Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin

Podor, Thomas J.; Peterson, Cynthia B.; Lawrence, Daniel A.; Stefansson, Steingrimur; Shaughnessy, Stephen G.; Foulon, D; Butcher, Martin; Weitz, Jeffrey I.

doi:10.1074/jbc.m908079199

Cited by 69 publications

(78 citation statements)

References 45 publications

(42 reference statements)

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“…Isolation of Vitronectin from Human Plasma-Native vitronectin was isolated from PPP by affinity chromotography using SAHVn IgG coupled to Affi-Gel affinity resin (Bio-Rad) (8,35). For some experiments, native vitronectin was converted to the oligomeric form by treatment with 6 M urea in phosphate-buffered saline (PBS), pH 7.4, at 37°C for 1 h, followed by dialysis against PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Dual wavelength images were acquired using an argon ion laser (488 nm excitation), a helium/neon ion laser (543 nm excitation) and two matched long pass barrier filters for FITC (515-525 nm emission) and TxR (575-640 nm emission) images. Immunofluorescence detection of PAI-1 in clots formed from normal plasma was conducted as previously described (35).…”

Section: Methodsmentioning

confidence: 99%

“…Vitronectin preparations were subjected to PAGE analysis in the absence or presence of SDS, and conformational changes were assessed by measuring their relative affinities for heparin-Sepharose and for the conformation-dependent monoclonal antibody 8E6. Fibrinogen was rendered vitronectin-free by immunoaffinity chromatography using immobilized SAHVn IgG and was characterized as described (35).…”

Section: Methodsmentioning

confidence: 99%

“…Immunohistochemical studies have localized vitronectin along fibrin strands of thrombi formed in vivo (33), and in vitro (34,35), suggesting that vitronectin binds to fibrin. Recently, we demonstrated that fibrin-associated vitronectin mediates the cooperative multivalent binding of PAI-1 to fibrin (35).…”

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confidence: 99%

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Incorporation of Vitronectin into Fibrin Clots

Podor¹,

Campbell²,

Chindemi³

et al. 2002

Journal of Biological Chemistry

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19788 -19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that ϳ20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from 125 I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K d of ϳ0.6 M. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the ␥A/␥ variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the ␥A/␥ fibrinogen fraction, and clots formed from ␥A/␥ fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.Vitronectin is a multifunctional plasma glycoprotein that participates in the regulation of coagulation, fibrinolysis, and the complement cascade (reviewed in Refs. 1 and 2). Vitronectin also regulates cell adhesion and pericellular proteolysis on surfaces of cells and extracellular matrices (1-5). Like fibrinogen, vitronectin is found in plasma at micromolar concentrations (6), and is stored in megakaryocyte and platelet ␣-granules (7-9). In plasma, vitronectin circulates as a native, monomeric form that is a mixture of 72-kDa single-chain and two-chain disulfide-linked species (10 -12). Under normal conditions, less than 3% of plasma vitronectin is comprised of more reactive oligomeric forms that display enhanced affinity for heparin or heparin-like molecules, and for the conformationsensitive monoclonal antibody 8E6 (10 -12).During acute phase response, plasma vitronectin levels increase (6), with a relative increase in the percentage of oligomeric vitronectin (13). Levels of the oligomeric forms of vitronectin in serum relative to plasma also increase; indicating the process of coagulation alters vitronectin structure and function (10 -12, 14). The altered, oligomeric form of vitronectin is generated, at least in part, by interactions with other plasma proteins such as thrombin-antithrombin complexes (11, 12, 14, 15, and complement C5b-9 complexes (11, 12, 16).A portion of vitronectin in plasma (17,18), and in platelets is complexed with type 1 plasminogen activator inhibitor (PAI-1) 1 (8,9,19), an interaction that induces the formation of higher order complexes (4,5,20,21) and influences the structure and function of both PAI-1 and vitronectin. Thus, when bound to vitronectin, PAI-1 is stabilized in its active conforma...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Incorporation of Vitronectin into Fibrin Clots

