FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the TF–FVIIa–FXa complex

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FVIIa PREVENTS THE PROGRESSIVE HEMORRHAGING OF A BRAIN CONTUSION BY PROTECTING MICROVESSELS VIA FORMATION OF THE TF–FVIIa–FXa COMPLEX

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QIANG YUAN, a DALONG ZHANG, b SIRONG WU, b JIAN YU, a LEI YU, c YIRUI SUN, a ZHUOYING DU, a ZHIQI LI, a LIANGFU ZHOU, a XING WU a*y AND JIN HU a*y

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a Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, PR China

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b Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou, PR China

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pathways. In summary, the present findings demonstrated that FVIIa prevented the progressive hemorrhaging of brain contusions by protecting microvessel endothelial cells via the formation of the ternary TF–FVIIa–FXa complex. These findings are novel and of great clinical significance because FVIIa is used to prevent the progressive hemorrhaging of brain contusions in humans. Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Institute of Biomedical Engineering and Technology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai 200062, PR China

Key words: FVIIa, TF–FVIIa–FXa complex, endothelial cell, progressive hemorrhage, brain contusion.

Abstract—Factor VII (FVII) plays a key role in the initiation of the coagulation cascade and, in clinical situations, recombinant activated FVII (rFVIIa) effectively prevents progressive hemorrhaging after a brain contusion. However, it remains unclear whether decreases in FVII activity directly lead to progressive hemorrhaging and, moreover, the precise mechanisms underlying this process are not yet known. The present study demonstrated that decreased FVII activity directly led to progressive hemorrhaging of the cerebral contusions. Administration of FVII prevented the progression of hemorrhaging from cerebral contusions by protecting microvessel endothelial cells in the penumbra of the contusion. The present study also showed that the ternary TF–FVIIa–FXa complex cleaved endogenous proteaseactivated receptor 2 (PAR2) on endothelial cells, activated the p44/42 mitogen-activated protein kinase (MAPK) signaling cascade, and inhibited p65 nuclear factor-jB (NF-jB) signaling. Furthermore, exposure to ternary TF–FVIIa–FXa protected endothelial cells from thrombin- or inflammatory cytokine-induced apoptosis. Although activation of the p44/42 MAPK signaling pathway is endothelial cell protein C receptor (EPCR)-dependent, inhibition of the p65 NF-jB signaling pathway is EPCR-independent; thus, the regulation mechanism underlying the effects of TF–FVIIa–FXa in vascular endothelial cells appears to be multiple signaling

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*Corresponding authors. Address: Department of Neurosurgery, Huashan Hospital, Fudan University, 12 Wulumuqi Zhong Road, Shanghai 200040, PR China. E-mail addresses: [email protected] (X. Wu), [email protected] (J. Hu). y Xing Wu and Jin Hu contributed equally to this work. Abbreviations: APC, anticoagulant protein C/activated protein C; CCI, controlled cortical impact; CT, computed tomography; ELISA, enzymelinked immunosorbent assay; EPCR, endothelial cell protein C receptor; FVII, factor VII; GPCR, G protein-coupled receptor; HUVECs, human umbilical vein endothelial cells; IgG, Immunoglobulin G; MAPK, mitogen-activated protein kinase; mFVII, mouse FVII; MRI, magnetic resonance imaging; NE, neutrophil elastase; NF-jB, nuclear factor-jB; PAR2, protease-activated receptor 2; PKC, protein kinase C; PR3, neutrophil proteinase-3; TBI, traumatic brain injury; TEs, thromboembolic events; TF, tissue factor. http://dx.doi.org/10.1016/j.neuroscience.2017.02.020 0306-4522/Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Traumatic brain injury (TBI) encompasses numerous types of insults to the brain, and one of the most severe is a hemorrhagic cerebral contusion (Kurland et al., 2012). TBI associated with cerebral contusion is a frequent cause of death and disability in trauma victims who reach the hospital alive (Alahmadi et al., 2010). When head trauma results in a contusion, the hemorrhagic lesion often expands or a new hemorrhagic lesion develops remotely (non-contiguously) from the original contusion within several hours of impact (Oertel et al., 2002; Smith et al., 2007; Narayan et al., 2008). In the case of a typical contusion, enough energy is deposited near the epicenter of the impact to shear tissues and fracture microvessels, which, in turn, results in an immediate hemorrhagic lesion (i.e., the initial contusion) (Flaherty, 2010). In contrast, the amount of energy deposited in the penumbra, which is the shell of tissue surrounding the contusion, is not sufficient to shear tissues and fracture microvessels, but it can activate mechanosensitive molecular processes (Patel et al., 2010). Subsequently, a series of these events will eventually lead to the delayed catastrophic structural failure of microvessels and the hemorrhagic progression of a contusion (Simard et al., 2009). It is widely known that plasma coagulation factor VIIa (FVIIa) initiates the coagulation cascade by binding to its cofactor, tissue factor (TF), on cell surfaces, and that this eventually leads to fibrin deposition and platelet activation (Mann et al., 1988; Davie et al., 1991). Although clinical studies have reported that decreases in FVII activity is closely related to the occurrence of progressive hemorrhagic injury in patients with isolated TBI (Wu et al., 2014), the causal relationship has yet to be confirmed, and whether decreases in FVII

