A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

Weiler-Guettler, Hartmut; Christie, Patricia D.; Beeler, David; Healy, Aileen M.; Hancock, Wayne W.; Rayburn, Helen; Edelberg, Jay M.; Rosenberg, R D

doi:10.1172/jci2006

Cited by 240 publications

(246 citation statements)

References 41 publications

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“…Thrombomodulin null mice die during embryonic development (27) but can be rescued by homologous recombination to insert Q387P mutant thrombomodulin into the thrombomodulin locus. Mice with the Q387P mutation are normal but have higher levels of fibrin deposition than wild-type littermates (28). Previously we had shown (17) that thrombomodulin with the Q387P mutation did not enhance activation of protein C. Here we show that TAFI activation is reduced to the same extent as that of protein C activation, suggesting that the rescue of thrombomodulin null mice by the Q387P mutant thrombomodulin is not due to its ability to activate TAFI.…”

Section: Discussionsupporting

confidence: 55%

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Wang¹,

Nagashima²,

Schneider³

et al. 2000

Journal of Biological Chemistry

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Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombinactivable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca 2؉ -dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mM Ca 2؉ . Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a 60-kDa plasma protein that circulates in plasma at concentration of about 75 nM (1). It is a zymogen that is activated to a carboxypeptidase B-like enzyme by a single thrombin-catalyzed cleavage at arginine 92 (2-4). The enzyme, designated TAFIa, catalyzes removal of carboxyl-terminal arginine and lysine residues in fibrin as it undergoes fibrinolysis (5). As a consequence, feedback up-regulation of plasminogen activation is eliminated, and the process of fibrinolysis is suppressed. The activation of TAFI by thrombin and subsequent actions of TAFIa define a molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses activity in the latter (6). Although thrombin at the high levels generated after the clotting of fibrin occurs can activate sufficient TAFI to suppress fibrinolysis, thrombin by itself is a relatively weak activator (2, 7). Thrombin bound to thrombomodulin, however, activates TAFI with a catalytic efficiency 1250-fold greater than that of thrombin alone (6). Thus, the thrombin-thrombomodulin complex is probably the physiologic ac...

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Section: Discussionsupporting

confidence: 55%

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Wang¹,

Nagashima²,

Schneider³

et al. 2000

Journal of Biological Chemistry

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“…Fibrin was localized using a grading scale 25 : 1, fibrin deposition limited to intravascular space; 2, fibrin deposition in the intravascular lumen and the perivascular space; and 3, fibrin lattices in the extravascular or parenchymal tissue only. For immunostaining, anti-mouse fibrin II antibody (NYB-T2G1, Accurate Chemical Science Corp) 26 (1:1000 dilution) was used. Routine control sections included deletion of primary antibody, deletion of secondary antibody, and the use of an irrelevant primary antibody.…”

Section: Histopathologymentioning

confidence: 99%

“…The supernatant was aspirated and the sediment dissolved at 65°C in reducing SDS buffer, subjected to SDS-polyacrylamide gel electrophoresis (8%), and transferred to a polyvinylidine difluoride membrane (Immobilon-P; Millipore Corp) by electroblotting. 26 Fibrin was visualized with anti-mouse fibrin II antibody (given above) and an enhanced chemiluminescence system (Amersham Corp). Fibrin standards were prepared by clotting a known amount of murine fibrinogen (Sigma Chemical Co) with an excess of thrombin in the absence of calcium.…”

Section: Detection Of Fibrin In Brain Tissue Sections By Quantitativementioning

confidence: 99%

Tissue Plasminogen Activator (tPA) Deficiency Exacerbates Cerebrovascular Fibrin Deposition and Brain Injury in a Murine Stroke Model

Tabrizi

Wang

Seeds

et al. 1999

ATVB

118

105

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Abstract-Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPAϪ/Ϫ) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPAϪ/Ϫ and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPAϪ/Ϫ and genetically matched tPAϩ/ϩ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2-and 6.7-fold in tPAϪ/Ϫ versus tPAϩ/ϩ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (PϽ0.05) and impaired motor neurological score by 70% (PϽ0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research. (Arterioscler Thromb Vasc Biol.