Podor¹,

Campbell²,

Chindemi³

et al. 2002

Journal of Biological Chemistry

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“…Thus, it appears that the complexes observed in the ultracentrifugation experiments can readily form in the vicinity of a blood clot. In fact, recent studies by Podor and colleagues (49) indicate that vitronectin binds directly to fibrin clots, 4 and the fibrin-associated vitronectin mediates the high-affinity binding of PAI-1 to fibrin.…”

Section: Figmentioning

confidence: 99%

New Insights into the Size and Stoichiometry of the Plasminogen Activator Inhibitor Type-1·Vitronectin Complex

Podor¹,

Shaughnessy²,

Blackburn³

et al. 2000

Journal of Biological Chemistry

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Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M r ϳ 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.Vitronectin is a versatile glycoprotein that is found in circulation, in the extracellular matrix of endothelial cells, in platelets, and within various tissues of the human body. Circulating at micromolar levels, vitronectin participates in the regulation of humoral responses such as coagulation, fibrinolysis, and the complement cascade (reviewed in Ref. [1][2][3][4]. Other functions of the protein that are confined to surfaces or tissues include cell-adhesion and regulation of pericellular proteolysis. Interactions with an assortment of biological molecules are responsible for the multiple functions exhibited by vitronectin. Defining the binding sites for these various biomolecules, along with determining the molecular mechanism of regulation, constitutes an active area of research on the protein. Work to date has focused on binding sites for several ligands, including heparin, PAI-1, 1 and integrins; a working model representing the domain structure of vitronectin has been recently reported from this laboratory (5).Arguably, one of the most important interactions known for vitronectin occurs with the serine protease inhibitor, PAI-1. PAI-1 is the physiological inhibitor of plasminogen activators, both tPA and uPA, the enzymes responsible for generating plasmin from...

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An experimental and theoretical study on the dissolution of mural fibrin clots by tissue‐type plasminogen activator

Wootton

Popel

Alevriadou

2002

Biotech & Bioengineering

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During thrombolytic therapy and after recanalization is achieved, reduction in the volume of mural thrombi is desirable. Mural thrombi are known to contribute to rethrombosis and reocclusion. The lysis rate of mural thrombi has been demonstrated to increase with fluid flow in different experimental models, but the mechanisms responsible are unknown. An experimental and a theoretical study were developed to determine the contribution of outer convective transport to the lysis of mural fibrin clots. Normal human plasma containing recombinant tissue-type plasminogen activator (tPA; 0.5 microg/mL) was (re)perfused over mural fibrin clots with fluorescently labeled fibrin at low arterial, arterial, or higher wall shear stresses (4, 18, or 41 dyn/cm(2), respectively) and lysis was monitored in real time. Flow accelerated lysis, but significantly only at the highest shear stress: The average lysis front velocity was 3 x 10(-5) cm/s at 41 dyn/cm(2) vs. almost half of that at the lower shear stresses. Confocal microscopy showed fibrin fibers dissolving only in a narrow region close to the surface when permeation velocity was predicted to be low. A heterogeneous transport-reaction finite element model was used to describe mural fibrinolysis. After scaling the effects of outer and inner convection, inner diffusion, and chemical reactions, a simplified inner diffusion/reaction model was used. Correlation to fibrin lysis data in purified systems dictated higher rates of plasmin(ogen) and tPA adsorption onto fibrin and a decreased catalytic rate of plasmin-mediated fibrin degradation, compared with published parameters. At each shear stress, the model predicted a temporal pattern of lysis of mural fibrin (similar to that observed experimentally), and protease accumulation in a narrow fibrin region and significant lysis inhibition by plasma alpha(2)-antiplasmin (according to the literature). Increasing outer convection accelerated mural fibrinolysis, but the model did not predict the big increase in lysis rate at the highest shear stress. At higher than arterial flows, additional mechanisms not accounted for in the current model, such as fibrin collapse at the fibrin front, may regulate the lysis of mural clots and determine the outcome of thrombolytic therapy.

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Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin

Cited by 69 publications

References 45 publications

Incorporation of Vitronectin into Fibrin Clots

Incorporation of Vitronectin into Fibrin Clots

New Insights into the Size and Stoichiometry of the Plasminogen Activator Inhibitor Type-1·Vitronectin Complex

An experimental and theoretical study on the dissolution of mural fibrin clots by tissue‐type plasminogen activator

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