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activity directly lead to the occurrence of progressive hemorrhaging remains unclear. In addition to triggering the coagulation cascade, FVII is also involved in many other physiological and pathological mechanisms in vascular endothelial cells (Mackman, 2009; Pendurthi and Rao, 2008, 2010). For example, FVIIa alone binds to endothelial cell protein C receptor (EPCR), which is a receptor for anticoagulant protein C/activated protein C (APC), and activates protease-activated receptor (PAR)-1 to protect against thrombin-induced barrier disruption (Sen et al., 2011). Following brain injury, exposure to TF activates FVIIa, which causes the TF/FVIIa complex to bind to and cleave zymogen Factor X into FXa, which is the active protease. Formation of ternary TF–FVIIa–FXa is the major pathophysiological trigger underlying thrombin generation and blood coagulation (Davie et al., 1991); however, the protective nature of the ternary complex is now understood to be too complex to involve only a single independent factor. PARs comprise a class A G protein-coupled receptor (GPCR) family with currently four members, PAR1, PAR2, PAR3 and PAR4 that mediate the cellular effects of proteinases. Numerous proteinases have been shown to cleave and activate PAR1 including factor Xa, plasmin, kallikreins, APC, matrix metalloproteinase-1 (MMP1), neutrophil elastase (NE), and neutrophil proteinase-3 (PR3). As will be seen, this activation can result from exposure of a variety of ‘tethered ligands’ that can drive a variety of signaling pathways. PAR2, like PAR1, can also be activated by many serine proteinases including trypsin, NE, PR3, mast cell tryptase, TF/factor VIIa/factor Xa, human kallikreinrelated peptidases (KLKs) and membrane-tethered serine proteinase-1/matriptase 1 as well as by parasite cysteine proteinase, but is insensitive to thrombin. Like other GPCRs, the PARs signal via a variety of G proteins, including Gq, Gi and G12/13 but not directly via Gs. For G protein-mediated signaling, the receptor acts as a ligand-triggered guanine nucleotide exchange factor, stimulating the exchange of GTP for GDP in the Ga subunit of the heterotrimeric G protein oligomer. This exchange enables the ‘release’ of the Ga subunit from its tight binding to the Gbc dimer subunit. Each of the G protein moities (Ga-GTP and Gbc) are then independently able to interact with downstream signaling effectors like phospholipase C (Gq) or ion channels (Gbc). This ‘dual effector’ signaling, resulting in principle from the same PAR-activated G protein heterotrimer (e.g. GqGbc), can converge for complex downstream signaling, for instance leading to nuclear factor-jB (NFjB) activation and intracellular adhesion molecule-1 (ICAM-1) transcription by the engagement of parallel Gq/protein kinase C (PKC)- and Gi/phosphatidylinositol 3-kinase (PI3K) pathways that converge. Alternatively, as already indicated, via a ‘biased signaling’ process, PARs can be activated to affect selectively mitogenactivated protein kinase (MAPK) signaling via a G12/13triggered process, without causing a Gq-mediated calcium signaling event.

PAR-2 does not appear to be activated by thrombin or FVIIa, but FXa may activate this receptor (Coughlin, 2005; Feistritzer et al., 2005). A recent study demonstrated that the interaction of FX with endothelial cells leads to dissociation of EPCR from caveolin-1 and the recruitment of PAR-1 to a protective pathway (Disse et al., 2011). Additionally, the activation of FX by FVIIa on TF-bearing endothelial cells initiates protective signaling responses independent of EPCR mobilization via the activation of PAR-2 (Bae et al., 2010). However, it has also been shown that FXa plays a similar proinflammatory role in in vitro cellular models (Riewald and Ruf, 2001; Ahamed et al., 2006; Borensztajn et al., 2008; Krupiczojc et al., 2008). At present, the role of the ternary TF–FVIIa–FXa complex after brain injury remains unclear. Therefore, the primary goals of the present study were to examine whether decreased FVII activity would directly lead to the occurrence of progressive hemorrhaging in mice and whether administration of FVIIa would prevent the delayed catastrophic structural failure of microvessels and the progressive hemorrhaging of brain contusions by protecting vascular endothelial cells via formation of the ternary TF–FVIIa–FXa complex. Additionally, the present study aimed to identify the regulatory mechanism underlying the effects of ternary TF–FVIIa– FXa on vascular endothelial cells and to determine whether this effect is EPCR- or PAR-dependent.

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EXPERIMENTAL PROCEDURES

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Reagents

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For the present study, recombinant human activated FVII (rFVIIa) was obtained from Novo Nordisk, human FX and human thrombin were obtained from HYPHEN BioMed, tumor necrosis factor a (TNF-a) and interleukin (IL)-1b were purchased from Cell Signaling Technology, and the InvivofectamineÒ 2.0 Reagent was obtained from Life Technologies. Additionally, human umbilical vein endothelial cells (HUVECs) and 293T cells were purchased from ScienCell Research Laboratories (California, USA).

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In vivo FVII-targeting siRNA interference siRNA products corresponding to mouse FVII (mFVII) were targeted, and the oligonucleotide sequences were designed and synthesized as follows: miR-1- inhibitionF; 50 - rGrGrA fUfCrA fUfCfU fCrArA rGfUfC fUfUrA fCTT-30 and miR-1-inhibition-R; 50 - rGfUrA rArGrA fCfUfU rGrArG rAfUrG rAfUfC fCTT-30 . The InvivofectamineÒ 2.0 Reagent (Life Technologies) was used to deliver mFVII siRNA that targeted liver tissue to the mice via a tail vein injection according to the manufacturer’s instructions. The mice were randomly assigned to five groups based on the different doses of siRNA: sham and 1, 3, 5, and 10 mg/kg (n = 5/group). The sham group was given the same volume of a scrambled siRNA-InvivofectamineÒ 2.0 mixture. The efficacy of the FVII knockdown was evaluated at the

Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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mRNA level and by assessing reductions in protein activity in the plasma 2 days post-injection. Plasma samples were isolated and assayed to determine FVII concentrations using an enzyme-linked immunosorbent assay (ELISA), and FVII activity was assessed using a chromogenic substrate. FVII mRNA levels in the liver were determined with real-time polymerase chain reaction (PCR) assays.

