Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites

Abstract

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 μm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm(-1) s(-1). Interestingly, this value increases to 3.85 μm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.

FIGURE 1.

Binding of 5IAF-Glu-Pg (A) or 5IAF-Lys-Pg (B) to untreated (○) or TAFIa-treated (●) QSY-FDPs. 5IAF-Glu-Pg and 5IAF-Lys-Pg bound to QSY-FDPs with K_d values of 175 and 101 nm, respectively. Upon treatment with TAFIa, the K_d values were increased to 1.06 and 0.91 μm, respectively. An alternative interpretation indicates that 5IAF-Glu-Pg bound TAFIa-treated QSY-FDPs with a K_d of 8.34 μm. The data presented here are for one preparation of QSY-FDPs, but they are consistent with data from all other preparations. Data are presented as the mean ± S.E. (n = 3).

FIGURE 2.

Binding of TAFIa to 5IAF-Glu-Pg or 5IAF-Lys-Pg. Binding of TAFIa to 5IAF-Glu-Pg (●) or 5IAF-Lys-Pg (○) was determined by fluorescence. The K_d values of the interaction with TAFIa were determined to be 900 nm for 5IAF-Glu-Pg and 425 nm for 5IAF-Lys-Pg. The maximum extents of fluorescence quenching were 62% with 5IAF-Glu-Pg and 50% with 5IAF-Lys-Pg. Data are presented as the mean ± S.E. (n = 2).

FIGURE 3.

Kinetics of TAFIa in removing Glu-Pg- or Lys-Pg-binding sites from QSY-FDPs. The kinetics of 5IAF-Glu-Pg-binding site removal by TAFIa were determined by fitting the data by nonlinear regression to the Michaelis-Menten equation. A shows that the average k_cat and K_m values of Glu-Pg-binding site removal were 2.30 s⁻¹ and 142 nm, respectively, implying a catalytic efficiency of 16.21 μm⁻¹ s⁻¹. B shows that the average k_cat and K_m values of Lys-Pg-binding site removal were 0.89 s⁻¹ and 96 nm, respectively, which implies a catalytic efficiency of 9.23 μm⁻¹ s⁻¹. Data are presented as the mean ± S.E. (n = 3 for each TAFIa concentration).

FIGURE 4.

Effect of Glu-Pg on lysine cleavage by TAFIa. The kinetics of lysine cleavage by TAFIa were analyzed in the presence of Glu-Pg (●) or in the absence of plasminogen (■). The k_cat did not vary to any great extent whether plasminogen was added or not; however, the K_m was decreased from 2.87 to 0.95 μm in the presence of Glu-Pg. This resulted in an increase in the catalytic efficiency from 1.10 μm⁻¹ s⁻¹ in the absence of plasminogen to 3.85 μm⁻¹ s⁻¹ in the presence of Glu-Pg, respectively. Data are presented as the mean ± S.E. (n = 4).

SCHEME 1.

Equilibrium binding model of lysine cleavage by TAFIa. TAFIa (T) binds either FDPs (F) or plasminogen (P) to form the corresponding binary complexes, TF or TP. These then bind either plasminogen or FDPs to form the ternary TFP complex. The binary complex yields TAFIa plus cleaved FDPs (F′) with rate constant k₁, and the ternary complex TFP yields F′ and TP with rate constant k₂. The constants k₁ and k₂ are k_cat values. The binding interactions are characterized by equilibrium constants K_TF, K_TP, K_TFP, and K_TPF.