Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage.
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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 temperature-dependent 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). (+info)
Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.
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We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa. (+info)
Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis.
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Thrombin activatable fibrinolysis inhibitor (TAFI, or procarboxypeptidase B) is the precursor of a recently described carboxypeptidase that potently attenuates fibrinolysis. Therefore, we hypothesized that elevated plasma TAFI levels induce a hypofibrinolytic state associated with an increased risk for venous thrombosis. To evaluate this hypothesis, we developed an electroimmunoassay for TAFI antigen and used this assay to measure TAFI levels in the Leiden Thrombophilia Study, a case-control study of venous thrombosis in 474 patients with a first deep vein thrombosis and 474 age- and sex-matched control subjects. In 474 healthy control subjects, an increase of TAFI with age was observed in women but not in men. Oral contraceptive use also increased the TAFI concentration. TAFI levels above the 90th percentile of the controls (> 122 U/dL) increased the risk for thrombosis nearly 2-fold compared with TAFI levels below the 90th percentile (odds ratio, 1.7; 95% confidence interval, 1.1-2.5). Adjustment for various possible confounders did not materially affect this estimate. These results indicate that elevated TAFI levels form a mild risk factor for venous thrombosis. Such levels were found in 9% of healthy controls and in 14% of patients with a first deep vein thrombosis. Elevated TAFI levels did not enhance the thrombotic risk associated with factor V Leiden but may interact with high factor VIII levels. (Blood. 2000;95:2855-2859) (+info)
Aging, physical conditioning, and exercise-induced changes in hemostatic factors and reaction products.
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The influence of age on training-induced changes in resting and stimulated hemostatic potential was studied in three age categories (Cat I-III; 20-30 yr, 35-45 yr, and 50-60 yr, respectively) of sedentary men before and after 12 wk of training. Coagulation, fibrinolytic activity, and activation markers (reflecting fibrin formation and degradation) were determined. Physical conditioning resulted in a more pronounced increase in von Willebrand factor (vWF) and factor VIII clotting activity (FVIII:c) in Cat I and II and a more pronounced shortening of the activated partial thromboplastin time in all categories at maximal exertion and during recovery. Enhanced increases in tissue-type plasminogen activator (t-PA) antigen and activity and single-chain (sc) urokinase-type plasminogen activator (u-PA) at maximal exercise and 5 min of recovery were observed in all age groups after training. The effects on FVIII:c, vWF, and scu-PA were most pronounced in the youngest age group (Cat I). Increases in the marker of thrombin generation were highest in Cat III; no effect was seen on thrombin-antithrombin complex, plasmin-antiplasmin complex, and D-dimer in any of the age groups. We concluded that training enhances both coagulation and fibrinolytic potential during strenuous exercise. The effect on FVIII/vWF and t-PA/u-PA is most pronounced in younger individuals, whereas thrombin formation is most pronounced in older individuals. (+info)
Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation.
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Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombin-activable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca(2+)-dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mm Ca(2+). Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other. (+info)
Novel interactions between urokinase and its receptor.
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Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction. (+info)
Potentiation of endogenous fibrinolysis and rescue from lung ischemia/reperfusion injury in interleukin (IL)-10-reconstituted IL-10 null mice.
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Little is known about interactions between endogenous anti-inflammatory paradigms and microvascular thrombosis in lung ischemia/reperfusion (I/R) injury. Interleukin (IL)-10 suppresses macrophage activation and down-regulates proinflammatory cytokine production, but there are no available data to suggest a link between IL-10, thrombosis, and fibrinolysis in the setting of I/R. We hypothesized that hypoxia/ischemia triggers IL-10 production, to dampen proinflammatory cytokine and adhesion receptor cascades and to restore vascular patency by fibrinolytic potentiation. Studies were performed in a mouse lung I/R model. IL-10 mRNA levels in lung were increased 43-fold over base line by 1 h of ischemia/2 h of reperfusion, with a corresponding increase in plasma IL-10. Expression was prominently localized in bronchial epithelial cells and mononuclear phagocytes. To study the link between IL-10 and fibrinolysis in vivo, the induction of plasminogen activator inhibitor-1 (PAI-1) was evaluated. Northern analysis demonstrated exaggerated pulmonary PAI-1 expression in IL-10 (-/-) mice after I/R, with a corresponding increase in plasma PAI/tissue-type plasminogen activator activity. In vivo, IL-10 (-/-) mice showed poor postischemic lung function and survival after I/R compared with IL-10 (+/+) mice. Despite a decrease in infiltration of mononuclear phagocytes in I/R lungs of IL-10 (-/-) mice, an increased intravascular pulmonary fibrin deposition was observed by immunohistochemistry and Western blotting, along with increased IL-1 expression. Recombinant IL-10 given to IL-10 (-/-) mice normalized the PAI/tissue-type plasminogen activator ratio, reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury. Since recombinant hirudin (direct thrombin inhibitor) also sufficed to rescue IL-10 (-/-) mice, these data suggest a preeminent role for microvascular thrombosis in I/R lung injury. Ischemia-driven IL-10 expression confers postischemic pulmonary protection by augmenting endogenous fibrinolytic mechanisms. (+info)
Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy.
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Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease. (+info)