Abstract
We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.
Highlights
Boxypeptidase U [3]) has illuminated a novel molecular linkage between these cascades
Effect of the Thermal Instability of TAFIa on Proteolytic Cleavage—We have previously reported that the competitive TAFIa inhibitors guanidinoethylmercaptosuccinic acid (GEMSA) and ⑀-ACA prevent the thermal decay of TAFIa [6]. ⑀-ACA prevents proteolytic cleavage of TAFIa [2]
By using site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in mammalian cells, we have identified Arg-302 as the thrombin cleavage site in TAFIa (Fig. 2)
Summary
Materials—The synthetic carboxypeptidase substrates hippuryl-Llysine (Hipp-Lys) and N-[3-(2-furylacryloyl)]-L-alanyl-L-lysine (FA-AlaLys) as well as ⑀-aminocaproic acid (⑀-ACA) and Sepharose CL-4B was. To obtain preparative amounts of recombinant TAFI (rTAFI), stably expressing cell lines were cultured in triple flasks (500 cm; Nunc, Roskilde, Denmark) in Opti-MEM containing 0.1% (v/v) UltroSer G, 1% (v/v) PSF, and 40 M ZnCl2. Timed aliquots were removed from the incubations, and the TAFIa activity in them was measured using FA-Ala-Lys. In some experiments, TAFIa was formed as described above, and the thrombin was quenched by the addition of PPAck to 100 nM. TAFIa was formed as described above, and the thrombin was quenched by the addition of PPAck to 100 nM At this time, aliquots of TAFIa were transferred to wet ice (0 °C) or to a water bath thermostated at 37 °C. The TAFI-deficient plasma (TdP) was aliquoted and stored at Ϫ70 °C
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