi is also critical in the onset of adverse vascular events. Various factors such as vascular wall thrombogenicity, local hemorheology, systemic thrombogenicity and fibrinolytic activity modulate thrombus formation and propagation. Although the activation mechanisms of platelets and the coagulation cascade have been intensively investigated, the underlying mechanisms of occlusive thrombus formation on disrupted plaques remain obscure. Pathological findings derived from humans and animal models of human atherothrombosis have uncovered pathophysiological processes during thrombus formation and propagation after plaque disruption, and novel factors have been identified that modulate the activation of platelets and the coagulation cascade. These findings have also provided insights into the development of novel drugs for atherothrombosis.

Background Tumor stroma, of which fibroblasts are the most abundant cell, resembles a non‐healing wound, where a procoagulant environment creates a permissive milieu for cancer growth. We aimed to determine if tumor expression of coagulation factors (procoagulant phenotype), and systemic hypercoagulability, occur at the preinvasive (ductal carcinoma in situ; DCIS) stage and correlate with breast cancer subtype, disease‐free survival (DFS), and overall survival (OS). Methods In a prospective cohort of early breast cancer (DCIS, n = 76; invasive, n = 248) tumor, normal breast and plasma were examined. Fibroblast and epithelial expression of Tissue Factor (TF), thrombin, PAR1, PAR2, and plasma thrombin‐antithrombin (TAT) and D‐dimer were correlated with clinicopathological data, and 5‐year survival. Results Fibroblast expression of TF, thrombin, and PAR1 was increased in DCIS and invasive cancer compared to normal breast fibroblasts (P ≤ .003, all). Fibroblast TF, thrombin, PAR1, and PAR2 was increased in cancers with high Ki67, high grade, ER‐ (vs ER+), and HER2+ (vs HER2‐) (all P 3‐fold mortality risk compared to low TAT. Conclusion This demonstrates procoagulant phenotypic changes occur in fibroblasts at the preinvasive stage. Fibroblast procoagulant phenotype is associated with aggressive breast cancer subtypes and reduced survival. Coagulation may be a therapeutic target in breast cancer., In a large prospective cohort study (n = 324) of breast cancer patients, increased expression of markers of coagulation (Tissue Factor, thrombin and their respective receptors PAR2 and PAR1) are localized to fibroblasts, increased in aggressive breast cancer subtypes, and correlate with reduced overall survival. The phenotypic change in fibroblasts to a procoagulant state occurs at the preinvasive (DCIS) stage, implying an epithelial‐stromal communication across the basement membrane in DCIS creates a procoagulant stromal milieu and potentially an environment to facilitate invasion.

INTRODUCTION We have routinely used thrombin generation to investigate patients with unclassified bleeding disorder (UBD). AIMS To investigate haemostatic abnormalities in patients with UBD that had abnormal thrombin generation on at least one occasion. METHODS Investigation of 13 known UBD patients with thrombin generation and detailed haemostatic testing was undertaken including TFPI assays but also thrombomodulin and fibrinogen-γ. RESULTS 12 females and 1 male were included. No patient had a platelet function disorder or coagulation factor deficiency that explained the bleeding phenotype, though 2 patients had factor deficiencies; a factor X of 0.41 IU/mL and a factor XI of 0.51 IU/mL. ThromboGenomics revealed variants for these factors but no other abnormalities. Patients were included who previously had either prolonged lag time or decreased endogenous thrombin potential (ETP) via high dose tissue factor (5 pmol/L) or low dose tissue factor (1.5 pmol/L) with corn trypsin inhibitor (CTI). Tissue factor pathway inhibitor (TFPI) activity was significantly increased (P

Background Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter‐donor variability limit the application of such assays, especially with many patients on prophylaxis. Objective To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays. Methods Corn trypsin inhibitor (4 µg/mL)‐treated healthy blood was dosed with either anti–factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX‐V181T; hemophilia B model). Treated blood was perfused at 100 s−1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa). Results Anti‐FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti‐FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti‐FVIII‐treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient‐derived FVIII‐neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX‐V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti‐TFPI. Conclusion Two new microfluidic hemophilia A and B models demonstrate the potency of anti‐TF pathway inhibitor, emicizumab, and a patient‐derived inhibitory antibody. Using collagen/FXIa‐coated surfaces resulted in reliable and highly sensitive hemophilia models.

