To cite this article: Rijken DC, Lijnen HR. New insights into the molecular mechanisms of the fibrinolytic system. J Thromb Haemost 2009; 7: 4-13.See also Lijnen R. Retirement of Dé siré Collen. This issue, pp 2-3; Van de Werf FJ, Topol EJ, Sobel BE. The impact of fibrinolytic therapy for ST-segment-elevation acute myocardial infarction. This issue, pp 14-20; Loges S, Roncal C, Carmeliet P. Development of targeted angiogenic medicine. This issue, pp 21-33.Summary. Fibrinolysis is regulated by specific molecular interactions between its main components. Activation of plasminogen by tissue-type plasminogen activator (t-PA) is enhanced in the presence of fibrin or at the endothelial cell surface. Urokinase-type plasminogen activator (u-PA) binds to a specific cellular u-PA receptor (u-PAR), resulting in enhanced activation of cell-bound plasminogen. Inhibition of fibrinolysis occurs at the level of plasminogen activation or at the level of plasmin. Assembly of fibrinolytic components at the surface of fibrin results in fibrin degradation. Assembly at the surface of cells provides a mechanism for generation of localized cellassociated proteolytic activity. This review includes novel proteins such a thrombin-activatable fibrinolysis inhibitor (TAFI) and discusses new insights into molecular mechanisms obtained from the rapidly growing knowledge of crystal structures of proteins.

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 g/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 g/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 Ca 2؉ 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 Ca 2؉ 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 plasminogenbinding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

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The plasma activity level of the recently discovered fast-acting inhibitor of tissue-type plasminogen activator (t-PA) was found to be temporarily increased after surgery, myocardial infarction and severe trauma. Detailed analysis of the postoperative period revealed simultaneously increased t-PA antigen and inhibition and decreased t-PA activity only on the first postoperative day. These changes were more rapid than those in fibrinogen and C-reactive protein. It is concluded that t-PA inhibition shows the most rapidly changing pattern observed so far in response to trauma. The postoperative fibrinolytic shutdown in blood fibrinolytic activity can be ascribed to a primary increase in t-PA inhibitor levels.

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Binding of components of the fibrinolytic system to fibrin is important for the regulation of fibrinolysis. In this study, decomposition of the fibrin network and binding of plasminogen and plasminogen activators (PAs) to fibrin during lysis of a plasma clot were investigated with confocal microscopy using fluorescein-labeled preparations of fibrinogen, plasminogen, tissue-type PA (t-PA), and two-chain urokinase-type PA (tcu-PA). Lysis induced by PAs present throughout the plasma clot was accompanied by a gradual loss of fibrin content of fibers and by accumulation of plasminogen onto the fibers. Two sequential phases could be distinguished: a phase of prelysis, during which the fibrin network remained immobile, and a phase of final lysis, during which fibers moved with a tendency to shrink and eventually disappeared. The two phases occurred simultaneously but in different locations when lysis was induced by PAs present in the plasma surrounding the clot. The zone of final lysis was located within a 5-8 microns superficial layer, where fibers were mobile, a surface-associated fibrin agglomerates appeared. Plasminogen accumulated in these agglomerates up to 30-fold as compared with its concentration in the outer plasma. t-PA was also highly concentrated in the agglomerates, and tcu-PA bound to them slightly. The zone of prelysis, where plasminogen was moderately accumulated on the immobile fibers, was located deeper in the clot. This zone was much thinner in the case of t-PA-induced lysis than in the case of tcu-PA-induced lysis, reflecting the difference in penetration of the two PAs into the clot. We conclude that under conditions of diffusional transport of fibrinolytic enzymes from outside a plasma clot, extensive lysis is spatially restricted to a zone not exceeding 5-8 microns from the clot surface. In this zone the structure of the fibrin network undergoes significant changes, and strikingly high accumulation of fibrinolytic components takes place.

We conclude that the generation of potent surface-associated plasminogen-binding sites during thrombolysis results in a strikingly high plasminogen concentration at the dynamically changing surface of a lysing clot. The necessity of a continuous plasminogen supply from the plasma supports the use of fibrin-specific and plasminogen-sparing agents for thrombolytic therapy.

Summary. Background and objective: Several studies have suggested that thrombin-activatable fibrinolysis inhibitor (TAFI) levels are associated with the risk of arterial thrombosis, but results have been contradictory. We studied functional TAFI levels and TAFI gene polymorphisms in 124 patients with a recent ischemic stroke and 125 age-and sex-matched controls to establish the role of TAFI in ischemic stroke. Methods and results: Functional TAFI levels, defined as TAFI-related retardation (RT), the difference in clot lysis time (LT) in the absence or presence of a specific activated TAFI inhibitor (potato carboxypeptidase inhibitor [PCI]), were higher in patients than controls (19.5 ± 4.2 vs. 17.7 ± 3.7 min, P < 0.005). Clot LTs in the presence of PCI, which were independent of TAFI, were also increased in ischemic stroke patients. This indicates that in these patients fibrinolysis is impaired not only by high TAFI levels, but also by other mechanisms. Individuals with functional TAFI levels in the highest quartile had an increased risk of ischemic stroke compared with the lowest quartile [odds ratio (OR) 4.0, 95% confidence interval (CI): 1.6-9.8]. In an unselected group of 36 of the 125 stroke patients functional TAFI levels were also measured at 3 months, and were persistently high. This indicates that increased functional TAFI levels after stroke are not caused by an acute phase reaction. No difference was found between patients and controls with respect to TAFI genotype distribution. Conclusions: Increased functional TAFI levels, resulting in decreased fibrinolysis, are associated with an increased risk of ischemic stroke.