Summary
TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase
that upon activation inhibits the fibrinolytic process by removing the C-terminal
lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has
been suggested 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 not been properly investigated. We used an in vitro model consisting of
125I-fibrin blood clots submerged in auto-logous defibrinated plasma. Upon addition of
t-PA (125-5000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37° C, and clot lysis was measured at intervals
from the radioactivity released into solution. The role of TAFI was assessed either
by neutralizing the generated TAFIa with the specific inhibitor PTI (50 g/ml) or by
enhancing TAFI activation through the addition of recombinant soluble thrombomodulin
(solulin, 1 μg/ml). In our clot lysis model, activation of TAFI amounted to about
20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant
increase in the extent of lysis only at concentrations of t-PA equal to or lower than
250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of
TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis
was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in
those containing pharmacological concentrations of the activator (1000 and 5000 ng/ml).
Additional experiments suggest that loss of fibrin-dependence by elevated concentrations
of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data
indicate that, under our experimental conditions, clot lysis by pharmacological concentrations
of t-PA is not influenced by TAFIa even after maximal activation of this procarboxypeptidase.
Keywords
Thrombus - fibrinolysis - plasminogen activators - carboxypeptidase - thrombin
