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Keywords:

  • scuPA;
  • TAFI;
  • thrombi;
  • uPA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  The carboxypeptidase, TAFIa or CPU, is known to prolong plasma clot lysis by tissue plasminogen activator (tPA) and to have a role in thrombus stability in vivo. This current study examined lysis by urokinase (uPA) and single chain urokinase (scuPA) in addition to tPA. Further, we investigated the role of TAFIa in a model thrombus system, in which thrombi are formed under conditions of flow. We show that human thrombi, formed in vivo, and model thrombi both contain TAFI. No effect of thrombus TAFIa was observed in thrombus lysis assays, except when thrombi were bathed in plasma, in which case addition of potato tuber carboxypeptidase inhibitor (CPI) resulted in doubling of the rate of lysis. TAFIa inhibited lysis of model thrombi and plasma clots by uPA, scuPA in addition to lysis by tPA. The effect of TAFIa was more evident at high concentrations of plasminogen activator such as those used in thrombolytic therapy. Addition of plasminogen increased lysis and, in its presence, the enhancement by CPI was smaller. Thus the action of TAFIa could be partially overcome by plasminogen, whether lysis was by tPA, uPA or scuPA. This is consistent with TAFIa exerting its effect primarily through modifying the binding of plasminogen to fibrin and to a lesser extent through modification of the binding of tPA to fibrin.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fibrin formation and degradation is regulated by enzymes of the coagulation and fibrinolytic systems. Thrombin activatable fibrinolysis inhibitor (TAFI; EC 3.4.20.17) [1], also referred to as plasma procarboxypeptidase B (CPB) [2], procarboxypeptidase U (CPU) [3,4], or procarboxypeptidase R [5], provides a link between these processes. TAFI circulates in plasma as a zymogen, which is activated to yield an enzyme, TAFIa, with carboxypeptidase B-like activity [6].

TAFI can be activated by trypsin-like enzymes, including thrombin and plasmin [2,7]. High concentrations of thrombin are required to activate TAFI, but thrombomodulin (TM) enhances this activation 1250-fold [8], suggesting that thrombin–TM is the main physiological activator. Thrombin–TM can also activate protein C (PC) to form activated protein C, which down-regulates thrombin generation via factor V [9]. This implicates TM in the down-regulation of both fibrinolysis and coagulation. Potentiation of PC activation by TM requires epidermal growth factor (EGF)-like domains 4–6, whereas activation of TAFI also requires domain 3 [10]. Some reports have suggested that varying the concentration of TM stimulates the activation of PC and TAFI differentially, with preferential activation of TAFI at low TM concentrations (<5 nm) and activation of PC at high TM concentrations (>10 nm) [11,12].

A second carboxypeptidase, carboxypeptidase N (CPN), is found in plasma. CPN is a constitutively active enzyme, with specificity for carboxy-terminal arginine and lysine residues [3,4] but no effect on fibrinolysis [13]. TAFIa activity can be distinguished from CPN by the carboxypeptidase inhibitor (CPI) from potato tuber; TAFIa is completely inhibited while CPN is relatively unaffected [13]. No known physiological inhibitor of TAFIa has been found and regulation of this enzyme in vivo is thought to involve spontaneous degradation, as a result of an induced conformational change [14,15]. TAFIa inhibits fibrinolysis by removing carboxy-terminal lysine residues from fibrin [1]. Tissue plasminogen activator (tPA) and plasminogen bind with high affinity to lysine residues exposed on fibrin during plasmin degradation, potentiating plasmin generation at the thrombus surface [16,17]. Removal of these residues by TAFIa results in reduced plasminogen activation by tPA and a several-fold prolongation of clot lysis times [4,6,13,18].

