Eric S. Mullins

Early detection of thrombin activity in neuroinflammatory disease

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression. Ann Neurol 2014;75:303–308

Thrombomodulin is a determinant of metastasis through a mechanism linked to the thrombin binding domain but not the lectin-like domain

Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TM(Pro) mice expressing a mutant form of TM with reduced thrombin affinity and TM(LeD) mice lacking the N-terminal lectin-like domain. Studies of TM(Pro) mice revealed that TM is a powerful determinant of hematogenous metastasis. TM(Pro) mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TM(Pro) mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Genetic elimination of prothrombin in adult mice is not compatible with survival and results in spontaneous hemorrhagic events in both heart and brain

Mice carrying a conditional prothrombin knockout allele (fII(lox)) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII(lox/lox) mice or compound heterozygous mice carrying one fII(lox) allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII(lox) mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII(lox) allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short ( approximately 5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.

The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A

Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl₃ vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

Mice expressing a mutant form of fibrinogen that cannot support fibrin formation exhibit compromised antimicrobial host defense

Fibrin(ogen) is central to hemostasis and thrombosis and also contributes to multiple physiologic and pathologic processes beyond coagulation. However, the precise contribution of soluble fibrinogen vs insoluble fibrin matrices to vascular integrity, tissue repair, inflammation, and disease has been undefined and unapproachable. To establish the means to distinguish fibrinogen- and fibrin-dependent processes in vivo, Fib(AEK) mice were generated that carry normal levels of circulating fibrinogen but lack the capacity for fibrin polymer formation due to a germ-line mutation in the Aα chain thrombin cleavage site. Homozygous Fib(AEK) mice developed to term and exhibited postnatal survival superior to that of fibrinogen-deficient mice. Unlike fibrinogen-deficient mice, platelet-rich plasma from Fib(AEK) mice supported normal platelet aggregation in vitro, highlighting that fibrinogen(AEK) retains the functional capacity to support interactions with platelets. Thrombin failed to release fibrinopeptide-A from fibrinogen(AEK) and failed to induce polymer formation with Fib(AEK) plasma or purified fibrinogen(AEK) in 37°C mixtures regardless of incubation time. Fib(AEK) mice displayed both an absence of fibrin polymer formation following liver injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi following FeCl3 injury of carotid arteries. Fib(AEK) mice exhibited a profound impediment in Staphylococcus aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet Fib(AEK) mice displayed a significant infection dose-dependent survival advantage over fibrinogen-deficient mice following peritonitis challenge. Collectively, these findings establish for the first time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while simultaneously highlighting biologically meaningful contributions and functions of the soluble molecule.

Transglutaminase factor XIII promotes arthritis through mechanisms linked to inflammation and bone erosion

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration, irreversible cartilage and bone destruction, and exuberant coagulation system activity within joint tissue. Here, we demonstrate that the coagulation transglutaminase, factor XIII (fXIII), drives arthritis pathogenesis by promoting local inflammatory and tissue degradative and remodeling events. All pathological features of collagen-induced arthritis (CIA) were significantly reduced in fXIII-deficient mice. However, the most striking difference in outcome was the preservation of cartilage and bone in fXIIIA(-/-) mice concurrent with reduced osteoclast numbers and activity. The local expression of osteoclast effectors receptor activator of nuclear factor-κB ligand (RANKL) and tartrate resistant acid phosphatase were significantly diminished in CIA-challenged and even unchallenged fXIIIA(-/-) mice relative to wild-type animals, but were similar in wild-type and fibrinogen-deficient mice. Impaired osteoclast formation in fXIIIA(-/-) mice was not due to an inherent deficiency of monocyte precursors, but it was linked to reduced RANKL-driven osteoclast formation. Furthermore, treatment of mice with the pan-transglutaminase inhibitor cystamine resulted in significantly diminished CIA pathology and local markers of osteoclastogenesis. Thus, eliminating fXIIIA limits inflammatory arthritis and protects from cartilage and bone destruction in part through mechanisms linked to reduced RANKL-mediated osteoclastogenesis. In summary, therapeutic strategies targeting fXIII activity may prove beneficial in limiting arthropathies and other degenerative bone diseases.

