An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma

Kim, Paula Y.G.; Foley, Jonathan H.; Hsu, Grace; Kim, Paul Y.; Nesheim, Michael E.

doi:10.1016/j.ab.2007.09.034

Cited by 27 publications

(26 citation statements)

References 30 publications

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“…The assay was implemented as described by Kim et al. [18]. Eighty microliters of a solution in HBS of 1.0 μ m QSY9 C5‐maleimide‐conjugated high molecular mass fibrin degradation products and 50 n m recombinant S741C plasminogen labeled with 5‐iodoacetamidofluorescein was added to wells of a white opaque microtiter plate, and fluorescence intensity was monitored with excitation and emission wavelengths of 480 and 520 nm, respectively, with a 495‐nm emission cutoff filter, using a SpectraMax M2e fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Secretion and antifibrinolytic function of thrombin‐activatable fibrinolysis inhibitor from human platelets

Schadinger

Lin

Garand

et al. 2010

Journal of Thrombosis and Haemostasis

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To cite this article: Schadinger SL, Lin JHH, Garand M, Boffa MB. Secretion and antifibrinolytic function of thrombin-activatable fibrinolysis inhibitor from human platelets. J Thromb Haemost 2010; 8: 2523-9.Summary. Background: The thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen first characterized in human plasma that is activated through proteolytic cleavage by thrombin, thrombin in complex with thrombomodulin, or plasmin. Active TAFI attenuates fibrinolysis by removing Cterminal lysine residues from partially degraded fibrin, thereby inhibiting a potent positive feedback loop in the fibrinolytic cascade. The existence of a separate pool of TAFI within platelets has been described. Objectives and Methods: We aimed to confirm the presence of TAFI in the medium of washed, thrombin-stimulated platelets and to evaluate the characteristics of platelet TAFI by western blot analysis and with a quantitative assay for activated TAFI. We also assessed the ability of platelet TAFI to inhibit fibrinolysis in vitro, using a platelet-rich thrombus lysis assay. Results: Our data are consistent with the presence of TAFI in the a-granules of resting platelets. In contrast to previous reports, platelet TAFI is very similar in electrophoretic mobility to plasma-derived TAFI. We also show, for the first time, that platelet-derived TAFI is capable of attenuating platelet-rich thrombus lysis in vitro independently of plasma TAFI. Moreover, we demonstrate additive effects on thrombolysis of platelet-derived TAFI and TAFI present in plasma. Conclusions: Taken together, these observations indicate that the secretion of platelet-derived TAFI can augment the concentrations of TAFI already present in plasma to enhance attenuation of the fibrinolytic cascade. This could be significant in regions of vascular damage or pathologic thrombosis, where activated platelets are known to accumulate.

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Section: Methodsmentioning

confidence: 99%

Secretion and antifibrinolytic function of thrombin‐activatable fibrinolysis inhibitor from human platelets

Schadinger

Lin

Garand

et al. 2010

Journal of Thrombosis and Haemostasis

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“…This change in protocol allowed for a greater yield of fibrinogen. QSY-FDPs (fibrin degradation products that are covalently attached to the quencher, QSY9 C5-maleimide) and TAFIa standards used in the TAFIa assay were prepared as described [15,16] and recombinant human Pg (S741C) and the fluorescein derivative (5IAF-Pg) were prepared as described by Horrevoets et al [17]. S525C-prothrombin was purified and fluorescently labeled with 5-iodoamidofluorescein (5IAF) as previously described by Brufatto et al [18].…”

Section: Methodsmentioning

confidence: 99%

Soluble thrombomodulin partially corrects the premature lysis defect in FVIII‐deficient plasma by stimulating the activation of thrombin activatable fibrinolysis inhibitor

Foley¹,

Nesheim

2009

Journal of Thrombosis and Haemostasis

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To cite this article: Foley JH, Nesheim ME. Soluble thrombomodulin partially corrects the premature lysis defect in FVIII-deficient plasma by stimulating the activation of thrombin activatable fibrinolysis inhibitor. J Thromb Haemost 2009; 7: 453-9.Summary. Background: Previous work by others has shown that premature clot lysis occurs in plasmas deficient in components of the intrinsic pathway, due to a failure to activate thrombin activatable fibrinolysis inhibitor (TAFI). This suggests the hypothesis that bleeding in hemophilia is due not only to defective coagulation but also enhanced fibrinolysis. These studies were carried out to quantify the extent of TAFI activation over time in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP) and to determine whether soluble thrombomodulin (sTM) can correct the lysis defect in FVIII-DP. Methods: The time courses of TAFI activation in both NP and FVIII-DP were monitored after clotting with thrombin, PCPS and Ca 2+ , ± sTM. Clotting and lysis were measured turbidometrically and TAFIa using a functional assay. Results: Premature lysis that occurs in FVIII-DP is corrected by mixing deficient plasma with 10% NP. However, this does not fully correct the defect in TAFI activation. FVIII-DP must be mixed with up to 50% NP to attain the same TAFIa potential as NP. In FVIII-DP, sTM can correct the defect in TAFIa-dependent prolongation of lysis at low tPA concentrations and partially correct this defect at high tPA concentrations. Conclusions: TAFI activation increases as the concentration of FVIII increases. FVIII at a level of 10% fully corrects the lysis defect in spite of the extent of TAFI activation being only one half that obtained with 100% FVIII. In addition, sTM increases TAFI activation sufficiently to correct the premature lysis defect in FVIII-DP.

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“…Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) was measured using a functional assay. 20 Fibrin clot lysis time (CLT) was measured as described elsewhere. 21 Briefly, citrated plasma was mixed with 15 mmol/L calcium chloride, 10,000-diluted human tissue factor (Innovin, Dade Behring), 12 μmol/L phospholipid vesicles, and 60 ng/mL recombinant tissue-type plasminogen activator (Boerhinger Ingelheim, Ingelheim, Germany).…”

Section: Inflammatory and Hemostatic Markersmentioning

confidence: 99%

Modulation of Inflammatory and Hemostatic Markers in Obstructive Sleep Apnea Patients Treated with Mandibular Advancement Splints: A Parallel, Controlled Trial

Niżankowska-Jędrzejczyk

Almeida

Lowe

et al. 2014

Journal of Clinical Sleep Medicine

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BRIEF SUMMARYCurrent Knowledge/Study Rationale: The aim of our study was to assess the effi ciency of fi brinolysis and its determinants, in particular thrombin activatable fi brinolysis inhibitor (TAFI), in OSA patients, since until now plasminogen activator inhibitor-1 and D-dimer have been the only fi brinolytic parameters studied in this disease. This is the fi rst study to investigate the effects of mandibular advancement splints on hemostasis in OSA patients. Study Impact: The study demonstrates antifi brinolytic mechanisms in OSA patients, largely mediated by increased TAFI. MAS treatment in OSA patients is associated with favorable alterations in hemostasis, including improved fi brinolysis.

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An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma

Cited by 27 publications

References 30 publications

Secretion and antifibrinolytic function of thrombin‐activatable fibrinolysis inhibitor from human platelets

Secretion and antifibrinolytic function of thrombin‐activatable fibrinolysis inhibitor from human platelets

Soluble thrombomodulin partially corrects the premature lysis defect in FVIII‐deficient plasma by stimulating the activation of thrombin activatable fibrinolysis inhibitor

Modulation of Inflammatory and Hemostatic Markers in Obstructive Sleep Apnea Patients Treated with Mandibular Advancement Splints: A Parallel, Controlled Trial

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