TAFIa, PAI-1 and α2-antiplasmin: Complementary roles in regulating lysis of thrombi and plasma clots

Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK.
Journal of Thrombosis and Haemostasis (Impact Factor: 5.72). 05/2007; 5(4):812-7. DOI: 10.1111/j.1538-7836.2007.02430.x
Source: PubMed


PAI-1 and alpha(2)-antiplasmin (alpha(2)AP) are the principal direct inhibitors of fibrinolytic proteases. Thrombin activatable fibrinolysis inhibitor (TAFI), a plasma procarboxypeptidase activated by thrombin-thrombomodulin to form TAFIa, also regulates fibrinolysis by modulating fibrin. In this study, the relative contributions of PAI-1, alpha(2)AP and TAFIa to inhibition of lysis were assessed. In platelet-poor plasma clots, alpha(2)AP, TAFIa and PAI-1 all inhibited lysis, as shown by the addition of neutralizing antibodies to alpha(2)AP and PAI-1 +/- CPI, a potato carboxypeptidase inhibitor. alpha(2)AP played the largest role in regulating plasma clot lysis, but neutralization of inhibitors in combinations was more effective in shortening lysis times, with a maximal effect when all three inhibitors were neutralized. In platelet-rich clots, a larger contribution of PAI-1 was evident. Tissue plasminogen activator induced lysis of model thrombi, made from whole blood, was approximately doubled on incorporation of CPI, illustrating a substantial contribution of TAFIa to inhibition of thrombus lysis. Similar increases in thrombus lysis were observed on inclusion of neutralizing antibodies to PAI-1 and alpha(2)AP, with alpha(2)AP playing the dominant role. Maximal thrombus lysis occurred upon neutralization of all three inhibitors. These observations suggest that, despite the differences in concentrations and activities of inhibitors, and the different modes of action, the roles of the three are complementary in both plasma clot lysis and thrombus lysis.

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Available from: Nicola J Mutch, Nov 01, 2014
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    • "Complement and haemostatic mechanisms also interact at the inhibitor level, for instance, C1-inhibitor inhibits complement factors C1r, C1s, MASP1, and MASP2, as well as coagulation factors XIIa and XIa [153]. TAFI not only inhibits fibrinolysis by cleaving terminal lysine residues from fibrin, thereby preventing plasminogen binding and plasmin generation [154], but TAFI also cleaves terminal arginine residues from C3a and C5a to downregulate their proinflammatory activities [155]. Cross-talk between complement and coagulation cascades therefore occurs at multiple levels and appears to be important in coordination to haemostatic and immune responses. "
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    ABSTRACT: A wealth of evidence indicates a fundamental role for inflammation in the pathogenesis of cardiovascular disease (CVD), contributing to the development and progression of atherosclerotic lesion formation, plaque rupture, and thrombosis. An increasing body of evidence supports a functional role for complement activation in the pathogenesis of CVD through pleiotropic effects on endothelial and haematopoietic cell function and haemostasis. Prospective and case control studies have reported strong relationships between several complement components and cardiovascular outcomes, and in vitro studies and animal models support a functional effect. Complement activation, in particular, generation of C5a and C5b-9, influences many processes involved in the development and progression of atherosclerosis, including promotion of endothelial cell activation, leukocyte infiltration into the extracellular matrix, stimulation of cytokine release from vascular smooth muscle cells, and promotion of plaque rupture. Complement activation also influences thrombosis, involving components of the mannose-binding lectin pathway, and C5b-9 in particular, through activation of platelets, promotion of fibrin formation, and impairment of fibrinolysis. The participation of the complement system in inflammation and thrombosis is consistent with the physiological role of the complement system as a rapid effector system conferring protection following vessel injury. However, in the context of CVD, these same processes contribute to development of atherosclerosis, plaque rupture, and thrombosis.
    12/2012; 2012(4):402783. DOI:10.6064/2012/402783
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    • "Different explanations have been given for these discrepancies [8,15], but there is a need for a quantitative method that reveals the effect of cross-linking on fibrinolysis. Whole blood model thrombi formed under flow show a similar structure and protein distribution to thrombi formed in vivo [23], and have revealed the complementary nature of α2AP, PAI-1 and TAFI [24]. Here, we used model thrombi, and show that fibrinolysis is dramatically increased in FXIII deficiency, an effect that could be recapitulated by incorporating a non-reversible inhibitor of TGs. "
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    ABSTRACT: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve γ-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.
    Journal of Thrombosis and Haemostasis 09/2010; 8(9):2017-24. DOI:10.1111/j.1538-7836.2010.03963.x · 5.72 Impact Factor
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    • "In contrast, the flow conditions of the Chandler loop mimic the shear rates of those found in larger arteries (400–600 s À1 ), thus producing a thrombus with a biochemical and structural morphology that is markedly similar to that produced in vivo (Stringer et al., 1994; Robbie et al., 1997; Mutch et al., 2003). The subsequent lysis of these thrombi by rtPA can be monitored in detail over time and has recently been used to successfully evaluate the efficacy of endogenous inhibitors on fibrinolysis (Mutch et al., 2007) and in the study of fibrinolytic surfaces for use in biomaterials (McClung et al., 2007). The assay reliably detected the inhibitory effect of AMCA on fibrinolysis, thereby increasing confidence in the validity of the experimental technique. "
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    ABSTRACT: Recombinant tissue-type plasminogen activator (rtPA) is the only globally approved treatment for acute ischaemic stroke. Other potential treatments might be administered with rtPA, making it important to discover whether compounds interfere with rtPA-induced lysis. We evaluated methods for examining the effect of the neuroprotectant NXY-059 on the lytic property of rtPA. Plasma clot formation and lysis in the presence of rtPA and NXY-059 was measured as the change in plasma turbidity. The effect of NXY-059 on rtPA-induced lysis was similarly assessed on preformed clots. Lysis of the thrombus formed in a Chandler loop measured release of fluorescent-tagged fibrinogen that had been incorporated during thrombus formation. Thrombi were exposed to both rtPA and NXY-059 throughout lysis in the presence of 80% autologous plasma and the release of label during lysis was measured. Data interpretation is limited in the clot lysis experiments because either the rtPA was present during clot formation or the drug was added to a clot formed in static conditions. In contrast, thrombi were formed in dynamic flow conditions in the Chandler loop and the time course of lysis in plasma was examined. rtPA increased thrombolysis and the antifibrinolytic trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCA) inhibited lysis. Lysis induced by rtPA was unaltered by NXY-059. The Chandler loop method provides a reliable technique for examining the effect of compounds on rtPA-induced lysis in vitro and demonstrated that NXY-059 does not alter rtPA-induced lysis at clinically relevant concentrations of either drug.
    British Journal of Pharmacology 02/2008; 153(1):124-31. DOI:10.1038/sj.bjp.0707543 · 4.84 Impact Factor
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