Thrombin Drives Tumorigenesis in Colitis-Associated Colon Cancer

The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. On the basis of evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/- mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacologic inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration, and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, precancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression.

Thrombin promotes diet-induced obesity through fibrin-driven inflammation

Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fib&ggr;390–396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte &agr;M&bgr;2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fib&ggr;390–396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.

Plasminogen Deficiency Delays the Onset and Protects from Demyelination and Paralysis in Autoimmune Neuroinflammatory Disease

Multiple sclerosis (MS) is a neuroinflammatory, demyelinating disease of the CNS. Fibrinogen deposition at sites of blood–brain barrier breakdown is a prominent feature of neuroinflammatory disease and contributes to disease severity. Plasminogen, the primary fibrinolytic enzyme, also modifies inflammatory processes. We used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to evaluate the hypothesis that the loss of plasminogen would exacerbate neuroinflammatory disease. However, contrary to initial expectations, EAE-challenged plasminogen-deficient (Plg−) mice developed significantly delayed disease onset and reduced disease severity compared with wild-type (Plg+) mice. Similarly, pharmacologic inhibition of plasmin activation with tranexamic acid also delayed disease onset. The T-cell response to immunization was similar between genotypes, suggesting that the contribution of plasminogen was downstream of the T-cell response. Spinal cords from EAE-challenged Plg− mice demonstrated significantly decreased demyelination and microglial/macrophage accumulation compared with Plg+ mice. Although fibrinogen-deficient mice or mice with combined deficiencies of plasminogen and fibrinogen had decreased EAE severity, they did not exhibit the delay in EAE disease onset, as seen in mice with plasminogen deficiency alone. Together, these data suggest that plasminogen and plasmin-mediated fibrinolysis is a key modifier of the onset of neuroinflammatory demyelination. SIGNIFICANCE STATEMENT Multiple sclerosis is a severe, chronic, demyelinating disease. Understanding the pathobiology related to the autoreactive T-cell and microglial/macrophage demyelinating response is critical to effectively target therapeutics. We describe for the first time that deficiency of plasminogen, the key fibrinolytic enzyme, delays disease onset and protects from the development of the paralysis associated with a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Administration of a widely used, pharmacologic inhibitor of plasminogen activation, tranexamic acid, also delays the onset of neuroinflammation associated with EAE.

Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice

Thrombin-mediated proteolysis is central to hemostatic function but also plays a prominent role in multiple disease processes. The proteolytic conversion of fII to α-thrombin (fIIa) by the prothrombinase complex occurs through 2 parallel pathways: (1) the inactive intermediate, prethrombin; or (2) the proteolytically active intermediate, meizothrombin (fIIa(MZ)). FIIa(MZ) has distinct catalytic properties relative to fIIa, including diminished fibrinogen cleavage and increased protein C activation. Thus, fII activation may differentially influence hemostasis and disease depending on the pathway of activation. To determine the in vivo physiologic and pathologic consequences of restricting thrombin generation to fIIa(MZ), mutations were introduced into the endogenous fII gene, resulting in expression of prothrombin carrying 3 amino acid substitutions (R157A, R268A, and K281A) to limit activation events to yield only fIIa(MZ) Homozygous fII(MZ) mice are viable, express fII levels comparable with fII(WT) mice, and have reproductive success. Although in vitro studies revealed delayed generation of fIIa(MZ) enzyme activity, platelet aggregation by fII(MZ) is similar to fII(WT) Consistent with prior analyses of human fIIa(MZ), significant prolongation of clotting times was observed for fII(MZ) plasma. Adult fII(MZ) animals displayed significantly compromised hemostasis in tail bleeding assays, but did not demonstrate overt bleeding. More notably, fII(MZ) mice had 2 significant phenotypic advantages over fII(WT) animals: protection from occlusive thrombosis after arterial injury and markedly diminished metastatic potential in a setting of experimental tumor metastasis to the lung. Thus, these novel animals will provide a valuable tool to assess the role of both fIIa and fIIa(MZ) in vivo.