Evidence That Both Exosites on Thrombin Participate in Its High Affinity Interaction with Fibrin

Pospisil, Caroline H.; Stafford, Alan R.; Fredenburgh, James C.; Weitz, Jeffrey I.

doi:10.1074/jbc.m300545200

Cited by 73 publications

(113 citation statements)

References 44 publications

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“…39 -41 Therefore, fibrin deposition and its persistence at the injection site may result in an increased fibroblast proliferation and migration leading to enhanced collagen accumulation compared with PBS-injected sites. Furthermore, fibrin acts as a reservoir for growth factors including thrombin, 42 basic fibroblast growth factor, 43 platelet-derived growth factor, and TGF-␤ 3 suggesting there may be a continual supply of potent mediators stimulating a repair response at the fibrin injection site. In this regard, Coustry and colleagues 15 found that when fibroblasts were grown in collagen gels, they produced significantly less collagen in response to TGF-␤1 than when grown in fibrin gels with equivalent amounts of TGF-␤1, implying that fibrin provides a more suitable matrix for the availability of growth factors.…”

Section: Discussionmentioning

confidence: 99%

Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator

Giorgio-Miller

Bottoms

Laurent

et al. 2005

The American Journal of Pathology

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The deposition of fibrin is an integral part of the tissue repair process, but its persistence is also associated with a number of fibrotic conditions. This study addressed the hypothesis that reduced fibrinolysis and fibrin persistence are associated with an enhanced accumulation of collagen and the development of skin fibrosis. Decreased fibrinolysis was confirmed in fibrin gel cultures that contained human dermal fibroblasts plus the specific plasmin inhibitor ␣ 2 -antiplasmin or dermal fibroblasts isolated from plasminogen activator (PA)-deficient mice. Collagen accumulation was significantly increased in the presence of inhibitor and in tPA-deficient, but not uPAdeficient, fibroblasts compared with controls. These findings were also confirmed using a skin fibrosis model in which multiple injections of fibrin were given subcutaneously to PA-deficient mice. Injection sites from tPA-deficient mice displayed significantly increased collagen levels compared with uPA-deficient mice and wild-type controls. Up-regulation of fibroblast procollagen gene expression and reduced activation of pro-MMP-1 appeared to mediate the increase in collagen by human dermal fibroblasts in the presence of ␣2-antiplasmin. These findings suggest that persistent fibrin is associated with enhanced collagen accumulation that may result in the development of fibrotic skin disorders in which reduced fibrinolysis is a feature.

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Section: Discussionmentioning

confidence: 99%

Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator

Giorgio-Miller

Bottoms

Laurent

et al. 2005

The American Journal of Pathology

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“…Labeling of Proteins-␥Ј-Peptide was labeled with fluorescein isothiocyanate as previously described (9). ␣-Thrombin was radiolabeled by reaction with 125 I-labeled YPRck, and HRG was radiolabeled with Na 125 I (McMaster University Nuclear Reactor, Hamilton, ON) using Iodo-beads (Pierce) as described (29).…”

Section: Methodsmentioning

confidence: 99%

“…Interaction of HRG with Fluorescein-labeled ␥Ј-Peptide in the Absence or Presence of ZnCl 2 -The binding of 1.1 M HRG to 0.05 M fluorescein-␥Ј-peptide was monitored by fluorescence in the absence or presence of Zn 2ϩ using a PerkinElmer Life Sciences LS 50B luminescence spectrometer (9). Briefly, the base-line fluorescence (I o ) was determined at excitation and emission wavelengths (slit widths) of 492 (5 nm) and 532 nm (2.5 nm), respectively, and an emission filter at 515 nm.…”

Section: Surface Plasmon Resonance (Spr)-mentioning

confidence: 99%

“…The mixture was then titrated with aliquots of ZnCl 2 up to 20 M, and fluorescence intensity (I) was monitored after each addition. I/I o values were plotted against the concentration of ZnCl 2 , and the data were subjected to nonlinear regression analysis as previously described (9).…”

Section: Surface Plasmon Resonance (Spr)-mentioning

confidence: 99%

“…Exosite 1 of thrombin interacts with the central E-domain of fibrin, whereas exosite 2 binds only to the COOH terminus of the ␥Ј-chain. Consequently, thrombin binds ␥ A /␥ A -fibrin in a univalent fashion with a K d value of 2-4 M. In contrast, both exosites are engaged when thrombin binds to ␥ A /␥Ј-fibrin, resulting in a higher affinity interaction (K d value of 0.08 -0.18 M) (5,9). Fibrin-bound thrombin remains active, and the protease is protected from inhibition by fluid-phase inhibitors, such as antithrombin and heparin cofactor II (6).…”

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confidence: 99%

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Histidine-rich Glycoprotein Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain