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Controlled cortical impact model of brain contusion

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To implement the controlled cortical impact (CCI) model, a midline incision was made on the head of each mouse, the skin and temporal muscles were reflected to expose the skull, and a craniotomy was performed over the right parieto-temporal cortex using a portable drill and a 4-mm trephine. A CCI device (Pittsburgh Precision Instruments, Inc.) with the following characteristics was used on the brain: tip diameter = 3 mm, cortical contusion depth = 3 mm, and impact velocity = 1.5 m/s. The mice were randomly assigned to four groups: the FVII-inhibited group, which received in vivo administrations of FVII siRNA (3 mg/kg) via a tail vein injection to knock down the expression of FVII and then underwent the CCI model 2 days postinjection; the FVII-administered group, which received in vivo administrations of rFVIIa (0.5 mg/kg) immediately after undergoing the CCI model via a tail vein injection; the con-contusion injury group, which received in vivo administrations of normal saline (same volume as the other groups) and underwent the CCI model 2 days post-injection; and the invivofectamine alone group, which received in vivo administrations of invivofectamine alone (same volume as the other groups) and underwent the CCI model 2 days post-injection.

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Double-fluorescence immunohistochemistry

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Brain tissue samples were embedded with Tissue-Tek OCT mounting media and frozen in Isopentane cooled by liquid nitrogen. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using the In situ Cell Death Detection Kit (Roche Applied Science), and double-staining procedures for CD31/TUNEL and CD31/Cleaved Caspase-3 were performed on frozen tissue sections (20 mm) following fixation with ice-cold methanol for 10 min.

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Spectrophotometric assays of tissue blood

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The mice were sacrificed at various times after the CCI procedure (n = 4/group/time) and then perfused with heparinized saline to remove intravascular blood. The hemoglobin contents of the brains of mice subjected to the experimental procedures described above were quantified using a spectrophotometric assay with Drabkin’s reagent (Choudhri et al., 1997). Next, the concentration of hemoglobin was determined using the optical density (OD) of the solution at a wavelength of 550 nm.

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Magnetic resonance imaging analysis

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Magnetic resonance imaging (MRI) scans were performed using a high magnetic field micro-MR research scanner (7.0T Bruker PharmaScans, Bruker Biospin; Ettlingen, Germany). After being subjected to anesthesia with isoflurane, mice from each group underwent MRI scans to evaluate the occurrence of progressive hemorrhaging at 24, 48, and 72 h after the CCI injury. The estimated volume of the lesion was calculated using the following formula: A  B  C  0.5, where A and B represent the largest perpendicular diameters through the lesion area on the MRI scan and C represents the thickness of the lesion.

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Construction of the lentivirus and cell infection

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For the knockdowns of human EPCR, PAR1, and PAR2, shRNA sequences targeting the EPCR (GCAGTATGTGCAGAAACAT), PAR1 (CGGCAAGGTTTAAGTTATT), PAR2 (TCTTTGTAATGTGCTTATT), and negative control siRNA were designed by Shanghai GeneChem Company (Shanghai, China). HUVECs were infected with EPCR-specific, PAR1-specific, or PAR2-specific shRNA-expressing lentiviruses (EPCR KD, PAR1 KD, and PAR2 KD groups, respectively), and HUVECs infected with scrambled shRNA-expressing lentiviruses were used as negative controls (scrambled shRNA group).

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Flow cytometry detection of HUVEC apoptosis using Annexin V-APC single staining

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Apoptosis was determined using an Annexin-V-APC apoptosis detection kit (eBioscience; San Diego, CA, United States). The HUVECs were stimulated with TNFa and IL-1b and then treated with FVIIa or FVIIa + FX for different time intervals or a fixed time period. Next, the cells were stained with 10 mL of Annexin V-APC at room temperature for 15 min in the dark and transferred into flow cytometry tubes for detection with a Guava easyCyte HT (Millipore; USA). Cells positive for Annexin V-APC were considered apoptotic.

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Detection of HUVEC proliferation with MTT assays

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For the MTT assays, five 96-well plates were used, and observations were carried out for 5 d. Within the period from the second day of plating to 4 h prior to the termination of the culture, 20 mL of MTT (5 mg/mL; Genview) was added to each well without changing the medium. After 4 h, the culture medium was discarded and 100 mL of DMSO was added to each well to stop the reaction. After vortexing for 2–5 min, the OD values were detected at 490 nm using a microplate reader.

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Activations of p44/42 MAPK, p38 MAPK, and p65 NFjB signaling pathways

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The HUVECs were first stimulated with TNF-a and IL-1b and then treated with FVIIa or FVIIa + FX for different time intervals or a fixed time period. At the end of the treatment period, the supernatant was removed and the

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cells were lysed with a polyacrylamide gel electrophoresis sample buffer. Next, equal amounts of protein were subjected to polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and probed with phospho-specific and total p44/42 MAPK and p65 NF-jB antibodies (Cell Signaling Technology); the blots were developed using chemiluminescence. FVII activity in patients with brain contusion hemorrhages

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The present study evaluated 141 patients with brain contusion hemorrhages who were admitted to the Neurotrauma Center in the Department of Neurosurgery at Huashan Hospital, which is Affiliated with Fudan University, between April 2010 and April 2015. The inclusion criteria were Glasgow Coma Scale (GCS) score at admission less than 13, an extracranial Abbreviated Injury Scale (AIS) score less than 3, admission within 6 h after injury, brain contusion hemorrhages seen on head computed tomography (CT) at admission, two or more head CT scans obtained in the first 72 h. Exclusion criteria were use of anticoagulant (clopidogrel, aspirin, and coumarin, et al.), preexisting hypercoagulative state or thromboembolic events (TEs), known history of thrombocytopenia, or liver failure. Progressive hemorrhaging of the cerebral contusion was defined as those with a 15% or more increase in contusion size on follow-up CT scan (progression on CT scans could result from expansion of the hematoma or development of new lesions that were not present on initial scans). Within 8 h of admission, plasma samples from each patient were isolated and assayed with an ELISA to determine FVII concentration. The neuroradiologists interpreted CT scan blinded to levels of FVII.

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Statistical analysis

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Values are reported as mean ± SEM. Statistical significance (P value) of continuous variables for two groups was determined using the unpaired Student’s t-

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test. Continuous variables for more than two groups were compared using an analysis of variance (ANOVA) firstly. Then the LSD method was used for multiple comparison between any two groups. All graphs were plotted using the GraphPad Prism 5 or SPSS 20.0 software. For FVII activity in patients with brain contusion hemorrhages, Chi-square test or two-sided Fisher’s exact test was used for categorical variables. The relationship between FVII activity and the progressive hemorrhaging of the cerebral contusion was evaluated with a nonlinear regression analysis. Values of p < 0.05 were considered statistically significant.