Several models have been used to implicate TAFIa in the downregulation of fibrinolysis. Addition of TAFI to plasma or activation of plasma TAFI retards in vitro plasma clot lysis [1,6,7,13,18,19]. Inclusion of a TAFIa inhibitor during tPA thrombolysis enhances fibrinolysis 2–3-fold and results in a shorter time to patency [20,21]. This implicates TAFIa in the attenuation of fibrinolysis in vivo. Our study uses whole blood model thrombi [22] to shed light on the in vivo investigations. We used this system to assess the contribution of TAFIa to thrombus lysis by different plasminogen activators. We found that, despite the presence of TAFI antigen in thrombi, surrounding plasma was required to see an effect of CPI on thrombus lysis, suggesting that TAFIa influences fibrinolysis at the thrombus–plasma interface. This study also showed that thrombus lysis by both urokinase (uPA) and single chain urokinase (scuPA) is susceptible to modulation by TAFIa and that additional plasminogen partially overcame the effect of TAFIa. Thus our data point to an important role of TAFIa in controlling thrombus lysis by different activators, acting primarily through the modulation of binding of plasminogen, rather than of tPA, to partially degraded fibrin.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Blood collection and preparation of plasma

Blood samples were collected from normal healthy volunteers with their consent into 0.1 vol of 0.13 m trisodium citrate. Plasma that was essentially free of platelets was prepared by centrifugation at 1860 g for 30 min at 4 °C [23]. Normal pool plasma, used in clot lysis experiments, was from at least 20 normal donors.

Evaluation of CPI for anti-plasmin activity

Some commercially available CPI is reported to contain anti-plasmin activity [24,25], and we assessed our source of CPI (lot B38072; Calbiochem, Beeston, Notts, UK) for such activity. Purified fibrin clots [23] were prepared in duplicate and their lysis by 0.15–0.6 U mL−1 plasmin (Kabi Chromogenix, Quadratech, Epson, Survey, UK) ± CPI (6.25–400 µg mL−1) was measured. Lysis was found to be unaffected by CPI at any concentration (Fig. 1a); for clarity only the routinely used 25 µg mL−1 concentration is shown. For the chromogenic assay (S-2390) plasmin ± CPI (5–100 µg mL−1) was diluted in 50 mm Tris, 0.01% Tween 80 pH 8.3 to final concentrations of 0.025 and 0.05 U mL−1. The chromogenic substrate S-2390 was added at a final concentration of 1.5 mm, and the change in absorbance at 405 nm monitored over time. Consistent with the clot lysis data, plasmin activity on S-2390 was unaffected by CPI (Fig. 1b). Similarly mass spectrometry of CPI did not reveal any contamination (not shown).

image

Figure 1. Carboxypeptidase inhibitor (CPI) contains no anti-plasmin activity. (a) Fibrin clot lysis by plasmin at 0.15 U mL−1 (triangles), 0.3 U mL−1 (squares) and 0.6 U mL−1 (circles), in the absence (open symbols) and the presence (closed symbols) of CPI (25 µg mL−1). (b) Analysis of plasmin activity on S-2390 at 0.025 U mL−1 (squares), 0.05 U mL−1 (circles) plasmin, in the absence (open symbols) and presence (closed symbols) of CPI (25 µg mL−1). n = 4.

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Clot lysis assay

Normal pooled plasma in the presence and absence of CPI and of the plasminogen activators tPA (Genentech, San Francisco, CA, USA), uPA (NIBSC) or scuPA (Abbott, Maidenhead, Berks, UK), or plasmin (NIBSC, Potter's Bar, UK) was prepared in a final volume of 200 µL 10 mm Tris pH 7.5, 0.01% Tween 20 (Tris–Tween buffer). Two 80 µL aliquots from each tube were added to microtiter wells, each containing 20 µL CaCl2 to give final concentrations in the wells as follows: 30% plasma, 10.6 mm CaCl2; 180 pm tPA, 1 nm uPA, 8 nm scuPA or 1.25 U mL−1 plasmin; ± 25 µg mL−1 CPI. The plate was incubated at 37 °C and read continuously at 405 nm.

Conversion of scuPA to tcuPA/T ± TAFI

scuPA was incubated with human thrombin (Sigma, Dorset, UK), TM (American Diagnostics, Shield Diagnostics, Dundee, UK) and calcium (1.7 µm; 80 nm; 24 nm; 25 mm, respective final concentrations) in the presence or absence of 1.7 µm TAFI for 10 min at 37 °C, after which 200 µg mL−1 hirudin were added. Fibrin detector plates [26] were used to detect protease activity by loading 1 ng scuPA per well. After 18 h at 37 °C zones of activity appear as dark areas in the opaque plates.

