Summary. Objective: To elucidate the mechanism and the binding regions of monoclonal antibodies (MA) that interfere with thrombin-activatable fibrinolysis inhibitor (TAFI)/activated thrombin-activatable fibrinolysis inhibitor (TAFIa) activity. Results: Of 42 MA, 19 interfere with the TAFI activation/ TAFIa activity resulting in an inhibition of up to 92%. Characterization of the mechanism of inhibition revealed that 14 MA blocked the activation of TAFI by thrombin/thrombomodulin completely whereas five MA interfered directly with the enzymatic activity of TAFIa. Surprisingly, the former, except one, induced a significant reduction of clot lysis time whereas the latter did not. Affinity studies using a human/ murine TAFI chimer revealed that the binding region of the 14 activation blocking MA is located between AA1 and AA67. MA that inhibit exclusively the activation of TAFI by thrombin/thrombomodulin bind to Gly 66. A MA that inhibits the activation of TAFI by both thrombin/thrombomodulin and plasmin binds to Val 41 . The MA that interfere with the enzymatic activity bind to the TAFIa moiety. Conclusions: The current study reveals at least three different putative molecular targets in the search for pharmacologically active compounds to modulate TAFIa activity.
Objectives-To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. Methods and Results-A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; PϽ0.001) and TAFIa (112.1 versus 103.3; Pϭ0.03), and not of TAFI antigen (92.5 versus 87.9; Pϭ0.07) (results in % of plasma pooled from normolipidemic subjects). Conclusion-ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases. ; 36 kDa). Subsequently, TAFIa is inactivated through a conformational change to an inactive form (TAFIai), followed by proteolytic cleavage resulting in fragments of 25 and 11 kDa. 5 TAFIa exerts an antifibrinolytic effect by removing C-terminal lysine residues from fibrin, resulting in a decreased plasmin formation and a retardation of clot lysis. 6 TAFIa is highly unstable in vitro (half-life at 37°C between 8 to 15 minutes). [7][8][9][10] Different clinical studies have investigated the possible relationship between TAFI and cardiovascular events. A positive correlation was found between TAFI levels and the risk for coronary artery disease, 11 venous thrombosis, 12 angina pectoris, 13 and ischemic stroke. 14 However, another study revealed that elevated TAFI may be protective against myocardial infarction. 15 A possible explanation for these contradictory results was that the different studies used different methods for TAFI determination. 16 Silveira et al used quantitative activation of the zymogen followed by determination of the total enzymatic activity with a chromogenic assay. Van of TAFI antigen levels, as a putative risk marker for cardiovascular events, has been mainly focused on "total" antigen levels (either by ELISA or by activity assays after full activation of TAFI), reporting levels of 4 to 15 g/mL. 16,20 -22 However, it should be stressed that in most of these studies the used immunologic assays were not validated with respect to putative differential reactivities toward different isoforms and fragments...
Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change.
Announcing a TAFIa mutant with a 180-fold increased half-life and concomitantly a strongly increased antifibrinolytic potential Activated thrombin-activatable fibrinolysis inhibitor (TAFIa; 36 kDa), formed upon activation of TAFI (56 kDa) by thrombin-thrombomodulin, plays a pivotal role in fibrinolysis and inflammation [1][2][3]. Regulated by an intrinsic temperature-dependent instability (i.e., half-life of 10 min at 37°C, 45 min at 30°C, and several hours at 22°C, and stable at 0°C) [4], TAFIa is converted into an inactive conformation (TAFIai; 36 kDa). Furthermore, it has been shown that this intrinsic instability of TAFIa is of major importance for the in vivo downregulation of its activity [5][6][7]. The underlying structural mechanisms of this rapid and spontaneous loss of activity are still unclear, and this has complicated the study of the effect of TAFIa on fibrinolysis. In addition, attempts to crystallize human TAFI or TAFIa have been unsuccessful so far, probably due to glycosylation in the prosegment or to the intrinsic instability of the enzyme. Attempts have been made to increase the stability of TAFIa by mutagenesis [7] or by construction of chimeric variants [8].Although some variants exhibit an increased TAFIa stability [8], this effect is not associated with an increased antifibrinolytic effect. Recently, we reported the generation of the TAFI-S305C-T325I-T329I variant, harboring the naturally occurring Ile isoform at position 325 and two stabilizing mutations i.e., S305C and T329I [9]. We determined a half-life of 70 ± 3.0 min at 37°C for the TAFIa-S305C-T325I-T329I variant vs. 6.3 ± 0.3 min for wild-type TAFIa (TAFIa-wt) (harboring residue Thr325) (elevenfold increase) and observed a 3.0-fold increased antifibrinolytic potential vs. TAFIa-wt. Simultaneously, Knecht et al. [10] reported that the H333Y and H335Q mutations also increase TAFIa stability. TAFIa-H333Y-H335Q had a half-life of 90 min at 37°C vs. 12 min for TAFIa-wt (7.5-fold increase) and a concomitantly 7-fold increased antifibrinolytic potential vs. TAFIa-wt.In the current study, we combined all five previously reported stabilizing mutations, generating TAFI-S305C-T325I-T329I-H333Y-H335Q. The functional activity and stability, activation and enzyme kinetics and antifibrinolytic potential were determined and compared to those of TAFIa-wt.TAFIa activity and stability were determined as described previously [9], and similar activities of 32 ± 2.1 U mg )1 and 26 ± 3.3 U mg )1 (Table 1, P ¼ 0.06) for TAFIa-wt and TAFIa-S305C-T325I-T329I-H333Y-H335Q, respectively, were found. Both variants could be equally well inhibited by potato
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