Bajzar L, Manuel R, Nesheim M E
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1995 Jun 16;270(24):14477-84. doi: 10.1074/jbc.270.24.14477.
Previous studies demonstrated that tissue plasminogen activator-induced fibrinolysis in vitro is retarded in the presence of prothrombin (II) activation and that the anticoagulant-activated protein C appears profibrinolytic by preventing the formation of thrombin (IIa)-like activity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assay that is sensitive to the antifibrinolytic effect of II activation was developed and was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified components, is retarded when this protein is present and when II activation occurs in situ, the protein was named TAFI (thrombin-activatable fibrinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa product correlates with both prolongation of lysis time and CPB-like activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen but does not prolong the lysis of clots formed in the presence of Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive inhibitor of CPB, completely inhibits prolongation of lysis by activated TAFI in a purified system and the prolongation induced by II activation in barium-adsorbed plasma. This suggests that TAFI accounts for the antifibrinolytic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.
以往的研究表明，在凝血酶原（II）激活的情况下，组织型纤溶酶原激活剂诱导的体外纤维蛋白溶解会受到抑制，并且抗凝激活蛋白C通过在纤维蛋白溶解过程中阻止凝血酶（IIa）样活性的形成而表现出促纤维蛋白溶解作用。为了揭示IIa的产生与纤维蛋白溶解抑制之间的分子联系，开发了一种对II激活的抗纤维蛋白溶解作用敏感的溶解试验，并用于从人血浆中纯化一种60 kDa的单链蛋白。由于当这种蛋白存在且II激活在原位发生时，由纯化成分产生的凝块的溶解会受到抑制，因此该蛋白被命名为TAFI（凝血酶激活的纤维蛋白溶解抑制剂）。TAFI被IIa切割产生35 kDa、25 kDa和14 kDa的产物。氨基末端序列分析确定TAFI是血浆羧肽酶B（CPB）的前体。35 kDa产物的形成与溶解时间的延长和CPB样活性相关。溶解时间的延长在约125 nM TAFI时达到饱和。活化的TAFI抑制Glu-纤溶酶原的激活，但不会延长在Lys-纤溶酶原存在下形成的凝块的溶解时间。2-胍基乙硫基琥珀酸，一种CPB的竞争性抑制剂，在纯化系统中完全抑制活化的TAFI引起的溶解延长以及钡吸附血浆中II激活诱导的溶解延长。这表明TAFI解释了凝血酶原激活伴随的抗纤维蛋白溶解作用，并且活化蛋白C通过减弱TAFI激活而表现出促纤维蛋白溶解作用。
