Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. 778 9

TAFI (thrombin-activatable fibrinolysis inhibitor) is a recently discovered plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis. This work shows that the thrombin-thrombomodulin complex, rather than free thrombin, is the most likely physiologic activator. Thrombomodulin increases the catalytic efficiency of the reaction by a factor of 1250, an effect expressed almost exclusively through an increase in kcat. The kinetics of the reaction conform to a model whereby thrombin can interact with either TAFI (Km = 1.0 microM) or thrombomodulin (Kd = 8.6 nM), and either binary complex so formed can then interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex from which activated TAFI is produced with kcat = 1.2 s-1. This work also shows that activated TAFI down-regulates tPA-induced fibrinolysis half-maximally at a concentration of 1.0 nM in a system of purified components. This concentration of TAFI is about 2% of the level of the zymogen in plasma, which indicates that ample activated TAFI could be generated to very significantly modulate fibrinolysis in vivo. Therefore, TAFI in vitro and possibly in vivo defines an explicit molecular connection between the coagulation and fibrinolytic cascades, such that expression of activity in the former down-regulates the activity of the latter.
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PMID:TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. 866 47

Thrombin-activatable fibrinolysis inhibitor (TAFI) is the precursor of an exopeptidase that is identical to plasma procarboxypeptidase B. Upon activation by thrombin, activated TAFI (TAFIa) attenuates fibrinolysis, presumably by catalyzing the removal of C-terminal lysines from partially degraded fibrin. Activated protein C (APC) proteolytically inactivates the essential cofactor in prothrombinase, factor Va, and limits both the formation of thrombin and subsequent activation of TAFI, thereby appearing profibrinolytic. TAFI is able to reconstitute an APC-dependent shortening of lysis time in a purified system; however, it remained to be determined the extent to which TAFI is involved in the profibrinolytic effect of APC in a plasma-based system. To aid in addressing this question, two monoclonal antibodies (MoAbTAFI#16 and #13) and a polyclonal antibody were produced against purified TAFI. MoAbTAFI#16 was shown to inhibit TAFI activation and thereby appears to stimulate fibrinolysis. Furthermore, an enzyme-linked immunosorbent assay was developed using MoAbTAFI#13 and the polyclonal antibody. Through its use, the plasma concentration of TAFI was determined to be 73 nmol/L. In addition, a turbidity assay was used to determine the effect of APC on tissue plasminogen activator-induced fibrinolysis of clots produced from normal human plasma (NHP), plasma immunodepleted of TAFI (TdP), and TdP reconstituted with purified TAFI. APC shortened lysis time of clots produced from NHP in a saturable and concentration-dependent manner. However, APC had no effect on lysis time of clots formed from either TdP or NHP in the presence of 80 nmol/L MoAbTAFI#16. The APC effect could be reconstituted in TdP by the addition of purified TAFI. The lysis time in TdP was increased from 50 to 180 minutes in a TAFI concentration-dependent manner. The EC50 was 15 nmol/L and saturation was approached at physiologically relevant concentrations (60 nmol/L). The profibrinolytic effect of APC was also compared with that of MoAbTAFI#16 and two competitive inhibitors, an inhibitor of the carboxypeptidase A and B family purified from potato tubers and 2-Guanidinoethylmercaptosuccinic acid (GEMSA). All were able to reduce lysis time of clots formed from normal human plasma by 90 minutes, yielding respective EC50 values of 5 nmol/L, 15 nmol/L, 50 nmol/L, and 90 mumol/L. Therefore, the majority of the profibrinolytic effect of APC, in an in vitro plasma system, is dependent on TAFI. Because TAFIa dramatically influences lysis time, inhibitors of TAFIa or TAFI activation may prove to be important adjuvants for thrombolytic therapy.
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PMID:The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. 882 28

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.
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PMID:Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. 891 45

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.
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PMID:On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B. 916 90

To determine to what extent the Arg506 to Gln mutation in the factor V gene influences the fibrinolytic response after 20 min venous occlusion (VO) we investigated a population of APC resistant children (n = 60) and a group of age-matched healthy controls (n = 25). After 20 min VO, symptomatic (n = 30) carriers of the common factor V mutation showed significantly reduced t-PA activities compared with asymptomatic (n = 30) carriers (p <0.0001) and healthy controls (p <0.0001). In contrast, PAI 1 activity was significantly (p <0.0001) higher before and after VO in children with the factor V mutation compared with healthy children. No difference was found between symptomatic and asymptomatic probands. A significantly lower PAI 1 antigen decrease along with a lower t-PA antigen release was found in the APC resistant children compared with the controls. No significant difference was seen between individuals with and without previous vascular insults. As the lack of t-PA activity after VO in symptomatic carriers is the most conspicuous result, we suggest that the factor V gene mutation itself might induce the fibrinolytic impairment by increasing the thrombin levels and thus increasing the recently described thrombin-activable fibrinolysis inhibitor (TAFI).
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PMID:Arg506 to Gln mutation in the factor V gene causes poor fibrinolytic response in children after venous occlusion. 930 63

Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with kcat = 0.55 s-1, K(m) = 0.54 microM, and Kd = 6.0 nM for rTAFI and kcat = 0.61 s-1, K(m) = 0.55 microM, and Kd = 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37 degrees C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to approximately 62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (delta Ho++ approximately equal to 45 kcal/mol and delta So++ approximately equal to 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and epsilon-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.
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PMID:Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. 944 53

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.
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PMID:Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor. 944 87

Thrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.
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PMID:Thrombomodulin in human plasma contributes to inhibit fibrinolysis through acceleration of thrombin-dependent activation of plasma procarboxypeptidase B. 949 93

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.
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PMID:A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. 976 37


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