Abstract
Previous studies demonstrated that tissue plasminogen activator-induced fibrinolysis in vitro is retarded in the presence of prothrombin (II) activation and that the anticoagulant-activated protein C appears profibrinolytic by preventing the formation of thrombin (IIa)-like activity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assay that is sensitive to the antifibrinolytic effect of II activation was developed and was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified components, is retarded when this protein is present and when II activation occurs in situ, the protein was named TAFI (thrombin-activatable fibrinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa product correlates with both prolongation of lysis time and CPB-like activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen but does not prolong the lysis of clots formed in the presence of Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive inhibitor of CPB, completely inhibits prolongation of lysis by activated TAFI in a purified system and the prolongation induced by II activation in barium-adsorbed plasma. This suggests that TAFI accounts for the antifibrinolytic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.
Highlights
thrombin-activated and inhibits fibrinolysis (TAFI) activity was assessed by the prolongation oflysis time in the presence of factor Xa (FXa) relative to the lysis time in the absence of FXa
Shown in panel a are both the absolute lysis times (O,e) and relative lysis times of clots produced from fibrinogen solution containing 1.25 ILl of the TAFI activation solution, IIa (6.8 na), and both Ca 2 + (10.0) and type plasminogen activator (t-PA) (441 pv), The lysis times were measured in the absence (0) and presence (e) of FXa (100 pM, final)
Based on amino-te r minal sequence da ta we have identified TAFI as a plasma carboxypeptidase B (CPB), as described by
Summary
Materials and Methods-The recombinant t-PA, Activase, was generously provided by Dr Gordon Vehar of Genentech For each incubation time point a clot was produced by the addition of the fibrinogen solution to wells in which a 1.25-IJ.I aliquot of the TAFI activation medium had been placed, giving a final concentration of 40. Lysis times of clots prepared from 95-J.LI aliquots (see under "Lysis Assay") in the presence and absence of FXa (i.e. plus or minus II activation) were determined for each of the GEMSA concentrations. These experiments constituted an evaluation of the effect of GEMSA on TAFI-mediated prolongation of lysis. The species that were excised included Glu·Pgn, Lys·Pgn, and plasrnin-oy-antiplasmin complexes
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