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RESULTS

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FVII was knocked down by the in vivo delivery of FVIItargeting siRNA

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The injections of InvivofectamineÒ 2.0 complexed with FVII siRNA (1, 3, 5, and 10 mg/kg) significantly decreased the expression of FVII mRNA in the liver compared with the negative siRNA control group (Fig. 1A). At 48 h after the injections, 1, 3, 5, and 10 mg/ kg injections of FVII siRNA decreased the plasma concentrations of FVII from 0.90 ± 0.12 mg/mL to 0.70 ± 0.05 mg/mL, 0.48 ± 0.05 mg/mL (P < 0.01), 0.36 ± 0.03 mg/ mL (P < 0.01), and 0.06 ± 0.01 mg/ mL (P < 0.001), respectively (Fig. 1B). At 48 h after the injections, 1, 3, 5, and 10 mg/kg injections of FVII siRNA decreased plasma FVII activity levels to 64.1 ± 5.1% (P < 0.001), 30.8 ± 5.6% (P < 0.001), 14.6 ± 3.4% (P < 0.001), and 5.7 ± 0.1% (P < 0.001; Fig. 1C), respectively. These findings indicate that in vivo injections of the InvivofectamineÒ 2.0 Reagent complexed with siRNA that targeted FVII mRNA effectively knocked down the expression of FVII.

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Decreases in FVII levels directly led to the progressive hemorrhaging of brain contusions

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The in vivo expression of FVII was knocked down by injecting FVII-targeting siRNA (3 mg/kg) into mice; as in

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Fig. 1. FVII was knocked down in vivo by the delivery of FVII-targeting siRNA. Different doses of the InvivofectamineÒ 2.0-siRNA duplex mixture were slowly (20–40 lL/s) injected via the tail vein with a maximum volume of 300 lL. The 1 mg/kg siRNA dose corresponded to 1.5 lL/g of the InvivofectamineÒ 2.0-siRNA duplex mixture. The mice were randomly assigned to five groups based on the different doses of siRNA: sham and 1, 3, 5, and 10 mg/kg (n = 5/group). The efficacy of the FVII knockdown was evaluated at the mRNA level and by assessing reductions in protein activity in the plasma 2 days post-injection. (A) FVII mRNA levels in the liver were assessed with real-time PCR assays. (B) Plasma was isolated and assayed with an ELISA to determine FVII concentration. (C) FVII activity was represented with a chromogenic substrate. All data are presented as mean ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the sham group. Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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the FVII-inhibited group, the CCI brain model was imposed 48 h after the injection. Additionally, as in the FVII-administered group, 20 mg/kg of rFVIIa was injected into the mice immediately after they underwent the brain contusion model. The quantification of cerebral contusion hemorrhaging revealed that the estimated amounts of bleeding at 3, 24, and 72 h after the CCI injury were significantly higher in the FVII-inhibited group than in the FVII-administered group, con-contusion injury group and invivofectamine alone group. The estimated amount of bleeding at 72 h after the CCI injury was also significantly lower in the FVIIadministered group than in the con-contusion injury and invivofectamine alone groups. Additionally, the estimated amount of bleeding at 72 h after the CCI injury in the FVII-inhibited group was significantly greater than that at 3 and 24 h after the CCI injury, whereas the differences in bleeding among these time points did not significantly differ in the other three groups (Fig. 2A, B). The estimated volumes of the cerebral contusion hemorrhages were also evaluated using 7.0T MRI scans at 24, 48, and 72 h after the CCI injury. The estimated volumes of bleeding at 24, 48, and 72 h after the CCI injury in the FVII-inhibited group were significantly higher than those of the FVII-administered, con-contusion injury and the invivofectamine alone groups. The estimated volumes of bleeding at 48 and 72 h after the CCI injury in the FVII-administered group were also significantly lower than those of the concontusion injury and invivofectamine alone groups. Additionally, the estimated volume of bleeding at 72 h after the CCI injury in the FVII-inhibited group was significantly greater than those at 24 and 48 h after the CCI injury, whereas the differences in the estimated volumes of bleeding among these timepoints did not significantly differ in the other three groups (Fig. 2C, D). FVII decreased endothelial cell apoptosis in the penumbrae of brain contusions The penumbrae of the brain contusions (Fig. 3A) were examined using dual CD31/Cleaved Caspase-3 and dual CD31/TUNEL immunofluorescence analyses. The dual CD31/TUNEL staining revealed that endothelial cell apoptosis increased in the FVII-inhibited group and decreased in the FVII-administered group compared with in the con-contusion injury group (Fig. 3B). Furthermore, the level of staining for dual CD31/ Cleaved Caspase-3-specific labeled cells in the penumbra of the contusion was higher in the FVIIinhibited group and lower in the FVII-administered group than in the con-contusion injury group (Fig. 3C). Taken together, these findings indicate that the in vivo level of FVII was associated with vascular endothelial cell apoptosis in the penumbra of the contusion. Additionally, the FVII-inhibited group exhibited a greater degree of Immunoglobulin G (IgG) exudation in the penumbra of the contusion than did the concontusion injury group. This suggests that the decreased in vivo level of FVII was associated with increases in the vascular permeability in the penumbra

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of the contusion; thus, the in vivo level of FVII may be associated with injury to vascular endothelial cells (Fig. 3D).

