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ORAL PRESENTATIONS: Thrombin activatable fibrinolysis inhibitor (TAFI)
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28 Effect of TAFI activation on clot lysis by
pharmacological concentrations of t-PA *COLUCC1 M, *D 'APRILEAM, **GRESELEP, and *SEMERARON *Dept. of Biomedical Sciences, University of Bari, and **Inst. of Internal and Vascular Medicine, University of Perugia, Italy
TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that, upon activation by thrombin (TAFIa), inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. Activation of TAFI is supposed to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has been poorly investigated. We used an in vitro model consisting of tzsI-fibrin blood clot submersed in autologous citrated plasma. Upon addition of t-PA (0.1 - 5 lag/ml) and CaCI2 (25 mM), samples were incubated at 37°C, and TAFIa activity (with furoylacroleyl-alanil-arginine substrate) and clot lysis were measured at intervals. Under these conditions activation of TAFI amounted to 15-30% of inducible carboxypeptidase activity. Addition of PCI (carboxypeptidase inhibitor, 50 lag/ml) totally inhibited TAFIa but had vir-
tually no effect on clot lysis, a modest increase in the extent of lysis being only observed at concentrations of t-PA below 0.25 lag/ml. Addition of soluble thrombomodulin (solulin, 1 lag/ml, provided by Dr. J. Morser) to the plasma caused >75% activation of TAFI within 15 min but resulted in inhibition of clot lysis only in samples containing 0.1 or 0.25 lag/ml oft-PA (47.4% and 38.1% reduction in degree of lysis at 2 h, respectively). Surprisingly, solulin had no effect on clot lysis induced by 1 or 5 lag/ml of t-PA (10% variation in lysis). Neutralization of TAFIa by PCI enhanced clot lysis by low but not by pharmacological concentrations of t-PA, thus confirming that, in the latter condition, the activation of TAFI does not interfere at all with the rate of lysis. Interestingly, similar results were obtained when blood clots were replaced by Chandler thrombi, that better resemble in vivo formed thrombi. These data question the role of TAFI as an inhibitor of fibrinolysis during thrombolytic therapy.
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29 Thrombin-activatable fibrinolysis inhibitor
slows clot lysis in rats, rabbits and dogs WANG M, NAGASHIMA M, and MORSER J Berlex Biosciences, Richmond, CA, USA
Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma inducible carboxypeptidase B or carboxypeptidase U, has been shown to inhibit fibrinolysis in human plasma following in vitro thrombin-catalyzed activation. Its potential role in regulation of thrombolysis has been suggested based on an inverse correlation observed between time to recanalization of coronary vessels and plasma level of activated TAFI following t-PA induced thrombolysis in dog (Redlitz et al., 93, 1328-1330, 1996). In order to understand the importance of TAFI in fibrinolysis in various species, we studied the effect of activated TAFI directly in a clot lysis assay using plasma from human, rat, rabbit and dog. Clots were formed rapidly from citrated plasma in a microtiter plate by addition of~x-thrombin (10 nM) and calcium (20 raM). Extent and time of clot lysis induced by concomitant presence of tissue-plasminogen activator (tPA, up to 80 nM) were measured by monitoring turbidity at
405 nm. Effects of activated TAFI on clot lysis were demonstrated with thrombomodulin (TM), which potentiated TAFI activation by thrombin, and carboxypeptidase inhibitor from potato (CPI), which inhibited TAFI activity. In all plasma, clot lysis time was increased up to 3-fold upon addition of TM in a dose-dependent manner. While a much higher concentration of tPA was required to lyse clot in rat, compared with other species, effects of TM on clot lysis time were observed at various tPA concentrations. Prolongation of clot lysis time by activated TAFI was reversed by CPI, with an IC50 of around 5 nM in rabbit, and 30 to 130 nM in other species. In human and rabbit plasma, CPI had no effect on clot lysis time in the absence of exogenous TM. On the other hand, in rat and dog plasma, CPI reduced clot lysis time further in the absence of exogenous TM. Our data indicated that TAFI, upon activation, influences fibrinolysis through a similar mechanism in each species tested, but the extent of its involvement may differ depending on the level of available TM.
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30 Levels of carboxypeptidase B (thrombinactivatable fibrinolysis inhibitor: TAFI) following thrombolysis with streptokinase HUDSON I and DE BONO DP Division of Cardiology, University of Leicester, The GlenfieMHospital, Leicester, UK Background. Recently a thrombin-activatable fibrinolysis inhibitor
(TAFI) has been recognised which appears to be a precursor of plasma carboxypeptidase B (CpB) and which has CpB-like activity. Carboxyterminal lysine residues are exposed in the initial stages of fibrin proteolysis. These residues act as additional plasminogen binding sites and accelerate its activation and protect plasmin from physiological inhibitors. CpB removes these residues and can thus suppress fibrinolysis. The levels of CpB in human plasma are estimated at 2-9 U/L. Methods. We measured the levels of CpB following streptokinase
therapy of myocardial infarction (n= 12; ages 52-79, mean 68.8 years; 7 male; 8 anterior). Samples were taken prior to therapy and at 1, 2, 4, 12, 24, 36 and 48 hours. All samples were taken into citrate with aprotinin and hirudin. A dynamic chromogenic assay using furylacroleyl-
alanyl-arginine as substrate was employed. To obtain plasma CpB activity, the inhibitable portion of the total Cp activity was subtracted. The inhibitable portion is that which is inhibited by a specific CpB inhibitor: potato carboxypeptidase inhibitor (PCI). Results. CpB levels were within normal limits at the time of presentation (mean 6.14 U/L), fell at 1 hour (mean 3.58) and rose significantly to peak at 4 hours (mean 10.6 U/L). Levels of CpB then fell significantly from 4 to 12 hours (p=0.01) to a mean of 2.5 U/L. Beyond 12 hours levels were not significantly different from those at presentation. Conclusions. The rise in levels of CpB activity early following thrombolysis confirm in vitro studies which suggest plasmin induces activity of this enzyme. The fall in activity from 4 to 12 hours implies subsequent consumption of the enzyme. CpB (TAFI) could be of physiological importance during thrombolysis and thrombolytic therapy may itself induce the activation of inhibitors of fibrinolysis.