Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.
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.
Schneider
Mark
M
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Boffa
Michael
M
Stewart
Ronald
R
Rahman
Mona
M
Koschinsky
Marlys
M
Nesheim
Michael
M
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism; D001777:Blood Coagulation / Q000502:physiology; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism*; D015723:Gene Library; D014644:Genetic Variation; D006490:Hemostatics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives; D010949:Plasma / Q000737:chemistry; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D001391:Azo Compounds; D005343:Fibrinolytic Agents; D006490:Hemostatics; D011994:Recombinant Proteins; D005340:Fibrinogen; D010958:Plasminogen; D025901:Carboxypeptidase B2; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D008239:Lysine