Sickle cell disease (SCD) is the most prevalent inherited blood disorder in the world. But the clinical manifestations of the disease are highly variable. In particular, it is currently difficult to predict the adverse outcomes within patients with SCD, such as, vasculopathy, thrombosis, and stroke. Therefore, for most effective and timely interventions, a predictive analytic strategy is desirable. In this study, we evaluate the endothelial and prothrombotic characteristics of blood outgrowth endothelial cells (BOECs) generated from blood samples of SCD patients with known differences in clinical severity of the disease. We present a method to evaluate patient‐specific vaso‐occlusive risk by combining novel RNA‐seq and organ‐on‐chip approaches. Through differential gene expression (DGE) and pathway analysis we find that BOECs from SCD patients exhibit an activated state through cell adhesion molecule (CAM) and cytokine signaling pathways among many others. In agreement with clinical symptoms of patients, DGE analyses reveal that patient with severe SCD had a greater extent of endothelial activation compared to patient with milder symptoms. This difference is confirmed by performing qRT‐PCR of endothelial adhesion markers like E‐selectin, P‐selectin, tissue factor, and Von Willebrand factor. Finally, the differential regulation of the proinflammatory phenotype is confirmed through platelet adhesion readouts in our BOEC vessel‐chip. Taken together, we hypothesize that these easily blood‐derived endothelial cells evaluated through RNA‐seq and organ‐on‐chips may serve as a biotechnique to predict vaso‐occlusive episodes in SCD patients and will ultimately allow better therapeutic interventions.

Autoimmune protein S (PS) deficiency is a highly thrombotic, potentially life‐threatening disorder. Its pathophysiological relevance in the context of primary antiphospholipid syndrome (APS) is unclear. Here, we report the case of a 76‐year‐old woman, who presented with a painful reticular skin erythema caused by microvascular thromboses. Disseminated intravascular coagulation (DIC) with consumptive coagulopathy was controlled only by continuous anticoagulation. While significantly elevated IgM antibodies to cardiolipin and β2‐glycoprotein‐I were consistent with primary APS, a function‐blocking PS autoantibody of the IgG isotype was detected. Robust microvesicle (MV)‐associated tissue factor (TF) procoagulant activity (PCA) was isolated from patient plasma. Moreover, patient IgG, but not IgM, induced expression of TF PCA and release of TF‐bearing MVs by peripheral blood mononuclear cells from healthy donors. In primary APS, induction of monocyte TF in combination with an acquired PS inhibitor may provoke a deleterious imbalance of procoagulant and anticoagulant pathways with evolution of thrombotic DIC.

