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.

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1 ؎ 0.6 min, and that of wild type was 8.4 ؎ 0.3 min at 37°C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Because fibrin is commonly observed within arthritic joints, studies were undertaken to determine whether purified coagulation and fibrinolytic proteases degrade cartilage in vitro and to seek evidence for the activation of coagulation in arthritic joints through measurements of the levels of inhibitor-enzyme complexes and several other proteins associated with coagulation and fibrinolysis. The concentrations of 13 plasma proteins and complexes of thrombin and Factor Xa with antithrombin III were measured in synovial fluids recovered at the time of knee replacement surgery. All zymogens necessary to constitute the coagulation cascade were present. Thrombin and the combination of prothrombin plus prothrombinase induced proteoglycan release from both normal and arthritic cartilages. Factor Xa and plasmin induced release from diseased cartilage only, and urokinase, tissue plasminogen activator, and activated protein C were without effect at the levels used. At saturating levels of thrombin (-2.0 ,uM) 80% of the proteoglycan content of normal cartilage was released within 24 h. Thrombin, which is cationic, reversibly binds cartilage with Kd = 7.0±1.0 ,uM and B__ = 820±70 ng/mg of human cartilage. Levels of thrombin-antithrombin m complexes in synovial fluids and arthritis were 4-fold higher in osteo(OA) and 43-fold higher in rheumatoid(RA) than in controls (0.98 nM). Factor Xaantithrombin III complex levels were threefold lower in OA and fivefold higher in RA than in controls (0.24 nM). These elevated levels of enzyme-inhibitor complexes imply a history of activation of coagulation within the joint, especially in RA. Since thrombin degrades cartilage in vitro and had been generated in vivo, as inferred by the existence of thrombin-antithrombin m complexes, intraarticular activation of coagulation may both contribute to the pathology of arthritis and comprise a target for therapy and diagnosis. (J. Clin. Invest. 1994. 94:472-480.)

3187 Poster Board III-124 Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 60 kDa plasma protein that is activated to the enzyme TAFIa, by a single cleavage at Arg92 by thrombin, plasmin or trypsin. TAFIa is a carboxypeptidase B-like enzyme that attenuates fibrinolysis. Thrombomodulin (TM) is a cofactor which increases the overall efficiency of thrombin-mediated TAFI activation by 1250-fold. Thus, the thrombin-TM complex is believed to be the physiological TAFI activator. The minimal structure of TM required for efficient TAFI activation contains the EGF-like domains 3 through 6. New structure models have postulated that the C-loop of TM EGF-like domain 3 has a negatively charged molecular surface that could interact with several positively charged surface patches on TAFI. One positively charged surface patch of TAFI consists of the three consecutive lysine residues at positions 42, 43, and 44, which are unique to the TAFI activation peptide as no corresponding residues exist in rattus, bovine or human tissue procarboxypeptidases A and B. More interestingly, all three lysine residues are conserved in human, rattus, murine and canine TAFI, but not for bovine TAFI which only has a single lysine residue at position 42. We previously reported that when the three lysine residues are substituted by alanine residues (K42/43/44A), compared to the wild-type, the catalytic efficiencies for TAFI activation by thrombin-TM complex decreased 8-fold. In order to identify which residue(s) are key for TAFI activation by the thrombin-TM complex, combinations of mutations of the three lysine residues were constructed and expressed. TAFI wild-type or mutants were activated by thrombin for 10 minutes in the absence or presence of TM at varying levels. At this point, the levels of TAFIa formed were measured by adding the synthetic substrate AAFR containing PPAck and measuring the absorbance change at 349nm. The rates were used to determine the kinetic parameters of TAFI activation. The non-linear regression analysis with the NONLIN module of SYSTAT returned best fit values along with their asymptotic standard errors (A.S.E) for the kinetic parameters of TAFI activation (kcat, Km, and Kd). The value of Kd (the dissociation constant for the thrombin-TM interaction) is assumed to be the same for wild-type TAFI and the mutants, because all reactions have this interaction in common. The regression analysis yielded Kd = 22.4 ± 1.3 nM for this interaction. This value agrees favourably with a value of 22 nM measured directly and reported previously. The kcat values (1/sec) ranged from 1.06 ± 0.18 (K44A) to 1.19 ± 0.18 (K43A). The value for wild-type TAFI was 1.50 ± 0.63 (1/sec). Km values ranged from 1.14 ± 0.73 μM (WT) to 3.01 ± 2.17 μM (K42A). The kcat / Km ratios (1/sec/μM), which provides the best indication of overall catalytic efficiency, ranged from 1.43 ± 0.27 (WT) to 0.43 ± 0.17 (K42A). When the three lysine residues are individually substituted by alanine residues (K42A, K43A, and K44A), compared to the wild-type, their catalytic efficiencies (kcat / Km) for TAFI activation by the thrombin-TM complex decreased 3.3-fold for K42A, 1.83-fold for K43A, and 1.96-fold for K44A. When Lys43 and Lys44 are substituted by alanine residues simultaneously (K43/44A), its catalytic efficiency decreased 3.3-fold. Together, our data show that each of these lysine residues on the activation peptide of TAFI may contribute partially to the interactions of TAFI with the thrombin-TM complex that are needed for efficient activation. In addition, the effects of the mutations may be additive. Disclosures No relevant conflicts of interest to declare.