Western blotting

Human thrombi, obtained at autopsy within 24 h of death or removed during surgery, were extracted [27]. These extracts or recombinant TAFI (6.25–50 ng) were separated on 4–12% NuPAGE gels (Invitrogen, Paisley, UK) with MES running buffer, under non-reducing conditions, and transferred to nitrocellulose [27]. TAFI was detected with monoclonal antibody (1 µg mL−1) and rabbit antimouse IgG conjugated to alkaline phosphatase (Dako, Ely, Cambs, UK). Both TAFI and the monoclonal antibody were kindly supplied by M. Nesheim (Biochemistry, Queens, University, Ontario, Canada) [6]. For scuPA analysis, samples were separated on 10% SDS–PAGE, under reducing conditions, transferred to nitrocellulose and immunostained as described [27]. scuPA and its products were detected with goat antibody to uPA (5 µg mL−1; Biopool Stockport, Cheshire, UK) and rabbit antigoat IgG conjugated to alkaline phosphatase (Sigma).

Thrombus lysis

Citrated whole blood (0.9 mL), from normal donors, along with FITC-labeled fibrinogen (75 µg mL−1 final concentration; FITC : fibrinogen about 6 : 1 [28]) ± CPI (25 µg mL−1) was recalcified with 10.9 mm CaCl2 in a volume of 1.15 mL, and placed in the tubing. Model thrombi, formed by rotation at 30 r.p.m. for 90 min, were washed with 0.9% (w/v) NaCl and blotted on filter paper. Thrombi were then bathed in either Tris–Tween buffer, or 30% autologous plasma containing 25 mm calcium; the plasma did not clot. uPA, scuPA and tPA were included in the bathing solution at 18, 18 and 15.4 nm, respectively (all 1 µg mL−1), unless otherwise stated. Thrombi were incubated at 37 °C, and samples of supernatant (5 µL) were removed at time intervals and diluted 1/50 in Tris–Tween buffer. The fluorescence released, indicating fibrin degradation, was measured using a Cytofluor multiwell plate reader (series 4000; PerSeptive Biosystems, CA, USA) with an excitation wavelength of 485 nm and emission wavelength 530 nm. In some experiments Glu-plasminogen, purified from plasma by affinity chromatography on lysine-Sepharose [23], was added to the bathing plasma.

In situ zymography

Plasminogen activator activity was detected as previously reported [29]. Briefly, sections were overlaid with a fibrin-agarose film formed by clotting fibrinogen with bovine thrombin (1.7 NIH U mL−1) for 10 min at 37 °C. Slides were incubated at 37 °C and photographs taken at time intervals.

Immunohistochemistry

Thrombi were stained using the APAAP method [30]. Monoclonal antibodies to TAFI (M. Nesheim), thrombin (EST-7; American Diagnostics) and TM (American Diagnostics) were applied at final concentrations of 20 µg mL−1. Control slides, in which the primary antibody was omitted and replaced with buffer or mouse IgG1 isotype, were included in every series of slides.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

TAFIa modulates plasma clot lysis by uPA and scuPA

No lysis of plasma clots occurred in the presence of 1 nm uPA or 8 nm scuPA (t1/2 > 480 min) unless CPI (25 µg mL−1) was incorporated. This resulted in half lysis times, for both uPA and scuPA, of 42 min for the examples presented in Fig. 2a,b. The effect of CPI on lysis by uPA or scuPA was actually more marked than on that by 180 pm tPA (Fig. 2c). Plasmin, in the presence of CPI, lyzed 30% plasma clots only at very high concentrations (1.25 U mL−1; approximately 1 µm) (Fig. 2d). Lower concentrations of plasmin were ineffective and higher concentrations lyzed the fibrinogen and prevented clot formation.

image

Figure 2. Carboxypeptidase inhibitor (CPI) enhances uPA and scuPA-induced plasma clot lysis. Plasma clots formed by recalcification were lyzed by 1 nm uPA (a), 8 nm scuPA (b), 180 pm tPA (c) or 1.25 U mL−1 (approximately 1 µm) plasmin (d) in the absence (open symbols) and presence of CPI (closed symbols). n = 4.