Objective Alterations in coagulation could mediate functional outcome in patients with hyperglycemia after acute ischemic stroke (AIS). We prospectively studied the effects of intensive versus standard glucose control on coagulation markers and their relationships to functional outcomes in patients with AIS. Approach The Insights on Selected Procoagulation Markers and Outcomes in Stroke Trial measured the coagulation biomarkers whole blood tissue factor procoagulant activity (TFPCA); plasma factors VII (FVII), VIIa (FVIIa), and VIII (FVIII); thrombin‐antithrombin (TAT) complex; D‐dimer; tissue factor pathway inhibitor, and plasminogen activator inhibitor‐1 (PAI‐1) antigen in patients enrolled in the Stroke Hyperglycemia Insulin Network Effort trial of intensive versus standard glucose control on functional outcome at 3 months after AIS. Changes in biomarkers over time (from baseline ≈12 hours after stroke onset) to 48 hours, and changes in biomarkers between treatment groups, functional outcomes, and their interaction were analyzed by two‐way analysis of variance. Results A total of 125 patients were included (57 in the intensive treatment group and 68 in the standard treatment group). The overall mean age was 66 years; 42% were women. Changes from baseline to 48 hours in coagulation markers were significantly different between treatment groups for TFPCA (P = 0.02) and PAI‐1 (P = .04) and FVIIa (P = .04). Increases in FVIIa and decreases in FVIII were associated with favorable functional outcomes (P = .04 and .04, respectively). In the intensive treatment group, reductions in TFPCA and FVIII and increases in FVIIa were greater in patients with favorable than unfavorable outcomes (P = .02, 0.002, 0.03, respectively). In the standard treatment group, changes in FVII were different by functional outcome (P = .006). Conclusions Intensive glucose control induced greater alterations in coagulation biomarkers than standard treatment, and these were associated with a favorable functional outcome at 3 months after AIS.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is responsible for many pathological processes, such as altered vascular disease development, dysfunctional thrombosis, and a heightened inflammatory state. Although many clinical reports support these observations, there has been limited experimental work to determine the underlying mechanisms and cellular processes induced by exposure to SARS‐CoV‐2 structural proteins. Importantly, these structural proteins are conserved amongst all current known SARS‐CoVs. Our objective was to investigate the effects of the Spike (S), Nucleocapsid (N), and Membrane‐Envelope (M) SARS‐CoV‐2 structural proteins on inflammation, thrombosis, and diabetic disease markers of human aortic adventitial fibroblasts and human umbilical vein endothelial cells. We hypothesized that short‐term exposure to SARS‐CoV‐2 structural proteins would result in increased expression of inflammatory, thrombotic, and diabetic proteins in both cell types, which would support a mechanism for altered vascular disease progression. To test this, the cells were incubated independently with the three SARS‐CoV‐2 proteins for one hour, after which we analyzed the expression of gC1qR, ICAM‐1, tissue factor, RAGE, GLUT‐4, thrombomodulin, PECAM‐1, and Connexin‐43 (in their respective cell types), using an ELISA approach. All cells were monitored for maintenance of typical culture parameters using a live/dead cell cytotoxicity assay and the MTT assay (for metabolic activity). We observed that each of these markers were significantly up‐regulated after exposure to SARS‐CoV‐2 structural proteins as compared to fibroblasts or endothelial cells that were not exposed to these proteins. Interestingly, the extent of the expression of these markers was sometimes significantly different for each of the SARS‐CoV‐2 structural proteins. This suggests that each of the SARS‐CoV‐2 proteins interacts with these cells through different mechanisms, however, more work will need to be undertaken to determine the mechanisms by which these proteins interact with cells. Thus, these results indicate that the cellular response of vascular cells towards SARS‐CoV‐2 structural proteins promotes inflammatory, thrombotic, and vascular dysfunction. However, these interactions are regulated by complex and possibly different cellular receptors/signal transduction pathways that should be explored further.

Studies have shown that microRNAs (miRNAs) participate in almost all pathological and physiological processes including acute lower-extremity deep venous thrombosis (LEDVT). Here, this study was designed to elucidate the possible function of miR-103a-3p in acute LEDVT. Expression of miR-103a-3p and chemokine C-X-C motif ligand 12 (CXCL12) was initially quantified in plasma collected from 81 LEDVT patients. Then LEDVT mouse models were established by injection with 3% sodium pentobarbital. The interaction between miR-103a-3p and CXCL12 was identified by dual-luciferase reporter gene assay. After gain- and loss-of-function studies, interleukin-6 (IL-6) and IL-8 and tissue factor (TF) levels, and expression of plasminogen activator inhibitors (PAIs), von Willebrand factor (vWF), thromboxane A2 (TH-A2), F4/80, IL-12, Arginase-1 (Arg-1) and CD206 were determined using enzyme-linked immunosorbent assay (ELISA), reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. miR-103a-3p was downregulated, while CXCL12 was upregulated in patients and mice with LEDVT. miR-103a-3p targets CXCL12 and inhibited its expression. Overexpressed miR-103a-3p or downregulated CXCL12 decreased expression of IL-6, IL-8, TF, PAIs, vWF, TH-A2, M1 markers (IL-6 and IL-12), yet increased expression of M2 markers (Arg-1 and CD206) in LEDVT mice. Additionally, upregulated miR-103a-3p or silencing CXCL12 suppressed thrombosis in LEDVT mice. However, overexpression of CXCL12 reversed the tendency mentioned above. Altogether, miR-103a-3p can potentially downregulate CXCL12 expression to disrupt inflammatory response and thrombosis, ultimately preventing the development of LEDVT. Our findings underscore a possible alternative therapeutic strategy to limit LEDVT.

