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Rebuttal to: Effect of heparin on TAFI-dependent inhibition of fibrinolysis

2003; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês

10.1046/j.1538-7836.2003.00036.x

1538-7933

Ton Lisman, P G de Groot,

Hemophilia Treatment and Research

Dear Sir, We read with interest the recent paper by Colucci et al. [1Colucci M. Pentimone A. Binetti B.M. Cramarossa M. Piro D. Semeraro N. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.Thromb Haemost. 2002; 88: 282-7Crossref PubMed Scopus (25) Google Scholar], who studied the effect of unfractionated heparin on TAFI-dependent inhibition of fibrinolysis. The conclusion of their study was that heparin has no effect on TAFI-dependent clot lysis, as heparin only inhibits thrombin in solution and not clot-bound thrombin. The authors postulate that clot-bound thrombin plays a pivotal role in TAFI-dependent down-regulation of fibrinolysis, and that localized generation of activated TAFI by clot-bound thrombin is sufficient for inhibition of clot lysis. The conclusion by Colucci et al. that heparin, at concentrations which completely inhibit thrombin generation in solution, has no effect on clot lysis, is in sharp contrast with results obtained in our laboratory. Using a plasma-based clot lysis assay, which has been extensively used to study TAFI-dependent down-regulation of fibrinolysis in a variety of pathologies including factor XI deficiency [2Von Dem Borne P.A. Bajzar L. Meijers J.C.M. Nesheim M.E. Bouma B.N. Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis.J Clin Invest. 1997; 99: 2323-7Crossref PubMed Scopus (207) Google Scholar], hemophilia [3Mosnier L.O. Lisman T. Van Den Berg H.M. Nieuwenhuis H.K. Meijers J.C.M. Bouma B.N. The defective downregulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration.Thromb Haemost. 2001; 86: 1035-9Crossref PubMed Scopus (115) Google Scholar, 4Lisman T. Mosnier L.O. Lambert T. Mauser-Bunschoten E.P. Meijers J.C.M. Nieuwenhuis H.K. De Groot P.G. Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe haemophilia A.Blood. 2002; 99: 175-9Crossref PubMed Scopus (149) Google Scholar], liver cirrhosis [5Lisman T. Leebeek F.W. Mosnier L.O. Bouma B.N. Meijers J.C. Janssen H.L. Nieuwenhuis H.K. De Groot P.G. Thrombin-activatable fibrinolysis inhibitor deficiency in cirrhosis is not associated with increased plasma fibrinolysis.Gastroenterology. 2001; 121: 131-9Abstract Full Text PDF PubMed Scopus (236) Google Scholar], and hemostatic changes associated with the use of oral contraceptives [6Meijers J.C. Middeldorp S. Tekelenburg W. Van Den Ende A.E. Tans G. Prins M.H. Rosing J. Buller H.R. Bouma B.N. Increased fibrinolytic activity during use of oral contraceptives is counteracted by an enhanced factor XI-independent down regulation of fibrinolysis: a randomized cross-over study of two low-dose oral contraceptives.Thromb Haemost. 2000; 84: 9-14Crossref PubMed Scopus (114) Google Scholar], we also studied the effect of heparin on TAFI-mediated down-regulation of fibrinolysis. In short, in a 96-wells microtitre plate, pooled normal plasma to which different concentrations of unfractionated heparin (Leo pharmaceutical products, Weesp, The Netherlands) was added, was allowed to clot by the addition of tissue factor (Innovin, Dade Behring, 1 : 1000 times diluted), calcium chloride (17 mmol L−1), and phospholipid vesicles (10 µmol L−1), fibrinolysis was induced by addition of tPA (30 U mL−1; Chromogenix, Mölndal, Sweden). Coagulation and clot lysis were monitored by continuous turbidity measurements at at a wavelength 405 nm, and clotting times and clot lysis times were determined as described [4Lisman T. Mosnier L.O. Lambert T. Mauser-Bunschoten E.P. Meijers J.C.M. Nieuwenhuis H.K. De Groot P.G. Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe haemophilia A.Blood. 