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Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

Author: Walker, J.B. · Hughes, B. · James, I. · Haddock, P. · Kluft, C. · Bajzar, L.
Institution: Gaubius Instituut TNO
Source:Journal of Biological Chemistry, 11, 278, 8913-8921
Identifier: 237005
doi: doi:10.1074/jbc.M205006200
Keywords: Biology · Biomedical Research · Chemical activation · Tissue · Competitive inhibitors · Biochemistry · 2 guanidinoethylmercaptosuccinic acid · Potato tuber carboxypeptidase inhibitor · Thrombin activatable fibrinolysis inhibitor · Tissue plasminogen activator · Unclassified drug · 3 (2 guanidinoethyl) 2 mercaptosuccinic acid · Carboxypeptidase · Carboxypeptidase inhibitor, plant · Succinic acid derivative · Vegetable protein · Blood clotting · Competitive inhibition · Concentration response · Enzyme kinetics · Enzyme stability · Half life time · In vitro study · Potato · Protein stability · Binding competition · Chemistry · Dose response · Drug antagonism · Metabolism · Theoretical model · Solanum tuberosum · Binding, Competitive · Carboxypeptidase U · Carboxypeptidases · Dose-Response Relationship, Drug · Enzyme Inhibitors · Fibrinolysis · Humans · Kinetics · Models, Theoretical · Plant Proteins · Protein Binding · Succinates · Temperature · Time Factors · Tissue Plasminogen Activator


Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged, or had no effect on lysis in vitro. The inhibitor-induced effects were both tissue-type plasminogen activator (tPA) and TAFIa concentration-dependent. Inhibitor-dependent prolongation was favored at lower tPA concentrations. The magnitude of the prolongation increased with TAFIa concentration, and the maximal prolongation observed at each TAFIa concentration increased saturably with respect to TAFIa. A theoretical maximal prolongation of 20-fold was derived from a plot of the maximum prolongation versus TAFIa. This represents, for the first time, a measurement of the maximal antifibrinolytic potential of TAFIa in vitro. Because TAFIa spontaneously decays, the stabilization of TAFIa was investigated as a mechanism explaining the inhibitor-dependent prolongation of lysis. Both inhibitors stabilized TAFIa in a concentration-dependent, non-saturable manner. Although their KI values differed by three orders of magnitude, TAFIa was identically stabilized when the fraction of inhibitor-bound TAFIa was the same. The data fit a model whereby only free TAFIa decays. Therefore, the variable effects of competitive inhibitors of TAFIa on fibrinolysis can be rationalized in terms of free TAFIa and lysis time relative to the half-life of TAFIa.