• Thrombophilia
• Coagulation Defects
• Purpurea
• Other Hemorrhagic Conditions

• Thrombophilia
• Coagulation Defects
• Purpurea
• Other Hemorrhagic Conditions
• Hematology

Assays for the activated form of thrombin activatable fibrinolysis Inhibitor(a) (TAFIa) can measure the extent of TAFI activation in patient populations. TAFI is a human plasma zymogen that is related to pancreatic carboxypeptidase B (CPB). The active form of TAFI (TAFIa), formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal of the carboxyl-terminal lysine residues of partially degraded fibrin that stimulate plasminogen activation. TAFI is encoded by the CPB2 gene (chromosome 13) [Boffa MB et al. Biochemistry 1999]. Ann Gils, PhD, Katholieke Universiteit, Leuven, Leuven, Belgium, presented insights into the function and regulation of TAFI.

During the cloning of TAFI cDNA from several human liver cDNA libraries, Zhao and colleagues [Thromb Haemost 1998] identified a single nucleotide polymorphisms (SNP) at position 505A/G in the coding region of the TAFI gene that resulted in a substitution of alanine for threonine at residue 147. Brouwers et al. [Blood 2001] identified another SNP, 1040C/T, in the coding region of the TAFI gene by comparing published sequences (GenBank no. NM_001872 and NM_016413). This SNP also resulted in an alanine substitution (Thr32511e) at residue 325.

It was previously shown that commercially available antigen assays underestimate the TAFI-Ile325 concentration (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively) [Gils A et al. Arterioscler Thromb Vase Biol 2003]. On the other hand, studies using recombinant proteins have demonstrated a functional effect of the Thr325lle substitution on the stability of activated TAFI resulting in altered antifibrinolytic activity [Brouwers GI et al. Blood 2001].

Quantifying and detecting TAFI, TAFIa, and TAFIai can be accomplished via direct (ELISA or functional assay) or indirect measurement. The latter includes, among others, animal models and the Chandler Loop (Figure 1) [Vercauteren E et al. J Thromb Haemost 2013; Mutch NI et al. J Thromb Haemost 2003].

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Figure 1.

The Chandler Loop System

Reproduced with permission from A Gils, PhD.

According to Prof. Gils, many pharmaceutical companies are attempting to inhibit TAFIa using TAFI-LMW, including 2-mercaptocyclopentanecarboxylic acid WO 10/0330064 and aryl guanidinic TAFIa inhibitors WO 03/080631.

Conclusions from mouse monoclonal antibodies in the pharmacologic inhibition of TAFI-Ab show that activation of TAFI as well as TAFIa can be targeted. T/TM as well as plasmin-mediated TAFI activation seems important. In vivo studies are scarce due to lack of cross-reactivity with mouse or rat TAFI.

Pharmacologic inhibition of TAFI-Nb has shown that TAFI and the activation of TAFI and TAFIa can be targeted. Further, due to their size, VHH has better clot penetration. The short half-life and lack of cross-reactivity with mouse and rat TAFI hampers studies in mouse models. TAFI also plays a role in inflammation, with TAFIa inactivating C3a, C5a, thrombin cleaved osteopontin, and bradykinin.

Prof. Gils explained that in time the toolbox of TAFI(a) inhibitors allows to investigate the role of TAFI inhibition using animal models whilst the availability of assays to measure the extent of TAFI activation allows to explore the role of TAFI (activation) in different pathologies.

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