Fibrin promotes oxidative stress and neuronal loss in traumatic brain injury via innate immune activation.

BACKGROUND: Traumatic brain injury (TBI) causes significant blood-brain barrier (BBB) breakdown, resulting in the extravasation of blood proteins into the brain. The impact of blood proteins, especially fibrinogen, on inflammation and neurodegeneration post-TBI is not fully understood, highlighting a critical gap in our comprehension of TBI pathology and its connection to innate immune activation. METHODS: We combined vascular casting with 3D imaging of solvent-cleared organs (uDISCO) to study the spatial distribution of the blood coagulation protein fibrinogen in large, intact brain volumes and assessed the temporal regulation of the fibrin(ogen) deposition by immunohistochemistry in a murine model of TBI. Fibrin(ogen) deposition and innate immune cell markers were co-localized by immunohistochemistry in mouse and human brains after TBI. We assessed the role of fibrinogen in TBI using unbiased transcriptomics, flow cytometry and immunohistochemistry for innate immune and neuronal markers in Fggγ390-396A knock-in mice, which express a mutant fibrinogen that retains normal clotting function, but lacks the γ390-396 binding motif to CD11b/CD18 integrin receptor. RESULTS: We show that cerebral fibrinogen deposits were associated with activated innate immune cells in both human and murine TBI. Genetic elimination of fibrin-CD11b interaction reduced peripheral monocyte recruitment and the activation of inflammatory and reactive oxygen species (ROS) gene pathways in microglia and macrophages after TBI. Blockade of the fibrin-CD11b interaction was also protective from oxidative stress damage and cortical loss after TBI. CONCLUSIONS: These data suggest that fibrinogen is a regulator of innate immune activation and neurodegeneration in TBI. Abrogating post-injury neuroinflammation by selective blockade of fibrin's inflammatory functions may have implications for long-term neurologic recovery following brain trauma.

Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

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

Novel Strategy to Combat the Procoagulant Phenotype in Heparin-Induced Thrombocytopenia Using 12-LOX Inhibition.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling.

Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots which assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and the insoluble character of fibrin clots, have restricted our ability to develop novel treatments for clotting diseases. The, so far resolved, disparate structural details have provided insights into linear elongation; however, molecular details like the C-terminal domain of the α-chain, the heparin-binding domain on the ß-chain, and other functional domains remain elusive. To illuminate these dark areas, we applied cross-linking mass spectrometry (XL-MS) to obtain biochemical evidence in the form of over 300 distance constraints and combined this with structural modeling. These restraints additionally define the interaction network of the clots and provide molecular details for the interaction with human serum albumin (HSA). We were able to construct the structural models of the fibrinogen α-chain (excluding two highly flexible regions) and the N termini of the ß-chain, confirm these models with known structural arrangements, and map how the structure laterally aggregates to form intricate lattices together with the γ-chain. We validate the final model by mapping mutations leading to impaired clot formation. From a list of 22 mutations, we uncovered structural features for all, including a crucial role for ßArg'169 (UniProt: 196) in lateral aggregation. The resulting model can potentially serve for research on dysfibrinogenemia and amyloidosis as it provides insights into the molecular mechanisms of thrombosis and bleeding disorders related to fibrinogen variants. The structure is provided in the PDB-DEV repository (PDBDEV_00000030).

[Clinicopathological features of fibrin-associated diffuse large B-cell lymphoma: a report of six cases].

