An Inhibitor of Activated Thrombin-Activatable Fibrinolysis Inhibitor Potentiates Tissue-Type Plasminogen Activator-Induced Thrombolysis in a Rabbit Jugular Vein Thrombolysis Model

Nagashima, Mariko; Werner, Matthias; Wang, Manping; Zhao, Lei; Light, David R.; Pagila, Rene; Morser, John; Verhallen, P.

doi:10.1016/s0049-3848(00)00184-5

Cited by 126 publications

(115 citation statements)

References 33 publications

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“…TAFI constitutes a risk factor for thrombosis and coronary artery disease, and its inhibition leads to an enhancement of clot lysis (40,41). The doubleheaded architecture provides TCI with a 5-fold lower K i toward TAFI compared with potato carboxypeptidase inhibitor and a 10-fold acceleration of fibrinolysis with respect to this singleheaded inhibitor used as a model in thrombolytic assays (42,43). In this way, and as previously discussed, the Nt domain could also contribute to improve the affinity/modulate the specificity of TCI toward CPBs, constituting a good target for the design of new TCI molecules to be used as thrombolytic drugs.…”

Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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Tick carboxypeptidase inhibitor (TCI) is a small, disulfiderich protein that selectively inhibits metallocarboxypeptidases and strongly accelerates the fibrinolysis of blood clots. TCI consists of two domains that are structurally very similar, each containing three disulfide bonds arranged in an almost identical fashion. The oxidative folding and reductive unfolding pathways of TCI and its separated domains have been characterized by kinetic and structural analysis of the acid-trapped folding intermediates. TCI folding proceeds through a sequential formation of 1-, 2-, 3-, 4-, 5-, and 6-disulfide species to reach the native form. Folding intermediates of TCI comprise two predominant 3-disulfide species (named IIIa and IIIb) and a major 6-disulfide scrambled isomer (Xa) that consecutively accumulate along the reaction and are strongly prevented by the presence of protein disulfide isomerase. This study demonstrates that IIIa and IIIb are 3-disulfide species containing the native disulfide pairings of the N-and C-terminal domains of TCI, respectively, and explains why the two domains of TCI fold sequentially and independently. Also, we show that the reductive unfolding of TCI undergoes two main independent unfolding events through the formation of IIIa and IIIb intermediates. Together, the comparison of the folding, stability, and inhibitory activity of TCI with those of the isolated domains reveals the reasons behind the two-domain nature of this protein: both domains contribute to the specificity and high affinity of its double-headed binding to carboxypeptidases. The results obtained herein provide valuable information for the design of more potent and selective TCI molecules.The mechanism by which an unfolded protein achieves its native state is one of the most complex problems in structural biology. Understanding the sequence of folding events in proteins may not only help to predict protein structures from amino acid sequences but also provide invaluable information to a variety of related fields, such as protein design or protein misfolding associated with pathological diseases (1, 2). A number of studies concerning folding have been focused on small, disulfide-rich proteins, given that the chemistry of disulfide bond formation allows the trapping and subsequent characterization of the folding intermediates that accumulate, something difficult to attain with disulfide-free proteins (3, 4). In oxidative folding, reduced and denatured proteins are allowed to recover both its native disulfide bonds and native structures in the absence or presence of redox agents. Bovine pancreatic trypsin inhibitor, ribonuclease A, lysozyme, ␣-lactalbumin, and hirudin have been extensively investigated using this methodology (5-9). The heterogeneity and structures of the intermediates that occur during the process define their respective folding landscapes (10).Although most of the analyzed proteins comprise a small, single-domain fold containing three or four disulfide bonds, a great diversity of folding mechanisms is observ...

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Section: Discussionmentioning

confidence: 99%

Characterizing the Tick Carboxypeptidase Inhibitor

Arolas

Bronsoms

Ventura

et al. 2006

Journal of Biological Chemistry

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“…Numerous in vivo studies (30,31,33,43) have shown that pCPB is an inhibitor of fibrinolysis, which depends primarily upon its capacity to remove carboxyterminal lysines from partially degraded fibrin, thereby eliminating an amplifying loop for Plg activation (10,12). Some in vivo studies using inhibitors (5,9,12,28) have also pointed to a physiological role for pCPB in regulating the fibrinolytic functions of Plg.…”

Section: Discussionmentioning

confidence: 99%

“…This observation is consistent with the hypothesis forwarded by Von dem Borne et al (32) that activation of factor XI generates thrombin, which protects fibrin clots from lysis by activating propCPB. Other in vivo studies have shown that inhibition of pCPB with PCI potentiated thrombolysis (33,34). On the other hand, activated protein C (APC) was shown to prevent death in a thrombin-induced thromboembolism model in mice (35).…”