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Ternary TF–FVIIa–FXa complex-induced activation of p44/42 MAPK and p65 NF-jB signaling and antiapoptotic effects in endothelial cells

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The mRNA expression levels of EPCR, PAR1, and PAR2 in the negative control group were higher than those in the EPCR-, PAR1-, and PAR2-knockdown groups, respectively (Fig. 4A); these findings demonstrate that the gene knockdowns were effective. The results of the Annexin V-APC single staining procedure showed that both the HUVEC and EPCR-knockdown HUVEC groups exhibited significant decreases in the rate of apoptosis after administration of either FVIIa or FVII + FX (Fig. 4B); thus, EPCR expression did not alter the antiapoptotic effects of the FVIIa–TF–FXa complex. The MTT assay revealed that FVIIa and FVII + FX both led to significant increases in the cellular growth of HUVECs in the HUVEC and EPCR-knockdown HUVEC groups (Fig. 4C). Under the conditions of no stimulation, HUVECs did not express TF. However, stimulation by either thrombin (16 NIH/mL), TNF-a, or IL-1b resulted in a significant increase in the expression of TF in HUVECs (Fig. 4D). To determine the effects of the ternary TF–FVIIa–FXa complex-induced activation of p44/42 MAPK and p65 NF-jB signaling in HUVECs, thrombin, TNF-a, or IL-1b was used to stimulate the expression of TF in the cells, and the cells were then treated with FVIIa or FVIIa + FX. The treatment of thrombin-stimulated HUVECs with FVIIa + FX attenuated p65 NF-jB phosphorylation but induced p44/42 MAPK phosphorylation (Fig. 5B). Similar results were observed in TNF-a- and IL-1bstimulated HUVECs; FVIIa and FX decreased p65 NFjB phosphorylation but enhanced p44/42 MAPK phosphorylation. On the other hand, FVIIa alone did not induce either p44/42 MAPK or p65 NF-jB phosphorylation (Fig. 5A). The present study also investigated whether the FVIIa + FX-induced increase in p44/42 MAPK phosphorylation and/or the attenuation in p65 NF-ΚB phosphorylation were/was dependent on EPCR (Fig. 5C, D). The EPCR knockdown inhibited FVIIa + FX-induced p44/42 MAPK phosphorylation but had no effect on the attenuation of p65 NF-jB phosphorylation. These results clearly indicate that the activation of p44/42 MAPK signaling by ternary TF– FVIIa–FXa was EPCR-dependent and that the inhibition of p65 NF-jB signaling by ternary TF–FVIIa–FXa was EPCR-independent. Taken together, these findings suggest that the regulatory mechanism underlying the effects of ternary TF–FVIIa–FXa in vascular endothelial cells involves multiple signaling pathways. Furthermore, the silencing of PAR2 by an shRNAexpressing lentivirus inhibited the FVIIa + FX-induced increases in p44/42 MAPK phosphorylation and the decreases in p65 NF-jB phosphorylation (Fig. 6), whereas the silencing of PAR1 had no effect on the FVIIa + FX-induced changes. These data clearly

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Fig. 2. Decreased FVII levels directly led to the progressive hemorrhaging of brain contusions. Mice were randomly assigned to four groups: the FVII-inhibited group, which received in vivo administrations of FVII siRNA (3 mg/kg) to knock down the expression of FVII and then underwent the CCI model 2 days post-injection; the FVII-administered group, which received in vivo administrations of rFVIIa (0.5 mg/kg) immediately after undergoing the CCI model; the con-contusion injury group, which received in vivo administrations of normal saline (same volume as the other groups) and underwent the CCI model 2 days post-injection; and the invivofectamine alone group, which received in vivo administrations of invivofectamine alone (same volume as the other groups) and underwent the CCI model 2 days post-injection. (A) When examined at 3, 24, and 72 h after the CCI injury, both the surface and coronal views revealed a significantly greater progressive spread of the hemorrhagic lesions to deeper tissues in the FVII-inhibited group compared with in the con-contusion injury group and invivofectamine alone group; in contrast, the spread of the hemorrhagic lesions in the FVII-administered group did not significantly differ from that in the con-contusion injury group and invivofectamine alone group. (B) The hemoglobin contents in the cerebral contusions were quantified with a spectrophotometric assay. The estimated amounts of bleeding at 3, 24, and 72 h after the CCI injury were significantly higher in the FVII-inhibited group than in other three groups. The estimated amount of bleeding at 72 h after the CCI injury was also significantly lower in the FVII-administered group than in the con-contusion injury group and the invivofectamine alone group. *P < 0.05, **P < 0.01, ***P < 0.001 versus con-contusion injury group and invivofectamine alone group. Additionally, the estimated amount of bleeding at 72 h after the CCI injury in the FVII-inhibited group was significantly greater than the estimated amounts at 3 and 24 h after the CCI injury (#P < 0.05, ###P < 0.001), whereas the differences in bleeding among these timepoints did not significantly differ in the other three groups. (D) The estimated volumes of cerebral contusion hemorrhages were also evaluated using 7.0T MRI scans at 24, 48, and 72 h after the CCI injury. The estimated volumes of bleeding at 24, 48, and 72 h after the CCI injury in the FVII-inhibited group were significantly higher than those of the other three groups. The estimated volumes of bleeding at 48 and 72 h after the CCI injury in the FVII-administered group were also significantly lower than those of the con-contusion injury group and the invivofectamine alone group. *P < 0.05, ***P < 0.001 versus con-contusion injury group and the invivofectamine alone group. Additionally, the estimated volume of bleeding at 72 h after the CCI injury in the FVII-inhibited group were significantly greater than the estimated volumes at 24 and 48 h after the CCI injury (#P < 0.05, ###P < 0.001). (C) Brain hemorrhages, depicted as low-density areas in the T2-weighted, T1-weighted, and T2 Flash images (white arrow), were evident in the percussion site after brain contusion. Brain edema, depicted as high-density areas in the T2-weighted images (black arrow), were evident in the percussion site after brain contusion. The low-intensity area expanded to a very wide region emanating from the contusion site in the FVII-inhibited group. Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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Fig. 3. (A) Rectangular areas showing the penumbra region of a representative brain contusion sampled for immunofluorescence analysis. (B) The penumbrae of the brain contusions were examined with dual CD31/Cleaved Caspase-3 and dual CD31/TUNEL immunofluorescence analyses. The dual CD31/TUNEL staining revealed that endothelial cell apoptosis increased significantly in the FVII-inhibited group and decreased in the FVIIadministered group compared with in the control group. (C) The level of staining for dual CD31/Cleaved Caspase-3-specific labeled cells in the penumbra of the contusion was higher in the FVII-inhibited group and lower in the FVII-administered group than in the control group. (D) The FVIIinhibited group exhibited a greater degree of Immunoglobulin G (IgG) exudation in the penumbra of the contusion than did the con-contusion injury group and FVII-administered group. This suggests that the decreased in vivo level of FVII was associated with increases in the vascular permeability in the penumbra of the contusion. Scale bar = 100 mm.