Objective: To investigate the clinical, pathological and immunophenotypic features, molecular biology and prognosis of fibrin-associated large B-cell lymphoma (LBCL-FA) in various sites. Methods: Six cases of LBCL-FA diagnosed from April 2016 to November 2021 at the Beijing Friendship Hospital, Capital Medical University, Beijing, China and the First Affiliated Hospital, Wenzhou Medical University, Wenzhou, China were collected. The cases were divided into atrial myxoma and cyst-related groups. Clinical characteristics, pathological morphology, immunophenotype, Epstein Barr virus infection status, B-cell gene rearrangement and fluorescence in situ hybridization of MYC, bcl-2, bcl-6 were summarized. Results: The patients' mean age was 60 years. All of them were male. Three cases occurred in atrial myxoma background, while the others were in cyst-related background, including adrenal gland, abdominal cavity and subdura. All cases showed tumor cells located in pink fibrin clot. However, three cyst-related cases showed the cyst wall with obviously fibrosis and inflammatory cells. All cases tested were non germinal center B cell origin, positive for PD-L1, EBER and EBNA2, and were negative for MYC, bcl-2 and bcl-6 rearrangements, except one case with MYC, bcl-2 and bcl-6 amplification. All of the 5 cases showed monoclonal rearrangement of the Ig gene using PCR based analysis. The patients had detailed follow-ups of 9-120 months, were treated surgically without radiotherapy or chemotherapy, and had long-term disease-free survivals. Conclusions: LBCL-FA is a group of rare diseases occurring in various sites, with predilection in the context of atrial myxoma and cyst-related lesions. Cyst-related lesions with obvious chronic inflammatory background show more scarcity of lymphoid cells and obvious degeneration, which are easy to be missed or misdiagnosed. LBCL-FA overall has a good prognosis with the potential for cure by surgery alone and postoperative chemotherapy may not be necessary.

Altered fibrinogen level and fibrin clot structure in myostatin homozygous mutant pig.

Obesity is associated with increased serum fibrinogen level. Myostatin (MSTN), a strong inhibitor of skeletal muscle growth, is recognized as a potential target for obesity. However, the effect of MSTN inhibition on fibrinogen is not largely known. The objective of the present study was to explore fibrinogen levels after MSTN inhibition. Fibrinogen levels and the fibrin clot structure of MSTN homozygous knockout (KO) and wild-type (WT) pigs (n = 4 in each group) were investigated. The protein expression of fibrinogen in the serum and liver of KO pigs decreased greatly (1.6-fold loss for serum and 2.5-fold loss for liver). KO pigs showed significantly decreased gene expression of fibrinogen chains: FGA (fibrinogen-α; 11-fold), FGB (fibrinogen-ß; 8-fold) and FGG (fibrinogen-γ; 7.4-fold). The basal transcriptional regulators of fibrinogen, HNF1 (hepatocyte nuclear factor 1) and CEBP-α (CCAAT/Enhancing-binding protein-alpha) were remarkably down-regulated after interruption of MSTN expression by siRNA (small interfering RNA) in cultured hepatocytes (about 2- and 4-fold, respectively). Compared with WT pigs, KO pigs displayed altered fibrin clot structure with thinner fibers, decreased turbidity and increased permeability. The findings indicate that the inhibition of MSTN could affect fibrinogen levels and the fibrin clot structure.

Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.

Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges.

Fibrinogen is one of the key molecular players in haemostasis. Thrombin-mediated release of fibrinopeptides from fibrinogen converts this soluble protein into a network of fibrin fibres that form a building block for blood clots. Thrombin-activated factor XIII further crosslinks the fibrin fibres and incorporates antifibrinolytic proteins into the network, thus stabilising the clot. The conversion of fibrinogen to fibrin also exposes binding sites for fibrinolytic proteins to limit clot formation and avoid unwanted extension of the fibrin fibres. Altered clot structure and/or incorporation of antifibrinolytic proteins into fibrin networks disturbs the delicate equilibrium between clot formation and lysis, resulting in either unstable clots (predisposing to bleeding events) or persistent clots that are resistant to lysis (increasing risk of thrombosis). In this review, we discuss the factors responsible for alterations in fibrin(ogen) that can modulate clot stability, in turn predisposing to abnormal haemostasis. We also explore the mechanistic pathways that may allow the use of fibrinogen as a potential therapeutic target to treat vascular thrombosis or bleeding disorders. Better understanding of fibrinogen function will help to devise future effective and safe therapies to modulate thrombosis and bleeding risk, while maintaining the fine balance between clot formation and lysis.

