Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics

Abstract

The blood coagulation protein fibrinogen is deposited in the brain in a wide range of neurological diseases and traumatic injuries with blood-brain barrier (BBB) disruption. Recent research has uncovered pleiotropic roles for fibrinogen in the activation of CNS inflammation, induction of scar formation in the brain, promotion of cognitive decline and inhibition of repair. Such diverse roles are possible in part because of the unique structure of fibrinogen, which contains multiple binding sites for cellular receptors and proteins expressed in the nervous system. The cellular and molecular mechanisms underlying the actions of fibrinogen are beginning to be elucidated, providing insight into its involvement in neurological diseases, such as multiple sclerosis, Alzheimer disease and traumatic CNS injury. Selective drug targeting to suppress the damaging functions of fibrinogen in the nervous system without affecting its beneficial effects in haemostasis opens a new fibrinogen therapeutics pipeline for neurological disease.

Figure 1 |. Fibrinogen at the nexus of the brain–vascular–immune axis.

a | Fibrinogen is a molecular mediator entering the CNS after blood–brain barrier (BBB) disruption that is causally linked with neuroinflammation, neuronal damage and immune cell recruitment in the nervous system. As a key component of the pathological lesion environment, fibrinogen is uniquely positioned to serve as an imaging and fluid biomarker as well as a therapeutic target for neurological diseases. b | A ‘fibrinogen toolbox’ of experimental models, pharmacological and genetic tools, molecular imaging probes and novel therapeutics has been developed to study fibrinogen’s contribution to neurovascular pathology in CNS disease. To study cellular responses and dissect molecular signalling mechanisms in vitro, fibrinogen and fibrin can be added to culture media, coated onto plates or formed into 3D gels^,,,,,,. Fibrin and/or fibrinogen can be detected in the diseased or injured nervous system with specific antibodies and through electron microscopy^, (TABLE 1). A number of pharmacological agents and genetic tools have been used to test the causal role of fibrinogen in neurological disease, including fibrinogen-depleting drugs^,,,,,,, inhibitors of fibrin formation^,, inhibitors of fibrin interactions with integrin receptors and other proteins^,, and fibrinogen-knockout and knock-in mice^,–,. Stereotactic fibrinogen injections and infusions into the CNS provide experimental models to test fibrinogen-induced cellular responses and signalling pathways in vivo^,,,,. Novel imaging tools also allow for real-time analysis of fibrinogen and coagulation dynamics at the neurovascular unit in health and disease^,,. Altogether, the fibrinogen toolbox can be employed to determine the role of fibrinogen in any neurological disease with BBB disruption and a dysfunctional brain–vascular–immune axis.

Figure 2 |. Fibrinogen structure, cellular targets and signalling networks in the nervous system.