Section: Introductionmentioning

confidence: 98%

In vivo regulation of plasminogen function by plasma carboxypeptidase B

Swaisgood

Schmitt²,

Eaton³

et al. 2002

J. Clin. Invest.

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The major functions of plasminogen (Plg) in fibrinolysis and cell migration depend on its binding to carboxy-terminal lysyl residues. The ability of plasma carboxypeptidase B (pCPB) to remove these residues suggests that it may act as a suppressor of these Plg functions. To evaluate this role of pCPB in vivo, homozygote pCPB-deficient mice were generated by homologous recombination, and the resulting pCPB–/– mice, which were viable and healthy, were mated to Plg–/– mice. Plg+/– mice show intermediate levels of fibrinolysis and cell migration compared with Plg wild-type and deficient mice, reflecting the intermediate levels of the Plg antigen in their plasma. Differences in Plg-dependent functions between pCPB+/+, pCPB+/–, and pCPB–/– mice were then analyzed in a Plg+/– background. In a pulmonary clot lysis model, fibrinolysis was significantly increased in mice with partial (pCPB+/–) or total absence (pCPB–/–) of pCPB compared with their wild-type counterparts (pCPB+/+). In a thioglycollate model of peritoneal inflammation, leukocyte migration at 72 hours increased significantly in Plg+/–/pCPB+/– and Plg+/–/pCPB–/– compared with their wild-type counterparts. These studies demonstrate a definitive role of pCPB as a modulator of the pivotal functions of Plg in fibrinolysis and cell migration in vivo

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“…90,92,93 The physiologic importance of TAFI in clot stability is unclear, however, since TAFI-deficient animals exhibit minimal to no bleeding. [94][95][96] Recombinant factor VIIa (rFVIIa, NovoSeven) is approved for the treatment of severe bleeds in hemophilic patients with inhibitory antibodies, and has demonstrated efficacy under these conditions and in patients with acquired hemophilia. 97 By binding to the surface of activated platelets, rFVIIa reestablishes platelet tenase activity and restores thrombin generation on the platelet surface.…”

Section: Hemophilia and Recombinant Factor Viiamentioning

confidence: 99%

Thrombin generation, fibrin clot formation and hemostasis

Wolberg

Campbell

2008

Transfusion and Apheresis Science

298

222

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Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro-and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo. Keywordsthrombin; fibrinogen; fibrin; plasmin; fibrinolysis; clot structure; hemophilia; thrombosis Fibrinogen and fibrin clot formationFibrinogen is a 450 Å, 340 kDa trinodular protein present at high (2 -4 mg/mL) concentrations in plasma. Fibrinogen consists of pairs of three different disulfide-linked polypeptide chains: Aα, Bβ, and γ. These six polypeptide chains are arranged with their N-termini converged in a central "E" domain of the molecule. The C-termini of the Bβ and γ chains extend outward into distal "D" domains. The C-termini of the Aα chains are globular and situated near the central E domain of fibrinogen where they interact intra-molecularly. 1 Comprehensive reviews on the biochemistry of fibrinogen and fibrin structure have been published. 2-4Mechanisms of fibrin production have been elucidated in studies conducted by adding exogenous thrombin to purified fibrinogen. These studies have shown that thrombin removes N-terminal peptides from the fibrinogen Aαl and Bβ chains, causing the spontaneous formation of half-staggered, double-stranded protofibrils followed by thickening of protofibril chains. 5 Initial formation of protofibrils occurs during a "lag" phase in which no turbidity increase is detected. Subsequent lateral aggregation of fibrin protofibrils causes a turbidity increase. The magnitude of the turbidity increase relates to the structure of the formed clot; formation of thicker fibers causes a greater increase in final turbidity. 6,7 Fibrin formation can be followed Address correspondence to: Alisa Wolberg, Ph. D., Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 815 Brinkhous-Bullitt Building, CB #7525, Chapel Hill, NC 27599-7525, phone: (919) 966-8430, fax: (919) 966-6718, email: alisa_wolberg@med.unc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production p...

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An Inhibitor of Activated Thrombin-Activatable Fibrinolysis Inhibitor Potentiates Tissue-Type Plasminogen Activator-Induced Thrombolysis in a Rabbit Jugular Vein Thrombolysis Model

Cited by 126 publications

References 33 publications

Characterizing the Tick Carboxypeptidase Inhibitor

Characterizing the Tick Carboxypeptidase Inhibitor

In vivo regulation of plasminogen function by plasma carboxypeptidase B

Thrombin generation, fibrin clot formation and hemostasis

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