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Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen.Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg 92 in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperaturedependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg 330 , but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg 302 . Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg 302 .Thrombin-activable fibrinolysis inhibitor (TAFI) 1 (1), also known as plasma procarboxypeptidase B (2) or procarboxypeptidase U (3, 4), provides an important link between coagulation and fibrinolysis (5, 6). TAFI is released into the circulation by the liver (2, 7) as a proenzyme of a carboxypeptidase B and can be activated (TAFIa) by thrombin, a process in which thrombomodulin acts as a cofactor (8). TAFIa down-regulates fibrinolysis presumably by removing C-terminal lysines from fibrin that is already partially degraded by plasmin. Those lysines act as ligands for the lysine-binding sites of plasminogen and tissue-type plasminogen activator (9). Removal of the lysines attenuates the fibrin cofactor function of tissue-type plasminogen activator mediated plasminogen activation, resulting in prevention of accelerated plasmin formation and subsequent down-regulation of fibrinolysis (10).Although the mechanism of TAFI regulation has been studied extensively, its mechanism of inactivation is still poorly understood. TAFI shows ϳ40% sequence homology to tissue procarboxypeptidases, that are activated as a result of a singlesite cleavage by trypsin-like enzymes that release the activation peptides (11-15). Upon incubation of TAFI with trypsin, TAFIa activity first increased and then decreased (2), in contrast to tissue carboxypeptidases which are not inactivated by trypsin. The trypsin-cleavage sites in TAFI were identified by SDS-PAGE and sequencing (2). TAFI was not only cleaved at Arg 92 but also at Arg 330 . The latter cleavage yielded a 25-kDa inactive fragment and a C-terminal polypeptide of 71 amino acids. Similar cleavage patterns were found upon incubation of TAFI with either plasmin or thrombin (2).When 125 I-TAFI was incubated with thrombin, the generation of a 35-kDa fragment became apparent. During the inactivation, this fragment was further proteolyzed into 25-and 14-kDa fragments (1). The N-terminal re...

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that after activation downregulates fibrinolysis. Platelets are known to contain antifibrinolytic factors that are secreted during platelet activation. Therefore, the presence of TAFI in platelets was analyzed. TAFI was identified in platelets in a concentration of about 50 ng/1 ؋ 10 9 platelets and was secreted on platelet activation. Thrombin-mediated activation of platelet-derived TAFI resembled that of plasma-derived TAFI with respect to stimulation by thrombomodulin and spontaneous loss of activity at 37°C. The different glycosylation of platelet-derived TAFI compared with plasma-derived TAFI suggests that platelet-derived TAFI is synthesized in the megakaryocyte. This suggestion was substantiated by the detection of mRNA in the megakaryocytic cell lines DAMI and CHRF, representing the intermediate and late stages of megakaryocyte development. These results establish the presence of TAFI in platelets and suggest a role for platelet-derived TAFI in the protection of the clot against fibrinolysis. IntroductionThrombin activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase U, EC 3.4.17.20) provides a balance between coagulation and fibrinolysis. 1 Because the concentration of TAFI in plasma (70-250 nM) is 10-to 30-fold below its Michaelis constant (K M ) for activation by thrombin, the amount of TAFIa formed during activation is dependent on the TAFI concentration. 2,3 TAFI levels vary considerably between individuals, and individual differences in clot lysis times could be attributed to variations in TAFI levels. 3 Increased TAFI levels are associated with thromboembolic disease, and polymorphic variations in the TAFI gene have been linked to plasma TAFI levels. [4][5][6][7][8] These observations indicate an important role for the concentration of TAFI in plasma on the regulation of fibrinolysis and the occurrence of thromboembolic disease.Because platelets are known to increase local concentrations of coagulation and fibrinolytic factors by releasing the contents of their ␣-granules on activation, this study was initiated to evaluate if TAFI is present in platelets and could contribute to the variations in plasma TAFI levels. Study design Immunofluorescence microscopyCytospins of gel-filtered platelets, fixed with 2% para-formaldehyde, were permeabilized with 1% saponine and subsequently incubated with monoclonal antibody (Moab) 9H10 against TAFI and a fluorescein isothiocyanate (FITC)-labeled secondary goat antimouse antibody. 3 Detection of TAFI in platelets by ELISAGel-filtered platelets were prepared as described. 9 Secretion of TAFI from gel-filtered platelets was determined by enzyme-linked immunosorbent assay (ELISA). 3 Determination of TAFIa activity in plateletsGel-filtered platelets were incubated for 60 minutes with thrombin (50 nM), thrombomodulin (10 nM), CaCl 2 (5 mM), and hippuryl-Arg (5 mM) after which the amount of hippuric acid was determined in the supernatant as ...