Introduction Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. Aim We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. Methods Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. Results Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. Conclusions These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Although direct adhesion of cancer cells to the mesothelial cell layer is considered to be a key step for peritoneal invasion of ovarian cancer cell masses (OCM), we recently identified a different strategy for the peritoneal invasion of OCM. In 6 out of 20 cases of ovarian carcinoma, extraperitoneal growth of the OCM was observed along with the neovascularization of feeding vessels, which connect the intraperitoneal host stroma and extraperitoneal lesions through the intact mesothelial cell layer. As an early step, the OCMs anchor in the extraperitoneal fibrin networks and then induce the migration of CD34‐positive and vascular endothelial growth factor A (VEGF‐A)‐positive endothelial cells, constructing extraperitoneal vascular networks around the OCM. During the extraperitoneal growth of OCM, podoplanin‐positive and α smooth muscle actin (αSMA)‐positive cancer‐associated fibroblasts (CAF) appears. In more advanced lesions, the boundary line of mesothelial cells disappears around the insertion areas of feeding vessels and then extraperitoneal and intraperitoneal stroma are integrated, enabling the OCM to invade the host stroma, being associated with CAF. In addition, tissue factors (TF) are strongly detected around these peritoneal implantation sites and their levels in ascites were higher than that in blood. These findings demonstrate the presence of neovascularization around fibrin net‐anchored OCMs on the outer side of the intact peritoneal surface, suggesting a novel strategy for peritoneal invasion of ovarian cancer and TF‐targeted intraperitoneal anti‐cancer treatment. We observed and propose a novel strategy for peritoneal implantation of ovarian cancer. The strategy includes the preinvasive growth of fibrin‐anchored cancer cells along with neovascularization on the outer side of the intact peritoneal surface.