Confocal Blood Flow Videomicroscopy of Thrombus Formation over Human Arteries and Local Targeting of P2X7.

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

A Novel Volume-Stable Collagen Matrix Induces Changes in the Behavior of Primary Human Oral Fibroblasts, Periodontal Ligament, and Endothelial Cells.

The aim of the present study was to investigate the influence of a novel volume-stable collagen matrix (vCM) on early wound healing events including cellular migration and adhesion, protein adsorption and release, and the dynamics of the hemostatic system. For this purpose, we utilized transwell migration and crystal violet adhesion assays, ELISAs for quantification of adsorbed and released from the matrix growth factors, and qRT-PCR for quantification of gene expression in cells grown on the matrix. Our results demonstrated that primary human oral fibroblasts, periodontal ligament, and endothelial cells exhibited increased migration toward vCM compared to control cells that migrated in the absence of the matrix. Cellular adhesive properties on vCM were significantly increased compared to controls. Growth factors TGF-ß1, PDGF-BB, FGF-2, and GDF-5 were adsorbed on vCM with great efficiency and continuously delivered in the medium after an initial burst release within hours. We observed statistically significant upregulation of genes encoding the antifibrinolytic thrombomodulin, plasminogen activator inhibitor type 1, thrombospondin 1, and thromboplastin, as well as strong downregulation of genes encoding the profibrinolytic tissue plasminogen activator, urokinase-type plasminogen activator, its receptor, and the matrix metalloproteinase 14 in cells grown on vCM. As a general trend, the stimulatory effect of the vCM on the expression of antifibrinolytic genes was synergistically enhanced by TGF-ß1, PDGF-BB, or FGF-2, whereas the strong inhibitory effect of the vCM on the expression of profibrinolytic genes was reversed by PDGF-BB, FGF-2, or GDF-5. Taken together, our data strongly support the effect of the novel vCM on fibrin clot stabilization and coagulation/fibrinolysis equilibrium, thus facilitating progression to the next stages of the soft tissue healing process.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

Modification with CREKA Improves Cell Retention in a Rat Model of Myocardial Ischemia Reperfusion.

Poor cell homing limits the efficacy of cardiac cellular therapy. The homing peptide, cysteine-arginine-glutamic acid-lysine-alanine (CREKA), targets fibrin effectively which is involved in the repair process of tissue injury. Here, we assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots (2.6- and 2.3-fold, respectively). CREKA-MSCs showed 6.5-fold higher accumulation than unmodified MSCs in injured rat myocardium one day after administration, resulting in better structural preservation and functional recovery. Fibrin is, therefore, a novel target for enhancing homing of transplanted cells to injured myocardium, and the delivery system of fibrin-targeting is on behalf of a universalizable platform technology for regenerative medicine. Stem Cells 2019;37:663-676.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype.

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bß, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bß, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.

Age-Related Differences in the Time Course of Coagulation and Fibrinolytic Parameters in Patients with Traumatic Brain Injury.

Coagulopathy and older age are common and well-recognized risk factors for poorer outcomes in traumatic brain injury (TBI) patients; however, the relationships between coagulopathy and age remain unclear. We hypothesized that coagulation/fibrinolytic abnormalities are more pronounced in older patients and may be a factor in poorer outcomes. We retrospectively evaluated severe TBI cases in which fibrinogen and D-dimer were measured on arrival and 3-6 h after injury. Propensity score-matched analyses were performed to adjust baseline characteristics between older patients (the "elderly group," aged ≥75 y) and younger patients (the "non-elderly group," aged 16-74 y). A total of 1294 cases (elderly group: 395, non-elderly group: 899) were assessed, and propensity score matching created a matched cohort of 324 pairs. Fibrinogen on admission, the degree of reduction in fibrinogen between admission and 3-6 h post-injury, and D-dimer levels between admission and 3-6 h post-injury were significantly more abnormal in the elderly group than in the non-elderly group. On multivariate logistic regression analysis, independent risk factors for poor prognosis included low fibrinogen and high D-dimer levels on admission. Posttraumatic coagulation and fibrinolytic abnormalities are more severe in older patients, and fibrinogen and D-dimer abnormalities are negative predictive factors.

