| Thrombin Activatable Fibrinolysis Inhibitor (TAFI, Plasma pro-carboxypeptidase B, carboxypeptidase U) is a single chain glycoprotein zymogen (Mr=60,000) synthesized in the liver and circulating at a plasma concentration of 50nM (1-4). Thrombin (plasmin, trypsin) cleavage of the zymogen releases a 92 amino acid N-terminal activation peptide containing 4 N-linked glycosylation sites (N22, N51, N63, N86) and the proposed plasminogen recognition site. The rate of thrombin catalyzed activation of TAFI is increased 1250 fold by formation of a ternary complex with thrombomodulin (5). The 309 amino acid C-terminal (Mr=35,783) catalytic domain (TAFIa, pCPB) displays the properties of a basic carboxypeptidase, hydrolyzing lysine and arginine from the C-terminal position of polypeptides. This portion of the molecule is homologous to tissue carboxypeptidase B and contains 7 conserved cystine residues (64,77,136,151,160,165,291), the active site Zn2+ coordination site (H67, E69, H196) and the basic C-terminal amino acid substrate binding pocket (D257, G244, S207). TAFI is proposed to play a key role in the interaction between procoagulant, anticoagulant and fibrinolytic systems (5-9). Effective fibrinolysis results from the formation of a ternary complex between tPA, plasminogen and C-terminal lysine residues on fibrin. Plasminogen bound to fibrin is more effectively converted to plasmin, thereby localizing the lytic activity to the area of the clot. Plasmin degradation of fibrin generates additional C-terminal lysine residues thereby amplifying the system locally. The ability of TAFI to bind specifically to plasminogen and to cleave C-terminal lysines on fibrin (and cell surfaces) results in down-regulation of fibrinolysis by reducing the number of plasminogen and tPA binding sites on fibrin. The activation of TAFI by the thrombin/thrombomodulin complex couples both the phenomenon of coagulation induced inhibition of fibrinolysis and the profibrinolytic effect of activated protein C. Applications: Suitable for use in ELISA and Immunoelectrophoresis. Other applications not tested. Recommended Dilutions: Optimal dilutions to be determined by the researcher. Rz Ratio (Reiheitszahl, A403/A280): 0.38 Storage and Stability: Store product at 4C if to be used immediately within two weeks. For long-term storage, aliquot to avoid repeated freezing and thawing and store at -20C. Aliquots are stable at -20C for 12 months after receipt. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Note: Sodium azide is a potent inhibitor of peroxidase and should not be added to HRP conjugates. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