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indicate that ternary TF–FVIIa–FXa mediated cell signaling in endothelial cells via the activation of PAR2.

Plasma FVII activity levels in patients with cerebral contusion hemorrhages were closely related to the occurrence of progressive hemorrhaging Of the 141 patients with cerebral contusion hemorrhages who were admitted to the Neurotrauma Center and assessed in the present study, 52 (36.9%) exhibited progressive hemorrhaging of the cerebral contusion. The median (interquartile) level of FVII activity in patients with progressive hemorrhaging of the cerebral contusion was 77.3% (62.0–92.3%), which was significantly lower than in patients without progressive hemorrhaging of the cerebral contusion (95.9% [69.3– 120.9%]; P < 0.001). After adjusting for other factors that could affect hemorrhaging, FVII activity and the occurrence of progressive hemorrhaging in cerebral contusions exhibited a curvilinear correlation (R2 = 0.245, area under the receiver operating characteristic [ROC] curve = 0.748; Fig. 7A). In particular, three TBI cases that presented with

progressive hemorrhaging of the cerebral contusion showed lower levels of plasma FVII activity (Fig. 7B–D).

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rFVIIa is approved for use in patients with hemophilia but recently has been used as an off-label treatment for coagulopathies and life-threatening bleeding in trauma patients (Dutton et al., 2003, 2004; Morey, 2005). Although rFVIIa is a safe and effective drug for the treatment of these issues in TBI patients, the correlation between in vivo FVII activity and the progressive hemorrhaging of brain contusions remains unclear and, importantly, the causal relationship between these two variables has yet to be confirmed. Based on the clinical and experimental findings of the present study, this is the first report to demonstrate that decreases in FVII activity directly lead to increases in contusion hemorrhaging and FVII could prevented the progression of hemorrhaging from cerebral contusions by protecting microvessel endothelial cells in the penumbra of the contusion. In vivo FVII activity is a key factor to consider when determining whether a cerebral contusion hemorrhage

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Fig. 4. Knockdown efficiencies of EPCR, PAR1, and PAR2 by EPCR-specific, PAR1-specific, and PAR2-specific shRNA-expressing lentiviruses in HUVECs. The EPCR, PAR1, and PAR2 mRNA expressions in the HUVECs were determined with a real-time PCR assay. (A) The mRNA expression levels of EPCR, PAR1, and PAR2 in the negative control group were higher than those in the EPCR-, PAR1-, and PAR2-knockdown groups, respectively; thus, the gene knockdowns were effective. ***P < 0.001. (B) HUVECs or HUVECs transfected with EPCR-specific shRNAexpressing lentiviruses were stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by treatment with either FVIIa (2 mg/mL) or FVIIa (2 mg/ mL) + FX (1 mg/mL) for 3 h. Cell apoptosis was determined using Annexin V-APC single staining; *P < 0.05, **P < 0.01, ***P < 0.001 compared with TNF-a and IL-1b stimulation alone. (C) HUVECs or HUVECs transfected with EPCR-specific shRNA-expressing lentiviruses were stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by treatment with either FVIIa (2 mg/mL) or FVIIa (2 mg/mL) + FX (1 mg/mL) for 3 h. Cell proliferation was determined using MTT assays. ***P < 0.001 for TNF-a + IL-1b + FVIIa stimulation compared with TNF-a + IL-1b stimulation alone. ###P < 0.001 for TNF-a + IL-1b + FVIIa + FX stimulation compared with TNF-a + IL-1b stimulation alone. (D) HUVECs were treated with FVIIa (2 mg/mL), FVIIa (2 mg/mL) + FX (1 mg/mL), TNF-a + IL-1b (20 ng/mL), TNF-a + IL-1b (20 ng/mL) + FVIIa (2 mg/mL), TNF-a + IL-1b (20 ng/mL) + FVIIa (2 mg/mL) + FX(1 mg/mL), or thrombin (4, 8, or 16 NIH/mL) for 3 h. TF expression was determined using Western blot analyses. *** P < 0.001 versus NC group, FVIIa group, FVIIa + FX group, thrombin (4 NIH/mL) group or thrombin (8 NIH/mL) group.

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will progress. However, the present study found that FVII activity and the occurrence of the progressive hemorrhaging of a cerebral contusion have a curvilinear relationship. When FVII activity was between 80% and 120%, the risk of progressive hemorrhaging dramatically increased as FVII activity decreased. These results suggest that the maintenance of in vivo FVII activity at levels above 120% may decrease the risk of progressive cerebral contusion hemorrhaging. However, when FVII activity was less than 80% or greater than 120%, the risk of progressive hemorrhaging of the cerebral contusion did not significantly change as FVII activity changed. Therefore, in clinical practice, a small dose of FVII may effectively prevent progressive hemorrhaging of a cerebral contusion, whereas a larger dose may increase the incidence of adverse events without having any ameliorative effects on the hemorrhaging. Previous studies from our research group have shown that the use of low-dose rFVIIa (20 mg/kg) effectively prevents the occurrence of progressive hemorrhagic injuries in patients with TBI without an increase in TEs (Yuan et al., 2015). Future research need to conduct full dose–response studies in animal models that include a pharmacokinetic analysis

of exogenously administered FVII to fully elucidate the relationship between circulating FVII and contusion size. The present study also revealed an important mechanism of FVII that was involved in the prevention of the progressive hemorrhaging of a cerebral contusion. In the cerebral contusion hemorrhage patients included in the present study, FVII not only restored hemostasis via the generation of thrombin but also protected the endothelium in the penumbra of the contusion against the delayed catastrophic structural failure of microvessels. Endothelial cell apoptosis increased in the FVII-inhibited group and decreased in the FVII-administered group compared with in the control group, which indicates that the in vivo FVII level was associated with vascular endothelial cell apoptosis in the penumbra of the contusion. Therefore, prophylactic administrations of low-dose FVIIa could prevent progressive hemorrhagic injury in patients with contusions by ameliorating early bleeding via the generation of thrombin and may also provide long-term benefits by protecting the integrity of the vascular endothelium. FVIIa is the protease of an extrinsic pathway that binds to the TF cell surface receptor on negatively

Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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Fig. 5. FVIIa- and FVIIa + FX-mediated activations of p44/42 MAPK and p65 NF-jB signaling in HUVECs stimulated or not stimulated by TNF-a/IL-1b analyzed using Western blot analyses with specific antibodies. (A) Lane 1: not treated control, Lane 2: FVIIa (2 mg/mL) for 3 h, Lane 3: FVIIa (2 mg/mL) + FX (1 mg/mL) for 3 h, Lane 4: cells stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h, Lane 5: cells stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by incubation with FVIIa (2 mg/mL) for 3 h, Lane 6: cells stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by incubation with FVIIa (2 mg/mL) + FX (1 mg/mL) for 3 h. (B) All procedures were the same as (A), except that the cells were stimulated with thrombin (16 NIH/mL) for 1 h instead of with TNF-a and IL-1b (20 ng/mL) for 6 h. (C) HUVECs and HUVECs transfected with EPCR-specific shRNAexpressing lentiviruses were stimulated with TNF-a and IL-1b followed by incubation with FVIIa or FVIIa + FX for 3 h. The expressions of p44/42 MAPK and p65 NF-jB were determined using Western blot analyses. Lane 1: HUVECs stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h, Lane 2: HUVECs stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by incubation with FVIIa (2 mg/mL) for 3 h, Lane 3: HUVECs stimulated with TNF-a and IL-1b (20 ng/mL) for 6 h followed by incubation with FVIIa (2 mg/mL) + FX (1 mg/mL) for 3 h, Lanes 4–6: the same as Lanes 1–3, respectively, except that HUVECs transfected with EPCR-specific shRNA-expressing lentiviruses were used as experimental cells instead of control HUVECs. (D) All procedures were the same as (C), except that the experimental cells were stimulated with thrombin (16 NIH/mL) for 1 h instead of with TNF-a and IL-1b. All data are presented as mean ± 95% CI; *P < 0.05, **P < 0.01, *** P < 0.001.

charged membrane phospholipids (Ruf et al., 2003). This factor induces the change of Factor X to FXa and forms the ternary TF–FVIIa–FXa complex, which initiates the clotting cascade. Although previous studies have reported that the binary FVIIa– TF complex activates PAR2 and elicits intracellular signaling responses in various cell types independent of its pro-coagulant activity (Ruf et al., 2003; Awasthi et al., 2007), the present study did not observe this phenomenon. This discrepancy may be due to the fact that the binary TF– FVIIa complex directly cleaves PAR2 and requires 10 nM of FVIIa, whereas subnanomolar concentrations of FVIIa mainly drive coagulation (Disse et al., 2011). In the present study, only lower concentrations of FVIIa that may be closer to physiological levels were used. On the other hand, the signaling of the ternary TF–FVIIa–FXa complex can be activated by lower concentrations of FXa. The present findings demonstrated that ternary TF–FVIIa–FXa cleaved endogenous PAR2 on endothelial cells, activated p44/42 MAPK signaling, and inhibited p65 NF-jB signaling. The exposure of endothelial cells to ternary TF–FVIIa–FXa protects them from thrombin- or inflammatory cytokine-induced apoptosis, and FXa can inhibit the barrier-disruptive effect of proinflammatory cytokines in HUVECs via the activation of PAR2 by a mechanism that is partly independent of the Gla-domain of the protease (Manithody et al., 2012; Rana et al., 2012). The physiological significance of FXa signaling in cellular models remains unclear because a high concentration of FXa (20 nM) was required to observe a signaling effect in these studies. However, the signaling of the ternary TF– FVIIa–FXa coagulation activation complex occurs at lower concentrations of FXa compared with reactions directly initiated by the addition of FXa, which is consistent with the present findings. In addition to the TF-dependent signaling that occurs via PAR2, FVIIa also activates PAR1 to initiate intracellular signaling responses in endothelial cells (Ghosh et al., 2007; Pendurthi and Rao, 2010; Sen et al.,

Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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Fig. 6. FVIIa- and FVIIa + FX-mediated activations of p44/42 MAPK signaling in HUVECs transfected with scrambled shRNA-expressing lentiviruses, PAR1-specific shRNA-expressing lentiviruses, or PAR2-specific shRNA-expressing lentiviruses as determined using Western blot analyses. (A) Lane 1: HUVECs transfected with scrambled shRNA-expressing lentiviruses that were not treated; Lane 2: HUVECs transfected with scrambled shRNA-expressing lentiviruses stimulated with thrombin (16 NIH/mL) for 1 h; Lane 3: HUVECs transfected with scrambled shRNAexpressing lentiviruses stimulated with thrombin (16 NIH/mL) for 1 h followed by incubation with FVIIa (2 mg/mL) for 3 h; Lane 4: HUVECs transfected with scrambled shRNA-expressing lentiviruses stimulated with thrombin (16 NIH/mL) for 1 h followed by incubation with FVIIa (2 mg/mL) + FX (1 mg/mL) for 3 h; Lanes 5–8: the same as Lanes 1–4, respectively, except that HUVECs transfected with PAR1-specific shRNA-expressing lentiviruses were used as experimental cells instead of HUVECs transfected with scrambled shRNA-expressing lentiviruses; Lanes 9–12: the same as Lanes 1–4, respectively, except that HUVECs transfected with PAR2-specific shRNA-expressing lentiviruses were used as experimental cells instead of HUVECs transfected with scrambled shRNA-expressing lentiviruses. (B) All procedures were the same as (A), except that the FVIIa- and FVIIa + FX-mediated activations of p65 NF-jB were analyzed using Western blot analyses. All data are presented as mean ± 95% CI; *P < 0.05, ** P < 0.01, ***P < 0.001.