2002; 99: 175-9Crossref PubMed Scopus (149) Google Scholar]. As shown in Fig. 1(A), clotting times dose-dependently increased on addition of heparin. No clot formation was observed at concentrations above 0.5 U mL−1. Clot lysis times dose-dependently decreased on addition of heparin (Fig. 1B, open circles). When carboxypeptidase inhibitor from potato (CPI, Calbiochem, La Jolla, CA), a specific inhibitor of activated TAFI, was added to the plasma, clot lysis time was significantly decreased compared to control values, indicating that TAFI activation contributes to inhibition of fibrinolysis in this assay. Addition of heparin did not affect clot lysis time in the presence of CPI (Fig. 1B, closed circles), indicating that the decrease in clot lysis time was attributable to decreased TAFI activation. The discrepancy between the experiments of Colucci et al. and our own, can probably be explained by substantial differences in experimental set-up. In the experiments performed by Colucci et al., first, a fibrin clot is made by adding thrombin and calcium to citrated plasma, which is subsequently incubated for 1 h at 37 °C. Next, the clot is washed with saline, and transferred to defibrinated plasma containing vehicle or heparin. Clot lysis is induced by adding tPA and calcium chloride. In contrast, in our assay, heparin is already present before clot formation. Thus, in the Colucci assay, the effect of heparin on lysis of a fully matured clot is investigated, whereas in our experiments, the effect of heparin on resistance against fibrinolysis on a developing clot is examined. In the assay performed by Colucci, the TAFI activation in the first part of the assay will already contribute in part to down-regulation of fibrinolysis in the second part of the assay, as some C-terminal lysine and arginine residues will become available during fibrinolysis induced by endogenous tPA. Moreover, and probably of more importance, the thrombin generated in the first part of the assay will bind to the clot, and will contribute to TAFI activation in the second part of the assay. TAFI activation via clot-bound thrombin formed in the first part of the assay, presumably will occur by factor XI-dependent enhancement of thrombin generation [7Von Dem Borne P.A. Meijers J.C.M. Bouma B.N. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis.Blood. 1995; 86: 3035-42Crossref PubMed Google Scholar]. In fact, heparin has been shown to enhance factor XI activation by fibrin-bound thrombin [8Von Dem Borne P.A. Meijers J.C. Bouma B.N. Effect of heparin on the activation of factor XI by fibrin-bound thrombin.Thromb Haemost. 1996; 76: 347-53Crossref PubMed Scopus (19) Google Scholar]. If this process remains associated to the clot, the generated thrombin will remain insusceptible to inhibition by heparin. Enhancement of thrombin generation via clot-bound thrombin is required for TAFI activation, as absence of calcium- or vitamin K-dependent factors enhances clot lysis (Fig. 3 in the paper by Colucci et al.) [1Colucci M. Pentimone A. Binetti B.M. Cramarossa M. Piro D. Semeraro N. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.Thromb Haemost. 2002; 88: 282-7Crossref PubMed Scopus (25) Google Scholar]. In contrast, in our assay, the presence of heparin in plasma before clot formation suppresses thrombin generation throughout the process of clot formation and subsequent lysis, and consequently diminishes the amount of clot-bound thrombin. In conclusion, we want to modify the conclusion drawn by Colucci et al. that heparin does not affect TAFI-mediated down-regulation of fibrinolysis in vitro. In our opinion, heparin does not affect resistance against fibrinolysis in a mature clot, but significantly affects clot stability of a developing clot by means of diminishing TAFI activation. If these data can be translated into the in vivo situation, this would mean that heparin administered after venous or arterial thromboembolism does not facilitate thrombolysis, as TAFI activation by clot-bound thrombin provides optimal lysis resistance. However, heparin would contribute to the prevention of reocclusion, as TAFI activation in a newly developing clot is efficiently inhibited, making the new thrombus more susceptable to breakdown by endogenous fibrinolysis.