Stafford

Leslie

et al. 2011

Journal of Biological Chemistry

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Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn2؉ -dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRGfibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn 2؉ -supplemented human plasma, a finding consistent with a high affinity interaction. Fibrinogen is the soluble precursor of fibrin, a critical component of blood clots that endows them with strength and elasticity. Fibrinogen is a glycoprotein composed of three pairs of polypeptide chains, termed A␣, B␤, and ␥, that are connected by disulfide bonds (1). Approximately 10 -15% of circulating fibrinogen has a variant ␥-chain termed the ␥Ј-chain, which results from differential processing of the ␥ A -chain mRNA transcript (2-4). The ␥Ј-chain is distinguished from the ␥ A -chain by the presence of an acidic 20-residue extension at its COOH terminus (2, 5).Thrombin catalyzes the conversion of fibrinogen to insoluble fibrin, and during this process some thrombin remains bound to the fibrin network (6). Exosites 1 and 2 are two regulatory domains that flank the active site of thrombin and mediate its binding to fibrin (7,8). Exosite 1 of thrombin interacts with the central E-domain of fibrin, whereas exosite 2 binds only to the COOH terminus of the ␥Ј-chain. Consequently, thrombin binds ␥ A /␥ A -fibrin in a univalent fashion with a K d value of 2-4 M. In contrast, both exosites are engaged when thrombin binds to ␥ A /␥Ј-fibrin, resulting in a higher affinity interaction (K d value of 0.08 -0.18 M) (5, 9). Fibrin-bound thrombin remains active, and the protease is protected from inhibition by fluid-phase inhibitors, such as antithrombin and heparin cofactor II (6). Because of its bivalent interaction with ␥ A /␥Ј-fibrin, thrombin bound to ␥ A /␥Ј-fibrin is more protected from inhibition by fluid-phase inhibitors than thrombin bound to ␥ A /␥ Afibrin (10).Like thrombin, histidine-rich glycoprotein (HRG) 3 binds to fibrinogen and is incorporated into fibrin clots (11). Although the plasma concentration of HRG ranges from 1.6 to 2 M, the concentration in platelet-rich thrombi may be higher because HRG is stored in the alpha granules of platelets and is released when platelets are activated (12, 13). A 75-kDa glycoprotein, HRG, is composed of two NH 2 -terminal cystatin-like domains, a central histidine-rich region (HRR) flanked by two prolinerich regions, and a COOH-terminal domain (14). In addition to fibrinogen, HRG also binds plasminogen, heparan sulfate, and divalent cations, such as Zn 2ϩ (12,15,16). Therefore, HRG is hypothesized to be an important accessory or adapter protein that brings different ligands together under specific conditions (14).HRG-deficient mice exhibit a shorter prothrombin time and accelerat...

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Anticoagulants

Henry

Desai

2010

Burger's Medicinal Chemistry and Drug Discovery

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Thrombotic disorders are a major cause of morbidity and mortality in humans. Examples of thrombotic disorders include pulmonary embolism, deep vein thrombosis, disseminated intravascular coagulation, acute myocardial infarction, unstable angina, and cerebrovascular thrombosis. Anticoagulants are the mainstay of treatment and prevention of these disorders. The most widely used anticoagulants include unfractionated heparin, low molecular weight heparins and warfarin. Newer agents introduced in the past decade include bivalirudin, fondaparinux, and argatroban. Mechanistically, these anticoagulants either directly inhibit thrombin or indirectly reduce the activity of thrombin and factor Xa. Several new molecules have been recently designed to directly inhibit thrombin and factor Xa with high potency and considerable selectivity, of which dabigatran and rivaroxaban were approved for clinical use in few countries in 2008. Molecules being pursued in clinical trials include AZD0837, LB‐30870, otamixaban, apixaban, YM‐150, and others. Ximelagatran, which was introduced in 2004 as a direct thrombin inhibitor, was taken off the market within 20 months due to significant hepatotoxicity. Indirect anticoagulants utilize the antithrombin‐mediated conformational activation and bridging mechanisms for inhibiting coagulation enzymes. Among the indirect anticoagulants that are currently being investigated in clinical trials are a biotinylated idraparinux and SR123781. Over the last two decades, major conceptual strides have been made including the design of the first anticoagulant based solely on factor Xa inhibition (fondaparinux) and the establishment of the paradigm that anticoagulation is possible without frequent laboratory monitoring (ximelagatran). Current work attempts to establish other concepts such as the dual inhibition of free and clot‐bound thrombin by a heparin‐based molecule (ORG42675) and the applicability of neutral P1‐group containing active site‐directed molecules as effective inhibitors of coagulation enzymes (rivaroxaban). To date, no molecule has been designed that satisfies all the properties of an ideal anticoagulant including a broad therapeutic window, absence of bleeding risk, no requirement for frequent coagulation monitoring, oral bioavailability, availability of an effective antidote, lack of hepatoxicity and absence other adverse effects. Yet, the newer agents are likely to be more attractive than the heparin‐ and coumarin‐based therapy. In this chapter, we review the structure, design, and mechanism of action of clinically used and new potential anticoagulants. Special emphasis is placed on the molecular interaction of anticoagulants with their targets.

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Evidence That Both Exosites on Thrombin Participate in Its High Affinity Interaction with Fibrin

Cited by 73 publications

References 44 publications

Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator

Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator

Histidine-rich Glycoprotein Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain

Anticoagulants

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