Abstract
Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated to TAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37 degrees C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30-50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant.
Highlights
Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a zymogen found in human plasma (1), which is known as plasma procarboxypeptidase B (2) and procarboxypeptidase U (3)
Less efficient plasminogen activation on the fibrin clot corresponds to prolongation of fibrinolysis, and in this way TAFIa can serve as a potent antifibrinolytic enzyme
Identification of a Novel Amino Acid Sequence Variant of TAFI—DNA sequence analysis of reverse transcriptase-PCR products obtained from a number of different human liver cDNA libraries revealed a single nucleotide difference from the cDNA sequence published by Eaton et al (21). This was a C to T mutation at position 1057 of the TAFI cDNA (numbering is as per Boffa et al (24)) and would result in the conversion of a Thr codon (ACU) to an Ile codon (AUU) at amino acid position 325
Summary
Materials—The synthetic carboxypeptidase substrate anisylazoformyllysine (AAFK) (16) was a generous gift from Dr William L. To determine the kinetics of TAFIa hydrolysis of AAFK, TAFIa was formed as described, diluted, and placed on ice. TAFIa (25 nM) was incubated with AAFK at various concentrations (0 – 4000 M) at 24 °C in a microtiter plate that had been presoaked in HBS with 1% Tween 80 (HBS/Tween 1%) and thoroughly rinsed with deionized distilled water. Clot Lysis Assays—All clot lysis assays were performed as described previously (8) in a final volume of 120 l in a plastic microtiter plate presoaked with HBS/Tween 1% and thoroughly rinsed with deionized distilled water. Fluorescence intensities were monitored for 10 min at 1-min intervals to obtain a starting value, after which clotting and plasminogen activation were initiated by the addition of 10 l of 100 nM thrombin and 3 nM t-PA, respectively. The concentration of free lysine in clot supernatants was determined by comparing decreases in NADH fluorescence after 4 h with those obtained in experiments using known concentrations of L-lysine (Sigma)
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