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  • 1
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 267-267
    Abstract: Thrombin is central in thrombus formation as both a positive mediator of thrombus formation through the proteolytic activation of PARs, fibrinogen, fXI and other prothrombotic substrates, and a negative modulator of the coagulation cascade through the activation of protein C. Detailed structure-function studies have revealed that thrombin can be redesigned to favor either procoagulant or anticoagulant substrates. The introduction of W215A/E217A substitutions in the murine thrombin active site (fIIWE) results in a pronounced “specificity switch” that reduces catalytic efficiency with fibrinogen by at least 3-orders-magnitude while only modestly reducing activity for protein C activation. To evaluate the effects of fIIWE activity in vivo, we have used a gene-targeting strategy to generate mice carrying the W215A/E217A mutations in the endogenous murine prothrombin gene. The mutant allele was transmitted through the germline and was found to support the expression of normal levels of hepatic fII mRNA and plasma fII in both heterozygous and homozygous neonates. Unlike fII knockout mice, homozygous fIIWE mice were observed at term with the expected Mendelian frequency. Nevertheless, homozygous fIIWE offspring uniformly succumbed to spontaneous bleeding events within days of birth. Heterozygous fIIWE/WT animals generally survived to adulthood, were capable of carrying multiple liters to term, and unchallenged mice displayed a hematological profile similar to wildtype mice. However, consistent with a predicted anticoagulant phenotype, adult fIIWE/WT heterozygotes exhibited significantly delayed thrombus formation following ferric chloride injury of mesenteric arterioles and extended bleeding times following tail tip excision relative to control mice expressing wildtype fII. Given that activated protein C has been shown to be efficacious in the treatment of sepsis, we explored whether the shift in thrombin specificity in heterozygous fIIWE/WT mice would confer the benefit of rendering animals tolerant to acute septic challenges. Kaplan-Meier analyses following intravenous administration of S. aureus revealed that fIIWE/WT mice exhibited a significant survival advantage over littermate wildtype animals challenged in parallel and tracked over a 7-day observation period. Notably, extended thrombus formation and bleeding times as well as resistance to sepsis was not simply a function of half normal wildtype fII expression. When these analyses were performed in animals carrying one wildtype allele and one null mutation allele, results were similar to wiltype. These studies further underscore the interplay between the hemostatic and inflammatory systems in vivo and highlight the possible therapeutic utility of recombinant (pro)thrombin derivatives with selected alterations in substrate specificity.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
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  • 2
    In: The Journal of Neuroscience, Society for Neuroscience, Vol. 37, No. 14 ( 2017-04-05), p. 3776-3788
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0270-6474 , 1529-2401
    Language: English
    Publisher: Society for Neuroscience
    Publication Date: 2017
    detail.hit.zdb_id: 1475274-8
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  • 3
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 580-580
    Abstract: Rheumatoid arthritis (RA) is a common and debilitating autoimmune disease characterized by chronic inflammation, synovial hyperplasia, edema, cartilage and bone erosion and loss of joint function. Increasing evidence suggests that the plasminogen activation (PA) system plays a fundamental role in the mechanisms mediating inflammatory joint disease pathogenesis. However, analysis of the precise contribution of PA system components to arthritis pathogenesis has been complicated by the use of gene-targeted mice on non-susceptible genetic backgrounds or experimental models that simultaneously induce wound trauma in conjunction with arthritis induction. To rigorously define the contribution of the urokinase-type plasminogen activator system to arthritis pathogenesis, previously generated genetic deficiencies in both uPA and uPA receptor (uPAR) were inbred for 7 generations (99% inbred) to the well-characterized, collagen-induced arthritis (CIA)-susceptible strain, DBA/1J. Our results indicate a near complete amelioration of joint disease in uPA-deficient mice that was also observed in uPAR-deficient mice. Limited disease development in both uPA- and uPAR-deficient mice correlated with significantly reduced local mRNA levels of key inflammatory mediators (e.g., TNFα, IL-1β, and IL-6) in these animals. To determine if development of inflammatory joint disease in CIA-challenged mice was dependent on the expression of uPAR by non-hematopoietic- or hematopoietic-derived cells, reciprocal bone marrow transplant studies were performed. Mice in which uPAR deficiency was limited to the bone marrow compartment elicited significantly reduced macroscopic and histopathological disease in the paws and knees compared to wild-type mice or mice in which only hematopoietic-derived cells express uPAR. Our results are the first to report in the context of the highly CIA susceptible DBA/1 background that both uPA and uPAR are key determinants of inflammatory joint disease pathogenesis. Furthermore, our findings indicate a fundamental role for uPAR expression by hematopoietic cells in driving arthritis incidence and progression. Thus, these findings suggest that cell-surface associated uPA/uPAR-mediated proteolysis and/or uPAR-mediated signaling events from bone-marrow derived cells are important in promoting inflammatory joint disease, and that disrupting this key proteolytic/signaling system may provide a novel therapeutic strategy to limit clinical arthritis. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
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  • 4
    Online Resource
    Online Resource
    American Society for Clinical Investigation ; 2007
    In:  Journal of Clinical Investigation Vol. 117, No. 11 ( 2007-11-1), p. 3224-3235
    In: Journal of Clinical Investigation, American Society for Clinical Investigation, Vol. 117, No. 11 ( 2007-11-1), p. 3224-3235
    Type of Medium: Online Resource
    ISSN: 0021-9738
    Language: English
    Publisher: American Society for Clinical Investigation
    Publication Date: 2007