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TAFI in thrombi

Extracts of thrombi were subjected to SDS–PAGE and analyzed by Western blotting with a monoclonal antibody to TAFI. A 58-kDa band of TAFI was observed in human thrombi (n = 6); two typical thrombi are illustrated in Fig. 3, which also shows a range of purified TAFI concentrations. By densitometry the thrombus extracts were in the linear range of the response and we estimate the concentration at 0.5 µg mL−1 extract, corresponding to 0.5 µg g−1 tissue. No activated TAFI was detected by blotting but it should be noted that the antibody used recognizes this form poorly. Positive staining for TAFI was observed in human thrombi and model thrombi, along fibrin strands and in platelet-rich areas (data not shown).

image

Figure 3. Human thrombi contain TAFI. Extracts of human thrombi and recombinant TAFI (6.25–50 ng) were separated on 4–12% NuPAGE gels under non-reducing conditions and the blot probed for TAFI. n = 3.

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TAFIa regulates thrombus lysis

Model thrombi were bathed in buffer containing uPA, and lysis was recorded as release of fluorescence over time (Fig. 4a). Incorporation of CPI into both thrombi and bathing buffer did not increase lysis. Similarly, in situ lysis of thrombi, in which a frozen thrombus section was overlaid with a fibrin–agarose film, was unaffected by CPI (not shown). Lysis of thrombi in 30% plasma by uPA was slower than in buffer (2.8 vs. 6.0 FU min−1) but addition of CPI doubled the rate of lysis (6.4 FU min−1; Fig. 4b). Similar results were obtained when thrombi were lyzed with scuPA and tPA (data not shown). The effect of CPI was observed more clearly at higher concentrations of tPA, uPA or scuPA (Fig. 5). The increase in lysis rate was 1.5-fold at 7.7 nm tPA and 9 nm uPA/scuPA and 2-fold at 15.4 nm tPA and 18 nm uPA/scuPA. Inclusion of CPI also increased the extent of lysis. The lack of effect at low activator concentration may be explained by the slow lysis at low concentrations masking any effect of TAFIa.

image

Figure 4. The effect of carboxypeptidase inhibitor (CPI) on thrombus lysis in buffer and plasma. Thrombi were bathed in buffer (a) or 30% plasma (b). CPI (25 µg mL−1) was incorporated into thrombi and bathing buffer or plasma, and lysis by uPA monitored in the absence (○) and presence of CPI (●) by fluorescence release over 2.5 h. The graph is representative of six experiments on different donors. Rates are given as fluorescence units (FU) per minute.

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image

Figure 5. The effect of increasing activator concentration on lysis of thrombi ± carboxypeptidase inhibitor (CPI). Thrombi were bathed in 30% plasma with uPA (a), scuPA (b) or tPA (c). Thrombi were lyzed in the absence (open symbols) and presence (closed symbols) of CPI (25 µg mL−1) at 90 pm (circles), 9 nm (triangles) and 18 nm (squares) uPA/scuPA or 77 pm (circles), 7.7 nm (triangles) and 15.4 nm (squares) tPA. Each graph is representative of experiments on at least three donors.

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It is interesting that scuPA was effective in the lysis of both clots and thrombi, despite its known inactivation by thrombin–TM, resulting in formation of tcuPA/T [31]. The activity of scuPA was indeed decreased (but not abolished) on treatment with thrombin–TM (Fig. 6a). TAFI protected against this loss of activity, observed most clearly at equimolar concentrations, as shown. This was confirmed by Western blotting, which showed less formation of tcuPA/T when TAFI was present (Fig. 6b), suggesting competition between TAFI and scuPA as substrates for thrombin–TM.

image

Figure 6. TAFI protects scuPA from thrombin inactivation. TAFI and scuPA were mixed at equimolar concentrations in the presence of thrombin and thrombomodulin (TM) for 10 min at 37 °C prior to analysis. n = 4. (a) Analysis of activity (1 ng load); top, preincubation; bottom, postincubation. (b) Western blot for uPA. Samples (300 ng) were run on 10% gels under reducing conditions and blotted for uPA.