Fibrin and fibrinogen interact with receptors on nervous system cells to activate downstream signalling, regulate basic cellular functions and influence inflammatory, neurodegenerative and repair processes in disease^,,,. Fibrinogen is composed of three distinct polypeptides, designated Aα, Bβ and γ, which contain multiple binding sites for cellular receptors and proteins. Grey-shaded areas designate the location of binding sites, with fibrinogen amino acid sequence numbers and the corresponding receptor or bound protein listed above the site. Fibrinogen chain Aα (blue) binds plasminogen/tissue-type plasminogen activator (tPA), fibronectin and latent transforming growth factor-β (TGFβ) and interacts with the integrin receptors α5β1 (REF. 199), αvβ3 (REF. 200) and αvβ8 (REF. 201) through its Arg–Gly–Asp (RGD) motif. Fibrinogen chain Bβ (green) binds vascular endothelial cadherin (VE-cadherin; also known as CDH5), very low-density lipoprotein receptor (VLDLR) and amyloid-β. Fibrinogen chain γ (red) binds tPA, the integrin receptors CD11b/CD18^, and αIIbβ3 (REF. 27); it also binds intercellular adhesion molecule 1 (not shown on figure). Upon activation of the coagulation, fibrinogen is converted into insoluble fibrin by thrombin, which cleaves fibrinopeptide A and B from fibrinogen (red triangles) to expose polymerization sites that facilitate clot formation. Fibrinogen, fibrin and fibrin degradation products produce different responses from CNS cells. Soluble fibrinogen can induce growth factor receptor pathway activation in astrocytes, oligodendrocyte progenitor cells (OPCs) and neurons to regulate scar formation, cell differentiation and inhibition of neurite outgrowth, respectively^,,. Fibrinogen and fibrin also exert biological effects on mature oligodendrocytes and pericytes (see note added in proof). Conversion of fibrinogen into fibrin or immobilization of fibrinogen to a substrate exposes cryptic epitopes, such as the γ377–395 epitope, which is the binding site for the CD11b/CD18 integrin receptor^,. Indeed, immobilized fibrinogen or fibrin activates microglia and macrophages^,,. Similar to soluble fibrinogen, immobilized fibrinogen inhibits OPC maturation. 3D fibrin gels have also been utilized for testing Schwann cell differentiation and migration^,. 3D fibrin gels are ideally suited for the study of mechanisms of fibrin degradation and the discovery of novel mechanisms in the CNS that regulate fibrinolysis. Indeed, the discovery of the low-affinity neurotrophin receptor p75NTR (NGFR) as a regulator of PLAT and plasminogen activator inhibitor 1 (PAI1) transcriptional regulation was facilitated by the culture of Schwann cells on 3D fibrin gels^,. Studies to methodically compare the responses of CNS cells to all the potential forms of fibrinogen, fibrin and fibrin degradation products are required to associate fibrinogen structure and function with an observed biological outcome. These studies can be influenced by the experimental setting, the purity of fibrinogen preparations, the methods of primary cell isolation and the stage of cell differentiation at the time of fibrinogen or fibrin treatment. ACVR1, activin A receptor type 1; P-AKT, phosphorylated serine/threonine-protein kinase; P-EGFR, phosphorylated epidermal growth factor receptor; P-SMAD2/3, phosphorylated MAD homologue 2/3; RHOA, transforming protein RhoA; ROS, reactive oxygen species; TGFR1, TGFβ receptor type 1.

Figure 3 |. Timeline of in vivo genetic and pharmacological evidence showing a causal role for fibrin and/or fibrinogen in the development of neurological disease.

Pharmacological depletion of fibrinogen with the defibrinogenating agents ancrod or batroxobin protected Lewis rats in multiple sclerosis (MS) models^,,; this finding was later confirmed in mouse MS models^,,. Akassoglou et al. provided the first genetic proof for a causal role for fibrin in a nervous system experimental model of nerve regeneration. Later studies demonstrated fibrin to be a critical determinant of neuroinflammation as fibrinogen-knockout mice showed reduced microglial activation and improved neurological function in animal models of MS^, and protection from neuropathology and cognitive deficits in Alzheimer disease (AD)^,. In addition, blocking the conversion of fibrinogen to fibrin with the thrombin inhibitor hirudin protected from experimental autoimmune encephalitis (EAE)^,. To selectively inhibit the inflammatory actions of fibrin in the CNS while preserving coagulation, Adams et al. blocked fibrin’s interaction with its high-affinity receptor CD11b/CD18 genetically by using Fgg^γ390−396A mice, in which fibrinogen has been mutated to lack the CD11b/CD18-binding motif, and pharmacologically by administering the γ377–395 peptide. Genetic and pharmacological inhibition of fibrin–CD11b/CD18 reduced paralysis, inflammation, microglia activation, axonal damage and demyelination in EAE^,. In AD mice, administration of a compound that specifically inhibits fibrin–amyloid-β (Aβ) interaction reduced vascular pathology, blocked neuroinflammation and protected from cognitive decline. Altogether, the genetic and pharmacological evidence point to a causal role for fibrin and/or fibrinogen in the CNS and may represent a novel target for therapeutic intervention in neurological disease. GFAP, glial fibrillary acidic protein; MOG, myelin-oligodendrocyte glycoprotein; PLP, myelin proteolipid protein; TNF, tumour necrosis factor.