    detail.hit.zdb_id: 2018375-6
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  • 5
    In: Blood, American Society of Hematology, Vol. 117, No. 23 ( 2011-06-09), p. 6326-6337
    Abstract: 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 (fIIWE), 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 fIIWE mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fIIWE/WE mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fIIWT/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, prothrombinWE expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombinWE 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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    In: Blood, American Society of Hematology, Vol. 125, No. 3 ( 2015-01-15), p. 427-437
    Abstract: Genetic elimination of the coagulation transglutaminase fXIII limits arthritis incidence and severity in mice. FXIII supports arthritis pathogenesis through distinct mechanisms linked to joint inflammation and osteoclastogenesis.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
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  • 7
    In: Blood, American Society of Hematology, Vol. 126, No. 17 ( 2015-10-22), p. 2047-2058
    Abstract: Mutation of the fibrinogen Aα chain in mice to selectively eliminate thrombin cleavage prevents fibrin polymer formation in vivo. Fibrin polymer formation drives antimicrobial function and supports host survival following S aureus peritoneal infection.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 64-64
    Abstract: Activation of the hemostatic system is a common pathological feature of severe inflammatory processes. In the context of inflammatory joint disease, articular fibrin deposition is a prominent feature that has been hypothesized to contribute to the derangements in immune/inflammatory processes and tissue reorganization that lead to debilitating and irreversible damage within arthritic joints. We directly examined the role of fibrin(ogen) in the progression of inflammatory joint disease using the well-established experimental setting of collagen-induced arthritis (CIA). Mice with genetically-imposed fibrinogen deficiency (Fib−/−) were found to exhibit significantly reduced macroscopic arthritic disease incidence, progression, and severity in the paws relative to littermate controls throughout the evaluation period. Consistent with the macroscopic observations, qualitative and quantitative histological analysis of distal- and knee-joint sections revealed a significant decrease in all joint disease parameters (e.g., inflammatory cell infiltrates, synovial hyperplasia, edema, and cartilage/bone loss) in Fib−/− mice as compared to fibrinogen-sufficient controls. Fibrin(ogen) has been shown to promote pro-inflammatory activity through engagement of the leukocyte integrin receptor αMβ2 (Mac-1). To determine if fibrinogen/αMβ2 interactions promote the pathogenesis of arthritis, we compared CIA progression in mice expressing fibrinogen that lacks the leukocyte integrin receptor αMβ2 binding motif (i.e., Fibγ390–396A mice). We observed decreased gross and histological inflammatory joint disease in Fibγ390–396A mice relative to control animals. This diminution in arthritic disease severity was comparable to that observed in Fib−/− mice despite the maintenance of normal clotting function in Fibγ390–396A animals. In comparison, CIA studies in mice expressing a mutant form of fibrinogen lacking the platelet integrin receptor αIIbβ3 binding motif showed that this feature of fibrinogen was not an essential driver of arthritis. The reduction in macroscopic and histological arthritic disease observed in CIA-challenged Fibγ390–396A mice was accompanied by reduced local mRNA levels of the pro-inflammatory cytokines TNF α, IL-6, and IL-1β in paws relative to paws from control animals. This data suggests that fibrin(ogen) may function upstream of local production of these critical inflammatory mediators. Consistent with this hypothesis, fibrinogen deficiency did not diminish arthritic disease driven by TNFα-overexpression. Together, these data support the view that local fibrin deposition is an important determinant of inflammatory joint disease and that one mechanism by which fibrin promotes arthritic disease is through leukocyte activation events downstream of αMβ2 engagement.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 9
    In: Arthritis & Rheumatology, Wiley, Vol. 66, No. 6 ( 2014-06), p. 1504-1516
    Abstract: A fundamental metric in the diagnosis of arthropathies is the pattern of joint involvement, including differences in proximal versus distal joints and patterns of symmetric or asymmetric disease. The basis for joint selectivity among arthritides and/or within a defined disease such as rheumatoid arthritis remains enigmatic. Coagulation and fibrinolytic activity are observed in both experimental animals with inflammatory joint disease and patients with inflammatory arthritis. However, the contribution of specific hemostatic factors to joint disease is not fully defined. We sought to determine the contribution of the fibrinolytic protease, plasminogen, to tumor necrosis factor α (TNFα)–driven arthritis in distinct joints in mice. Methods The impact of plasminogen and/or fibrinogen genetic deficiencies on arthritis progression was evaluated in Tg197 mice genetically predisposed to spontaneous, nonabating, and erosive polyarthritis due to exuberant human TNFα expression. Results Elimination of plasminogen in Tg197 mice significantly exacerbated the incidence and severity of arthritis within the paw joints, but simultaneously and dramatically diminished the entire spectrum of pathologies within the knee joints of the same animals. These opposing outcomes were both mechanistically linked to fibrin(ogen), in that superimposing fibrinogen deficiency reversed both the proarthritic phenotype in the paws and arthritis resistance in the knees of plasminogen‐deficient mice. Intriguingly, the change in disease severity in the knees, but not the paws, was associated with a plasminogen‐dependent reduction in matrix metalloproteinase 9 activity. Conclusion Plasminogen is a key molecular determinant of inflammatory joint disease capable of simultaneously driving or ameliorating arthritis pathogenesis in distinct anatomic locations in the same subject.
    Type of Medium: Online Resource
    ISSN: 2326-5191 , 2326-5205
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2014
    detail.hit.zdb_id: 2754614-7
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