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Our data show that TAFIa regulates fibrinolysis by uPA and scuPA, whereas the effect of TAFIa has been studied largely on tPA-mediated activity, its effect being explained as limiting the formation of ternary complex between fibrin, plasminogen and tPA [6,13,32] Addition of purified plasminogen to 30% plasma, in which thrombi were bathed, enhanced the rate and extent of lysis by uPA and scuPA (Fig. 7a,b). Inclusion of CPI showed the usual doubling of lysis rate except in the presence of added plasminogen, where the effect of CPI on lysis was decreased (Fig. 7c,d; Table 1). Thus plasminogen in excess can partially overcome the effect of TAFIa, consistent with TAFIa exerting its effect primarily on the binding of plasminogen to fibrin.

image

Figure 7. Addition of plasminogen during thrombus lysis. Thrombus lysis, in 30% plasma, by 18 nm uPA (a,c) or 18 nm scuPA (b,d) is shown. The effect of additional human plasminogen: 0 µm (circles), 0.5 µm (triangles), 1 µm (squares) and 2 µm (diamonds) on lysis by uPA (a) and scuPA (b) is shown with inclusion of carboxypeptidase inhibitor (CPI) (25 µg mL−1) (closed symbols) in (c) (uPA) and (d) (scuPA). n = 3.

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Table 1.  Effect of added plasminogen on lysis of model thrombi
Added plasminogen, µmLysis FU min−1Lysis + CPI FU min−1Fold increase
  1. Thrombus lysis, in 30% plasma, was induced using 18 nm uPA or scuPA. Plasminogen was added to the bathing fluid, 30% plasma, at the concentrations shown + carboxypeptidase inhibitor (CPI).

Lysis by uPA
01.42.82
0.53.44.11.2
13.63.91
25.15.91.1
Lysis by scuPA
02.64.71.8
0.54.94.91
16.571.1
27.78.31.1

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The current study addresses the contribution of TAFIa to thrombus lysis by the activators, scuPA and uPA, which do not bind fibrin. It extends the investigation of the effect of TM on fibrinolysis, which made the preliminary observation that uPA-dependent lysis is susceptible to TAFIa modulation [11]. Further, by the use of model thrombi, our study explores the status of TAFIa as a regulator of fibrinolysis in a system more complex than plasma clots, and sheds light on the interpretation of in vivo studies on thrombus formation and stability.

TAFIa studies have centered on the analysis of plasma clot lysis by tPA [1,6–8,12,13,18,19,33], but we show that TAFIa influences lysis of both thrombi and plasma clots by uPA and scuPA at least as well as that by tPA. This raised the question of how TAFIa modulates fibrinolysis by these activators. The usual model of TAFIa action on tPA-dependent lysis is explained by the known ternary complex between fibrin, plasminogen and tPA and the consequent stimulation of plasminogen activation in that setting [6,16,32]. Since uPA is active in the absence of fibrin, we explored whether TAFIa regulation of lysis was influenced by plasminogen concentration. Addition of Glu-plasminogen to our system at high concentrations increased the rate and extent of lysis. Addition of CPI further enhanced lysis but its effect became less obvious at higher plasminogen concentrations. These data agree well with the view that plasminogen can be the limiting factor in fibrinolysis [34] and with the elegant study showing that plasminogen binding to fibrin during lysis is diminished by TM, indicating a role for TAFIa in the prolongation of lysis [19]. We conclude that TAFIa probably affects uPA/scuPA-induced fibrinolysis primarily at the level of plasminogen binding and activation; we saw less effect on lysis by preformed plasmin. It is noteworthy that, while there is much emphasis on the fibrin specificity or selectivity of particular activators, the binding of plasminogen to fibrin is well established in catalyzing fibrinolysis. Our results showing the effect of TAFIa on lysis not only by tPA but also by uPA and scuPA are consistent with this view.

All our observations with uPA were echoed by those with scuPA, except for the characteristic lag phase with scuPA followed by a rapid rate of lysis. These activators differ in relation to dependence on fibrin. uPA does not require fibrin for activity, while the low intrinsic activity of scuPA [35] is greatly enhanced by fibrin fragment E [36]. In addition, the activity of plasmin, formed on fibrin, is protected from α2-antiplasmin (α2-AP) [37], accounting further for the fibrinolytic activity of scuPA. Our finding that lysis by scuPA is strongly affected by TAFIa echoes earlier observations that pancreatic carboxypeptidase B treatment decreased the activity of scuPA dramatically [38], consistent with its stimulation by fibrin fragment E, which has three C-terminal lysines [39].