Figure 4 |. Fibrinogen at the helm of CNS innate immune activation and neurodegeneration.

Fibrin is a key contributor to inflammatory demyelination, neurodegeneration and inhibition of CNS repair. a | In multiple sclerosis animal models, blood–brain barrier (BBB) disruption leads to increased coagulation activity and fibrin deposition in the brain, which binds to the CD11b/CD18 integrin receptor, thus driving early microglial activation, recruitment of peripheral macrophages and T cells and leading to demyelination and axonal damage^{, ,}. Fibrin-induced activation induces a gene transcription signature characterized by increased secretion of chemokines (such as CXC-chemokine ligand 10 (CXCL10) and monocyte chemoattractant protein 1 (MCP1)) to promote recruitment of T cells, increased antigen presentation and release of instructive signals (such as interleukin 12 (IL-12)) for inducing T helper 1 (TH1) cell differentiation to promote autoimmunity and demyelination. In parallel, perivascular microglia cluster at sites of fibrin deposition, which correlate with areas of reactive oxygen species (ROS) generation and axonal damage in vivo. Mechanisms and consequences of fibrin-induced ROS release in the CNS are not known (dotted lines). Fibrinogen also blocks oligodendrocyte progenitor cell (OPC) differentiation to myelinating cells by direct and immune-mediated mechanisms. b | In animal models of Alzheimer disease (AD), fibrin binds to amyloid-β (Aβ), which results in degradation-resistant clots^,,. Aβ further drives fibrin formation and the release of pro-inflammatory molecules^,. The mechanisms by which fibrin–Aβ interaction contributes to neuronal loss in AD models are not known (question marks and dotted lines). In addition, whether fibrin-induced microglia activation contributes to amyloid-driven neurodegeneration is unknown (question marks and dotted lines).

Figure 5 |. The coagulation cascade and its final product fibrin as clinically relevant biomarkers and potential therapeutic targets for neurological disease.

a | Fibrinogen and components of the coagulation cascade could serve as clinically relevant plasma and cerebrospinal fluid (CSF) biomarkers to diagnose and track disease progression in neurological disease. Upstream components of the coagulation cascade, including the contact system, coagulation factor X and prothrombin, are elevated in multiple sclerosis (MS)^, and/or Alzheimer disease (AD). Fibrinogen itself is elevated in the plasma or CSF from patients with MS and AD^,,– and often correlates with disease severity. Fibrin degradation products are also found in the CSF of traumatic brain injury (TBI) patients. Studies of animal models of AD and MS highlight the therapeutic potential of targeting coagulation for CNS disease. Coagulation factor XII depletion protects mice in the experimental autoimmune encephalitis (EAE) MS model and in AD models. The thrombin inhibitor hirudin blocks the conversion of fibrinogen into fibrin and is protective in EAE^,. Genetic and/or pharmacological depletion of fibrinogen is protective in animal models of MS^,,–,,, AD^, and nervous system injury^,. By contrast, tissue-type plasminogen activator (tPA)-knockout (KO) mice, which have impaired fibrin degradation, show increased severity of EAE and delayed recovery. Together, human biomarker and animal studies highlight the potential utility of monitoring and perhaps targeting the coagulation cascade in neurological disease. b | In the future, expanding the ‘fibrinogen toolbox’ will enable translation of novel therapeutics, biomarkers and imaging probes to human clinical trials to test the ‘fibrin hypothesis’ of neurological disease. ALS, amyotrophic lateral sclerosis; Apo-E, apolipoprotein E; ASO, antisense oligonucleotide; BBB, blood–brain barrier; CTE, chronic traumatic encephalopathy; GFAP, glial fibrillary acidic protein; GWAS, genome-wide association studies; HD, Huntington disease; PD, Parkinson disease; SAH, subarachnoid haemorrhage; TMEV, Theiler murine encephalomyelitis virus; TNF, tumour necrosis factor; 5×FAD and TgCRND8 are both transgenic mouse models of AD that develop severe amyloid pathology.