Cerium oxide nanoparticles (nanoceria [NC]) have attracted much attention in biomedicine due to their surface composition that confers interesting redox activities and regenerative properties. Studies have demonstrated that the application of NPs in biomedicine can influence components of hemostatic system, inducing blood clotting, alterations of blood cells, and endothelial cell functions. NC were tested in vitro to assess their hemocompatibility and anticoagulant, anti-inflammatory, and anti-senescence activity in human endothelial cells. Hemocompatibility has been evaluated in vitro looking at the impact of NC on coagulation times, fibrinogen, and platelet aggregation. The effect of NC on vascular endothelial cells were assayed by testing cell viability, antioxidant activity, anticoagulant (tissue factor [TF]-mRNA expression) and anti-inflammatory properties (VCAM-1 exposure, cytokine release), and senescence (telomere shortening). NC did not show significant effects on coagulation process, hemolysis, or platelet aggregation. In endothelial cells, NC did not affect cell viability, reduced oxidative stress, inhibited mRNA-TF expression, VCAM-1 expression, and cytokine release. Moreover, NC reduce telomere shortening, possibly counteracting premature senescence. The hemocompatibility combined with anticoagulant and anti-inflammatory phenotype and the ability of counteract the premature senescence in vascular cells make NC a promising therapeutic tool in oxidative stress-related conditions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Extracellular traps generated by neutrophils contribute to thrombus progression in coronary atherosclerotic plaques. It is not known whether other inflammatory cell types in coronary atherosclerotic plaque or thrombus also release extracellular traps. We investigated their formation by macrophages, mast cells, and eosinophils in human coronary atherosclerosis, and in relation to the age of thrombus of myocardial infarction patients. Coronary arteries with thrombosed or intact plaques were retrieved from patients who died from myocardial infarction. In addition, thrombectomy specimens from patients with myocardial infarction were classified histologically as fresh, lytic or organised. Neutrophil and macrophage extracellular traps were identified using sequential triple immunostaining of CD68, myeloperoxidase, and citrullinated histone H3. Eosinophil and mast cell extracellular traps were visualised using double immunostaining for eosinophil major basic protein or tryptase, respectively, and citrullinated histone H3. Single- and double-stained immunopositive cells in the plaque, adjacent adventitia, and thrombus were counted. All types of leucocyte-derived extracellular traps were present in all thrombosed plaques, and in all types of the in vivo-derived thrombi, but only to a much lower extent in intact plaques. Neutrophil traps, followed by macrophage traps, were the most prominent types in the autopsy series of atherothrombotic plaques, including the adventitia adjacent to thrombosed plaques. In contrast, macrophage traps were more numerous than neutrophil traps in intact plaques (lipid cores) and organised thrombi. Mast cell and eosinophil extracellular traps were also present, but sparse in all instances. In conclusion, not only neutrophils but also macrophages, eosinophils, and mast cells are sources of etosis involved in evolving coronary thrombosis. Neutrophil traps dominate numerically in early thrombosis and macrophage traps in late (organising) thrombosis, implying that together they span all the stages of thrombus progression and maturation. (c) 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PURPOSE To evaluate and correlate levels of various proteins involved in coagulation, inflammation and angiogenesis processes in the vitreous of patients with different vitreoretinal pathologies. METHODS Vitreous samples were collected from patients scheduled for pars plana vitrectomy for the treatment of rhegmatogenous retinal detachment (RRD), vitreous haemorrhage or tractional retinal detachment associated with proliferative diabetic retinopathy (PDR). Macular hole and epiretinal membrane served as controls. Levels of vascular endothelial growth factor, thrombin-antithrombin III complex, interleukin-8, tissue factor, thrombomodulin, P-selectin, D-dimer and tissue factor pathway inhibitor were compared among the vitreoretinal pathology groups. RESULTS Compared to controls, patients with PDR had significantly higher levels of thrombin-antithrombin III complex (p

INTRODUCTION The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII

Atherothrombosis is characterized by the inflammatory process of atherosclerosis combined with a hypercoagulable state leading to superimposed thrombus formation. In atherosclerotic plaques, cell signaling can occur via protease-activated receptors (PARs), four of which have been identified so far (PAR1-PAR4). Proteases that are able to activate PARs can be produced systemically, but also at the sites of lesions, and they include thrombin and activated factor X. After PAR activation, downstream signaling can lead to both proinflammatory effects and a hypercoagulable state. Which specific effect occurs depends on the type of protease and activated PAR, and the site of activation. Hypercoagulable effects are mainly exerted through PAR1 and PAR4, whereas proinflammatory responses are mostly seen after PAR1 and PAR2 activation. PAR signaling pathways contribute to atherothrombosis, suggesting that inhibition of these pathways possibly prevents cardiovascular events based on this pathophysiological mechanism. In this review, we highlight the pathways by which PAR activation leads to proinflammatory responses and a hypercoagulable state. Furthermore, we give an overview of potential pharmacological treatment targets that promote vascular protection.