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2011). In an interesting series of recent studies, Rao et al. (Ghosh et al., 2007; Sen et al., 2011) showed that EPCR functions as a true receptor for FVIIa and modulates the PAR1-dependent signaling specificity of FVIIa. Similar to APC, FVIIa binds with EPCR at a high affinity in both in vitro and in vivo model systems to effectively activate PAR1 and elicit protective signaling responses in endothelial cells (Ghosh et al., 2007; Pendurthi and Rao, 2010; Sen et al., 2011). However, the present results support only the role of ternary TF–FVIIa–FXa in the mediation of cell signaling in endothelial cells via the activation of PAR2. Under conditions of hemorrhaging, thrombin is generated in large amounts and can combine with and activate PAR1 but not PAR2. Although FVIIaand ternary TF–FVIIa–FXa-induced PAR1 signaling has been reported, PAR1 cleavage was detected only when thrombin was omitted from the reaction mixture. In contrast, the present study first administered thrombin to stimulate the expression of TF in cells and then treated the cells with FVIIa or FVIIa + FX. Therefore, PAR1 signaling was not detected in the presence of thrombin, which is closer to pathophysiological conditions. Although it has been shown that EPCR supports ternary TF–FVIIa–FXa signaling through the PARs (Disse et al., 2011), the authors of that study assessed only the p44/42 MAPK signaling pathway. The present findings clearly indicate that the activation of the p44/42 MAPK signaling pathway by ternary TF–FVIIa–FXa is

EPCR-dependent, which is consistent with previous reports. However, the present study also showed that the inhibition of the p65 NF-jB signaling pathway by ternary TF–FVIIa–FXa is EPCR-independent. Taken together, these findings suggest that the regulatory mechanism underlying the effects of ternary TF–FVIIa–FXa on vascular endothelial cells is a multi-channel process. In summary, the present findings demonstrated that decreased in vivo FVII activity directly led to increases in contusion hemorrhaging as well as to progressions in the level of hemorrhaging. However, FVIIa prevented this progression by initiating endothelial barrierprotective effects. Additionally, ternary TF–FVIIa–FXa activated PAR2-mediated cell signaling to initiate the endothelial barrier-protective effects. The regulatory mechanisms underlying the actions of ternary TF– FVIIa–FXa are multiple signaling pathways rather than completely EPCR-dependent. Future studies are required to determine the optimal medication time and dose of FVIIa that will prevent the progressive hemorrhaging of brain contusions and provide endothelial barrier-protective effects.

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Contribution: Q.Y. designed the research, performed most of the experiments described in the manuscript, analyzed the data, and compiled the figures; J.Y., S.R.

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Fig. 7. Relationship between FVII activity and the progressive hemorrhaging of cerebral contusions in TBI patients according to a nonlinear regression analysis. After adjusting for other factors that affect the progressive hemorrhaging of cerebral contusions, FVII activity and progressive hemorrhaging of the cerebral contusion exhibited a curvilinear correlation. (A) When FVII activity was between 80% and 120%, the risk of progressive hemorrhaging of the cerebral contusion significantly increased as FVII activity decreased. However, when FVII activity was less than 80% or greater than 120%, the risk of progressive hemorrhaging of the cerebral contusion did not significantly change as FVII activity changed. (B) Case 1 was a 57-year-old male who stumbled and suffered TBI 6 h prior to admission to our hospital. Emergency head CT scans showed multiple frontal and temporal cerebral contusions with a thin-layer subdural hematoma and traumatic subarachnoid hemorrhage (B1). Twenty-four hours after the injury, an additional head CT scan showed a significant increase in the volume of the right frontal contusion with a midline shift (B2); the patient’s FVII activity upon admission was only 61.3%. (C) Case 2 was a 65-year-old male who was knocked down by a car while riding a bicycle on the street 3 h prior to admission to our hospital. Emergency head CT scans showed bilateral frontal and left temporal cerebral contusion hemorrhages and traumatic subarachnoid hemorrhages (C1). Approximately 3 h after admission, an additional head CT scan showed a significant increase in the volumes of the bilateral frontal and left temporal cerebral contusion hemorrhages (C2); the patient’s FVII activity upon admission was only 67.1%. (D) Case 3 was a 47-year-old male who was knocked down by a car while riding a bicycle on the street 20 h prior to admission to our hospital; he was transferred from a local hospital. Emergency head CT scans showed a right frontal cerebral contusion hemorrhage and traumatic subarachnoid hemorrhage (D1). Three days after the injury, an additional head CT scan showed a significant increase in the volume of the right frontal cerebral contusion hemorrhage and a new left frontal cerebral contusion (D2); the patient’s FVII activity upon admission was only 50.6%.

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W. and D.R.Z. performed experiments involving mice and analyzed the data; Y.R.S., Z.Y.D. and Z.Q.L. performed experiments involving cell and analyzed the data; L.F.Z. and L.Y. contributed to the research design, guided experiments involving siRNAs, and participated in the preparation of manuscript; and X.W. and J.H. contributed to overall research design, analyzed the data, and wrote the manuscript. All authors read the manuscript and contributed to the preparation of the final version. Conflict-of-interest-disclosure: The authors declare no competing financial interests.

Acknowledgments—This work was supported by the National Natural Science Foundation of China (NSFC Grants 81271375 and 81471241).

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(Received 10 October 2016, Accepted 12 February 2017) (Available online xxxx)

Please cite this article in press as: Yuan Q et al. FVIIa prevents the progressive hemorrhaging of a brain contusion by protecting microvessels via formation of the tf–fviia–fxa complex. neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.02.020

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