Metabolomics Reveals Anaerobic Bacterial Fermentation and Hypoxanthine Accumulation for Fibrinous Pleural Effusions in Children with Pneumonia.

Fibrin formation in infectious parapneumonic effusion (IPE) characterizes complicated parapneumonic effusion and is important for providing guidelines for the management of IPEs that require aggressive interventions. We aim to identify metabolic mechanisms associated with bacterial invasion, inflammatory cytokines, and biochemical markers in cases of fibrinous infectious pleural effusions in children with pneumonia. Pleural fluid metabolites were determined by 1H nuclear magnetic resonance spectroscopy. Metabolites that contributed to the separation between fibrinous and nonfibrinous IPEs were identified using supervised partial least squares discriminant analysis ( Q2/ R2 = 0.84; Ppermutation < 0.01). IL-1ß in the inflammatory cytokines and glucose in the biochemical markers were significantly correlated with 11 and 9 pleural fluid metabolites, respectively, and exhibited significant overlaps. Four metabolites, including glucose, lactic acid, 3-hydroxybutyric acid, and hypoxanthine, were significantly correlated with plasminogen activator inhibitor type 1 in the fibrinolytic system enzymes. Metabolic pathway analysis revealed that anaerobic bacterial fermentation with increased lactic acid and butyric acid via glucose consumption and adenosine triphosphate hydrolysis with increased hypoxanthine appeared to be associated with fibrinous IPE. Our results demonstrate that an increase in lactic acid anaerobic fermentation and hypoxanthine accumulation under hypoxic conditions are associated with fibrin formation in IPE, representing advanced pleural inflammatory progress in children with pneumonia.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.

Sema4D/PlexinB1 inhibition ameliorates blood-brain barrier damage and improves outcome after stroke in rats.

The inflammatory process in stroke is the major contributor to blood-brain barrier (BBB) breakdown. Previous studies indicated that semaphorin 4D (Sema4D), an axon guidance molecule, initiated inflammatory microglial activation and disrupted endothelial function in the CNS. However, whether Sema4D disrupts BBB integrity after stroke remains unclear. To study the effect of Sema4D on BBB disruption in stroke, rats were subjected to transient middle cerebral artery occlusion and targeted injection of lentivirus-mediated clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene disruption of PlexinB1. We found that Sema4D synchronously increased with BBB permeability and accumulated in the perivascular area after stroke. Suppressing Sema4D/PlexinB1 signaling in the periinfarct cortex significantly decreased BBB permeability as detected by MRI and fibrin deposition, and thereby improved stroke outcome. In vitro, we confirmed that Sema4D disrupted BBB integrity and endothelial tight junctions. Moreover, we found that Sema4D induced pericytes to acquire a CD11b-positive phenotype and express proinflammatory cytokines. In addition, Sema4D inhibited AUF1-induced proinflammatory mRNA decay effect. Taken together, our data provides evidence that Sema4D disrupts BBB integrity and promotes an inflammatory response by binding to PlexinB1 in pericytes after transient middle cerebral artery occlusion. Our study indicates that Sema4D may be a novel therapeutic target for treatment in the acute phase of stroke.-Zhou, Y.-F., Li, Y.-N., Jin, H.-J., Wu, J.-H., He, Q.-W., Wang, X.-X., Lei, H., Hu, B. Sema4D/PlexinB1 inhibition ameliorates blood-brain barrier damage and improves outcome after stroke in rats.

Fibrin deposition following bile duct injury limits fibrosis through an αMß2-dependent mechanism.

Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMß2, we tested the hypothesis that disruption of the fibrin(ogen)-αMß2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMß2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMß2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMß2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.