BACKGROUND Viral infection of the bronchial epithelium disrupts the barrier properties of the epithelium in healthy individuals and those with lung disease. Repair of the bronchial epithelium is dependent of the formation of a provisional fibrin matrix and migration of epithelial cells to cover denuded areas, followed by proliferation and differentiation. OBJECTIVE The objective was to test the hypothesis that poly I:C, a model of viral infection, limits epithelial repair through the stimulated release of matrix metalloproteinase-13 (MMP-13). METHODS Confluent layers of cultured normal human primary bronchial epithelial cells (NHBE) and SV-40 virus-transformed 16HBE14o- bronchial epithelial cells were mechanically wounded, and video microscopy used to measure the rate of wound closure over 2 hours, in the absence and presence of poly I:C (1-20 μg/mL). MMP-13, tissue factor and endothelin release were measured by ELISA. The effect of inhibitors of MMP-13 activity and expression and a nonspecific endothelin receptor antagonist, bosentan, on the rate of epithelial repair was investigated. RESULTS Poly I:C limited the rate of epithelial repair, and NHBE were significantly more sensitive to poly I:C effects than 16HBE14o- cells. NHBE, but not 16HBE14o-, released MMP-13 in response to poly I:C. Inhibitors of MMP-13 activity (WAY 170523) and expression (dimethyl fumarate) significantly enhanced the rate of repair. Bosentan enhanced the rate of bronchial epithelial repair by a mechanism that was independent of MMP-13. CONCLUSIONS AND CLINICAL RELEVANCE Bronchial epithelial repair is limited by endothelin and by MMP-13, a protease that degrades coagulation factors, such as fibrinogen, and matrix proteins essential for epithelial repair. Further studies with primary cells from patients are needed to confirm whether repurposing bosentan and inhibitors of MMP-13 expression or activity, for inhalation may be a useful therapeutic strategy in diseases where repeated cycles of epithelial injury and repair occur, such as asthma and COPD.

Over the last three decades, the prognosis of osteosarcoma has remained unchanged; the prognosis for patients with lung metastasis is still poor, and the development of new treatments is urgently required. We previously showed that aggressive osteosarcoma cells express more tissue factor (TF) and demonstrate enhanced extrinsic pathway capacity. Furthermore, tumor growth can be suppressed with the anticoagulant low molecular weight heparin. However, the molecular mechanisms underlying TF regulation are still unclear. Here, we report that transforming growth factor-β (TGF-β) upregulates TF, which can occur via activated platelets. TF was found to be expressed on osteosarcoma cell surfaces, which mediated the production of Xa and thrombin. TF induction by TGF-β was observed in several osteosarcoma cells, and especially in MG 63 cells. Both TF expression by TGF-β and extrinsic pathway activity through TF were rapidly increased. This reaction was inhibited by a TGF-β type I receptor inhibitor and TGF-β neutralizing antibody. Although TGF-β was found to phosphorylate both Smad2 and Smad3, their roles were markedly disparate. Surprisingly, Smad2 knockdown resulted in no inhibitory effect, whereas Smad3 knockdown completely suppressed TGF-β-induced TF expression. Next, data suggested that platelets were the source of TGF-β. We confirmed that thrombin-activated platelets and osteosarcoma cells could release TGF-β, and that platelet-derived TGF-β could induce TF expression. These processes were also inhibited by a TGF-β type I receptor inhibitor and Smad3 knockdown. Moreover, CD42b, TF, TGF-β, Smad2/3, and p-Smad2/3 were also detected in a biopsy sample from an osteosarcoma patient. Collectively, these finding suggested that the interaction between osteosarcoma cells and platelets, via thrombin and TGF-β, results in a continuous cycle, and that anti-platelet or anti-TGF-β therapy could be a promising tool for disease treatment. © 2018 American Society for Bone and Mineral Research.

PD-1 blockade therapy activates T cells by blocking the interaction between PD-1 and PD-L1 and promotes IFN-γ and Th1 cytokine production. In turn, IFN-γ and Th1 cytokines produced by activated T cells promote TF synthesis in monocytes/macrophages, which results in hypercoagulability leading to thrombosis.

