Abstract-Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) exerts an antifibrinolytic effect by removing C-terminal lysines from partially degraded fibrin. These lysines are essential for a rapid conversion of plasminogen to plasmin by tissue type plasminogen activator. TAFI is heavily glycosylated at Asn 22 , Asn 51 , Asn 63 , and Asn 86 . Although the glycans occurring at the glycosylation sites have previously been identified, the biochemical role of these glycans is not known yet. Therefore, we have determined the biochemical importance of the glycosylation in TAFI. Four single, 6 double, 4 triple, and 1 quadruple mutant, in which asparagine was replaced by glutamine, were constructed and transfected into HEK293T cells. Based on the determination of antigen and activity levels on conditioned medium, 4 single and 1 triple mutant were purified and their biochemical properties were determined. The glycosylation knockout mutants did neither reveal an altered fragmentation pattern nor differences in TAFIa stability, but TAFI-N51Q, TAFI-N63Q, and TAFI-N22Q-N51Q-N63Q revealed a decreased TAFIa activity, an increased intrinsic catalytic activity of the zymogen, and a decreased antifibrinolytic potential compared with TAFI-wild-type, whereas TAFI-N22Q and TAFI-N86Q revealed an increased antifibrinolytic potential probably because of an increased catalytic efficiency toward the physiological substrate. From these data it can be concluded that mainly the glycosylation at Asn 86 contributes to the biochemical characteristics of TAFI. Furthermore we provide evidence that the activation peptide stays in close proximity to the TAFIa moiety after activation.
BelgiumTo cite this article: Hillmayer K, Macovei A, Pauwels D, Compernolle G, Declerck PJ, Gils A. Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI) -a comparative study assessing the biological equivalence of rat, murine and human TAFI. J Thromb Haemost 2006; 4: 2470-7.Summary. Background and objectives: Activated thrombinactivatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis. Although rat models to study the role of TAFI are available, the biochemical properties of rat TAFI are not well investigated and immunologic tools are lacking. Therefore, we have characterized recombinant rat TAFI-6His and compared its properties with those of human TAFI as well as of murine TAFI-V5-6His. Methods and results: TAFI from all three species is activatable by the thrombin-thrombomodulin complex, generating a highly unstable protein (TAFIa). Half-lives at 37°C are 8.5 ± 0.6 min, 3.4 ± 0.4 min and 2.2 ± 0.2 min for human, rat and murine TAFIa, respectively. The 50% clot lysis times are 6 ± 1 min for TAFI-depleted rat plasma and 137 ± 34 min, 62 ± 9 min and 50 ± 8 min when TAFIdepleted rat plasma is supplemented with 0.02 U of human, rat or murine TAFIa, respectively, which correlates with their halflives. Upon incubation with the thrombin-thrombomodulin complex, the 36-kDa fragment of rat and murine TAFI is not cleaved into 25-kDa and 11-kDa fragments. Upon incubation of rat TAFI and murine TAFI with plasmin, a 32-kDa fragment is formed due to cleavage at Arg147, in contrast to the formation of a 36-kDa fragment for human TAFI. Concomitantly, activity levels upon plasmin incubation are drastically reduced for rat and murine TAFI. Conclusions: Recombinant human, rat and murine TAFI have similar but not identical biochemical characteristics, suggesting a similar role during fibrinolysis in vivo.
Summary. Background: Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. Objective: To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA-RT13B2, MA-RT30D8, MA-RT36A3F5, MA-RT36B2 and MA-RT82F12). Methods and results: Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% ± 1%, 54% ± 4%, and 19% ± 2% in activity in the presence of a 16-fold molar excess of MA-RT13B2, MA-RT36A3F5, and MA-RT82F12, respectively whereas MA-RT30D8 and MA-RT36B2 had no direct inhibitory effect. Additionally, MA-RT13B2 and MA-RT36A3F5 reduced rat TAFIa half-life by 56% ± 2% and 61% ± 3%. Tissue-type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA-RT13B2, MA-RT30D8, MA-RT36A3F5, and MA-RT82F12 reduced clot lysis times by 86% ± 14%, 100% ± 5%, 100% ± 10%, and 100% ± 11%, respectively. , and Tyr 260 are involved in the binding of MA-RT30D8 and MA-RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA-RT13B2 and MA-RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA-RT30D8 and MA-RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. Conclusions: The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms.
We developed three highly specific and extremely sensitive sandwich-type ELISAs for the quantification of rat and murine TAFI in plasma. The described ELISAs will facilitate in vivo investigation on the pathophysiological role of TAFI.
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