It is of particular interest that TAFIa affects lysis by scuPA, because scuPA is inactivated by thrombin–TM to form tcuPA/T [31,40]. We found that this product retains some activity, consistent with the known stimulation of its activity on fibrin [41]. TAFI protected against formation of tcuPA/T, which we interpret as competition between scuPA and TAFI as substrates for added thrombin–TM complex. Normally TAFI circulates at a concentration about 4000-fold higher than scuPA [6,42] and we predict that TAFI would therefore prevent scuPA from inactivation by thrombin–TM. Their relative effectiveness as substrates may vary depending on local TM, echoing the differential activation of TAFI and PC [11,12]. The model thrombi and plasma lysis experiments shown were done without addition of TM, but clearly TAFIa was generated, consistent with soluble TM being sufficient for activation [43].

Human thrombi, formed in vivo, as well as model thrombi, contained TAFI, observed immunohistochemically, especially in fibrin-rich areas, consistent with its reported cross-linking to fibrin [44]. Extractable TAFI was present at about 0.5 µg g−1 of thrombus. This is consistent with other plasma proteins, including plasminogen, α2-AP and prothrombin, which are present in human thrombi at concentrations representing about 5–20% of their circulating concentration, based on assuming that 1 mL equates to 1 g [27,30]. TAFIa was not detected on Western blots. Similarly, it could not be clearly demonstrated in the functional assay of model thrombus lysis, as reported also for plasma clots [33]. The effect of TAFIa was clearly observed when thrombi were bathed in plasma, consistent with TAFIa being generated from the surrounding plasma. Thrombi are formed by rotating whole blood for 90 min at room temperature, and little TAFIa would be predicted to be active after thrombus formation, as a result of its lability [14,15]. In vivo, on the other hand, blood continually supplies the thrombus with fresh plasma and cellular components [27,30], which could penetrate the fibrin network. An alternative explanation for detecting TAFI, rather than TAFIa, in thrombus extracts is that TAFI in thrombi is not all activated. However, it may represent a pool of potential activity. Thrombi are a rich source of activated thrombin [27,45], which is protected by fibrin from inhibition [46]. Meizothrombin des F1, which is present in thrombi [27], can also activate TAFI [47]. This raises the possibility that local thrombin–TM activates TAFI on the fibrin surface [44]. The plasmin generated during lysis might also contribute to activation of TAFI [2,48,49].

This study highlights the role of TAFIa on lysis induced by activators other than tPA, which is of importance because uPA is the dominant plasminogen activator during endogenous thrombus lysis [28,29,50,51]. This paper does not directly address the issue of endogenous lysis, but there are conflicting reports on the role of TAFIa, with no observed effect of CPI on in vivo endogenous lysis [20] while, in contrast, both CPI and the thrombin inhibitor argatroban increased endogenous lysis in a rat model [52].

In order to mimic thrombolytic therapy we studied the effect of CPI on model thrombus lysis, using a wide range of activator concentrations. The increased lysis on inclusion of CPI was more easily observed at high activator concentrations, similar to those used in thrombolytic therapy [53]. The reduced effect at low activator concentrations may simply reflect the lack of plasmin-degraded fibrin, required to reveal the effect of TAFIa. The literature on this is conflicting: lysis of plasma clots was more affected by CPI at high tPA concentrations in one study [19], while another found a greater effect of TAFIa at low concentrations [33]. Thrombolysis of rabbit thrombi was significantly enhanced by co-administration of tPA and CPI [25,54]. Several studies have also shown that small direct thrombin inhibitors, co-administered with tPA, facilitate thrombolysis and prevent reocclusion [55–57]. These thrombin inhibitors effectively decreased TAFI activation [58]. Thus our studies on lysis of model thrombi, as well as the literature on in vivo experiments, support an important role for TAFIa in thrombolysis, not only by tPA but also by scuPA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr M. Nesheim for gifts of recombinant TAFI and a monoclonal antibody. This study was funded by the British Heart Foundation (PG 2001/005).

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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