Although cancer is associated with coagulation disorders, it is still unclear how the combination of tumor cell and host factors enhance the hypercoagulable state of cancer patients. Emerging evidence points to a central role for tumor endosomal and plasma membrane-derived vesicular components in the pathogenesis of cancer-related thrombosis. In particular, tumor cell membranes and extracellular vesicles (EV) harbor lipids and proteinaceous coagulation factors able to initiate multiple points within the coagulation matrix. The impact of chemotherapy upon a host already burdened with a hypercoagulable state increases the risk of pathological coagulation. We argue that chemotherapy-induced EV harbor the most active components for cancer related thrombosis and discuss how membrane components of the host and tumor act to initiate coagulation to enhance thrombotic risk in cancer patients.

Background Prostate cancer (PC) patients in advanced stages of the disease have high risk of blood coagulation complications. The procoagulant molecule Tissue factor (TF), and the fibrinolysis inhibitor plasminogen activator inhibitor-1 PAI-1 play important role in this complication. Extracellular vesicles (EV) shed from cancer cells may contribute to the regulation of TF and PAI-1. The procoagulant activity of EV can be associated with the oncogenic and metastatic characteristics of their cells. Methods We have expressed EGFRvIII in DU145 cells to assess the role of this oncogene in the procoagulant activity of EV. The intercellular exchange of TF via EV was assessed by downregulating its expression in DU145 cells using shRNA vector, and determining the transfer of TF via EV enriched with the protein. Two PC cell lines with different metastatic potential were used to assess the correlation between the procoagulant activity of EV and the metastatic potential of PC cells. Photometric assays were used to determine FXa-activity and thrombin generation as indicators for the procoagulant activity of EV. Double-tagged proteinase-activated receptor 1(PAR-1) expressed in CHO cells to assess its activation by EV. Results The expression of EGFRvIII in DU145 cells led to increased mRNA levels for TF and PAI-1, but the increase in these proteins expression was detected mostly in the EV. EV with enhanced levels of TF protein conferred higher TF procoagulant activity on the acceptor cells by intercellular exchange of this protein. Procoagulant activity of EV, assessed by FXa activity, and thrombin generation, was correlated with the oncogenic and metastatic potential of PC cells. The ability of EV to generate thrombin led to the activation of PAR-1, which was evident by the truncation of tagged-PAR-1. Conclusion The active oncogene EGFRvIII increases the concentration of TF and PAI-1 in EV. The procoagulant activity of EV is associated with the oncogenic and metastatic characteristics of their PC cells. Also, EV may contribute to the high procoagulant activity in the tumour microenvironment by the intercellular exchange of TF. Finally, through the generation of thrombin, EV can activate PAR-1, which evidently contributes to cancer progression, linking the coagulation system to tumor progression.

Introduction Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. Aim The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. Methods The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. Results Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. Conclusion The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.

Background and Purpose Protease-activated receptor 1 (PAR1) has been demonstrated to be involved in the pathogenesis of viral diseases. However, its role remains controversial. The goal of our study was to investigate the contribution of PAR1 to respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections. Experimental Approach Pharmacological approaches were used to investigate the role of PAR1 during RSV and hMPV infection, in vitro using epithelial A549 cells and in vivo using a mouse model of virus infection. Key Results In vitro, the PAR1 antagonist RWJ-56110 reduced the replication of RSV and hMPV in A549 cells. In agreement with these results, RWJ-56110-treated mice were protected against RSV and hMPV infections, as indicated by less weight loss and mortality. This protective effect in mice correlated with decreased lung viral replication and inflammation. In contrast, hMPV-infected mice treated with the PAR1 agonist TFLLR-NH2 showed increased mortality, as compared to infected mice, which were left untreated. Thrombin generation was shown to occur downstream of PAR1 activation in infected mice via tissue factor exposure as part of the inflammatory response, and thrombin inhibition by argatroban reduced the pathogenicity of the infection with no additive effect to that induced by PAR1 inhibition. Conclusion and Implications These data show that PAR1 plays a detrimental role during RSV and hMPV infections in mice via, at least, a thrombin-dependent mechanism. Thus, the use of PAR1 antagonists and thrombin inhibitors may have potential as a novel approach for the treatment of RSV and hMPV infections.