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“…16,17 In this model, analysis of fibrin formation can employ 3 complementary techniques: morphological and immunoblotting studies with monospecific polyclonal antibody to a neoepitope in fibrin gamma-gamma chain dimers (other investigators have used antibody to human fibrin beta chains with similar ability to detect murine fibrin), 16 electron microscopy, and deposition of radioiodinated fibrinogen. After exposure to hypoxia, vascular fibrin deposition occurs within approximately 6 hours.…”

Section: A Model Of Hypoxia-induced Fibrin Depositionmentioning

confidence: 99%

Hypoxia/Hypoxemia-Induced Activation of the Procoagulant Pathways and the Pathogenesis of Ischemia-Associated Thrombosis

Yan

Mackman

Kisiel

et al. 1999

ATVB

172

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Abstract-Although oxygen deprivation has long been associated with triggering of the procoagulant pathway and venous thrombosis, blood hypoxemia and stasis by themselves do not lead to fibrin formation. A pathway is outlined through which diminished levels of oxygen activate the transcription factor early growth response-1 (Egr-1) leading to de novo transcription/translation of tissue factor in mononuclear phagocytes and smooth muscle cells, which eventuates in vascular fibrin deposition. The procoagulant response is magnified by concomitant suppression of fibrinolysis by hypoxia-mediated upregulation of plasminogen activator inhibitor-1. These data add a new facet to the biology of thrombosis associated with hypoxemia/stasis and imply that interference with mechanisms causing Egr-1 activation in response to oxygen deprivation might prevent vascular fibrin deposition occurring in ischemia without directly interfering with other pro/anticoagulant pathways. Key Words: tissue factor Ⅲ hypoxia Ⅲ ischemia Ⅲ Egr-1 Ⅲ PAI-1 I n the 1850s, Virchow described the association between venous thrombosis and a triad of contributing factors, including hypercoagulability, vascular damage, and vascular stasis. 1 More recently, venous stasis has been linked to the rapid decline in intravascular oxygen tension and thrombus formation in veins of the lower extremities. [2][3][4] Definition of mechanisms through which low levels of oxygen cause blood to clot has been more elusive. The Wessler stasis model of venous thrombosis, 5,6 in which a rabbit vascular segment (typically the jugular vein) is occluded and a fibrin clot subsequently forms after addition of a procoagulant, demonstrated that acute lack of blood flow and/or hypoxemia, although necessary, were not sufficient, at least acutely, to trigger clot formation. Without inclusion of a strong procoagulant stimulus, such as activated clotting factors (eg, Factors IXa, Xa, or thrombin), fibrin deposition did not occur. These observations have been extended to different types of vessels, and this has led to the same conclusion; acute occlusion of normal vessels with their normal blood content does not by itself promote fibrin formation. This situation must be differentiated from that of an atherosclerotic or other pathologic vessel, in which abundant neointimal tissue factor, a maximal prothrombotic stimulus, is immediately exposed to blood contents, triggering rapid coagulation. In fact, the intact, healthy vessel wall has very low levels of tissue factor with an increasing gradient toward the adventitia. The cell type most uniformly accepted as being capable of expressing substantive amounts of tissue factor within the intravascular space in response to environmental stimuli is the mononuclear phagocyte, although polymorphonuclear leukocytes may also contribute, and endothelial cells have been shown to produce tissue factor in selected settings. 7,8 Thus it may not be surprising that a brief period of hypoxemia and/or stasis alone in a normal vessel are not sufficient to trigger ...

show abstract

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

Cited by 240 publications

References 41 publications

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Tissue Plasminogen Activator (tPA) Deficiency Exacerbates Cerebrovascular Fibrin Deposition and Brain Injury in a Murine Stroke Model

Hypoxia/Hypoxemia-Induced Activation of the Procoagulant Pathways and the Pathogenesis of Ischemia-Associated Thrombosis

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