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Keywords:

  • aPC;
  • coagulation;
  • fibrinolysis;
  • PAI-1;
  • thrombin

Abstract

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

Summary.  Several activated coagulation factors have been reported to enhance fibrinolysis by neutralizing plasminogen activator inhibitor type 1 (PAI-1) activity. We evaluated the physiological relevance of this mechanism using the euglobulin clot lysis time (ECLT) assay in the presence and absence of Ca2+, which is controlled by PAI-1 and mimics physiological thrombolysis. We found that the ECLT (18.5 ± 0.6 h) was shortened by Ca2+ (5 mm) (6.6 ± 0.1 h). A significant difference was observed in thrombin generation by the presence of Ca2+ in the euglobulin fraction. Prothrombin was almost fully converted to thrombin within 15 min in the presence of Ca2+, whereas essentially no conversion was observed without Ca2+. The presence of activated protein C (aPC) suppressed thrombin generation, and attenuated the shortening of ECLT in a dose-dependent manner, an effect enhanced by phospholipid and protein S. In the absence of Ca2+, aPC did not prolong the ECLT. After addition of biotin-labeled recombinant PAI-1 to the euglobulin fraction, PAI-1 was cleaved to lower molecular weight forms only in the presence of Ca2+. This cleavage did not occur in the presence of aPC, suggesting that thrombin was the catalyst for PAI-1 cleavage. The cleavage and inactivation of PAI-1 by generated thrombin is proposed to be responsible for the shortening of ECLT by Ca2+ and for coagulation-associated over-expression of fibrinolysis. Under such conditions, aPC appeared to suppress thrombin generation and to normalize highly activated fibrinolysis.


Introduction

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

Coagulation-associated enhancement of fibrinolysis is a well-known phenomenon [1], and plays an essential role in maintaining vascular patency by limiting over-accumulation of fibrin clots. Over-expression of fibrinolytic activity following disseminated intravascular coagulation is a pathological phenotype of this event. Several distinct mechanisms have been suggested to explain the phenomenon [2,3]. A more effective activation of plasminogen to plasmin on the fibrin surface is one of the mechanisms involved. Here a trimolecular complex formation among tissue plasminogen activator (t-PA), plasminogen, and fibrin, and a subsequent conformational alteration of Glu-plasminogen into a more activatable form are essential [4]. Inactivation of fibrin-bound plasmin, a process that is less effective than that of free plasmin, by α2-antiplasmin may be another contributing mechanism [5]. Besides these two well-established mechanisms, neutralization of plasminogen activator inhibitor type 1 (PAI-1) activity by activated coagulation factors is also suggested to be one such mechanism.

PAI-1 belongs to the serine protease inhibitor superfamily (SERPINS) [6], that also includes most of the protease inhibitors in plasma. In the vasculature, t-PA is the target protease for PAI-1, and its activity is directly determined by the equilibrium between t-PA and PAI-1 in plasma [7]. PAI-1 also forms a complex with active serine proteases other than plasminogen activators involved in the coagulation cascade, including factor XIa, kallikrein, factor (F)XIIa [8], thrombin [9,10], and Ca2+-bound FXa [11], especially in the presence of cofactors of heparin or vitronectin (Vn). As a consequence, the equilibrium between t-PA and PAI-1 in plasma is naturally modified, thereby increasing both the amount of free t-PA and the fibrinolytic activity [12,13]. The physiological relevance of this mechanism, however, remains to be elucidated.

Activated protein C (aPC) is an anticoagulant enzyme that is generated from its zymogen form by thrombomodulin-bound thrombin. This enzyme suppresses coagulation by inactivating FVa and FVIIIa by limited proteolytic cleavage [14]. aPC is believed to possess profibrinolytic properties, based on findings that it can neutralize PAI-1 activity [15] or attenuate the generation of a carboxypeptidase B-type enzyme, thrombin-activatable fibrinolysis inhibitor [16]. The importance of these profibrinolytic activities in hemostasis in vivo is not fully understood.

In order to gain a fuller appreciation of the role of aPC in physiological fibrinolysis, the relevance of the activated coagulation factor-dependent inactivation of PAI-1 was examined using the euglobulin clot lysis time assay (ECLT), which is dependent on PAI-1 and mimics physiological thrombolysis. Although the ECLT is essentially determined by the balance between t-PA and PAI-1, it is dramatically shortened by the physiological concentration of Ca2+[17]. The results of this study are reported here.

Materials and methods

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

Materials

Phospholipids (phosphatidylcholine from egg yolk and phosphatidylserine from bovine brain, both >98% pure) were from Sigma (St Louis, MO, USA).

Preparation of phospholipid vesicles

Phosphatidylcholine and phosphatidylserine in chloroform were mixed at 3 : 1 and dried under N2 stream in a glass tube. They were suspended in 50 mm Tris–HCl buffer pH 7.5 containing 100 mm NaCl at 4 mm, and dispersed by sonication in a bath-type sonicator for 30 min.

Proteins

aPC was provided by the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan) [18]. Its specific activity was assayed by the activated partial thromboplastin time (APTT) method, and it was found that aPC prolonged APTT 2-fold at a concentration of around 2.5 nm. Human protein S was purchased from ERL (South Bend, IN, USA). Human prothrombin and α-thrombin were purchased from Diagnostica Stago (Asnieres, France), and Sigma, respectively. Human plasma Vn was purchased from Promega (Madison, WI, USA). Human recombinant two-chain t-PA was provided by Sumitomo Seiyaku Co. (Osaka, Japan) and appeared to be >92% two-chain form. Its specific activity was 5.5 × 105 t-PA IU mg−1 protein. Soluble desAA-fibrinogen (DesafibR-X) was purchased from Biopool AB (Umea, Sweden).

Biotin-labeled PAI-1 Human recombinant prokaryotic PAI-1 (rpPAI-1) was expressed in Escherichia coli and purified as previously reported [19]. rpPAI-1 was labeled by biotin using a commercially available kit (ECL protein biotinylation module; Amersham Life Sciences, Little Chalfont, UK) according to the manufacturer's recommended procedure with the following modifications. To avoid possible conversion of active rpPAI-1 into its latent form during the procedure, a more acidic buffer (50 mm Na+–HEPES/0.5 m NaCl, pH 5.3) and a shorter incubation time (20 min) were used. At least two-thirds of the labeled material was confirmed to be active by its ability to form a high-molecular-weight complex with t-PA (data not shown).

Euglobulin clot lysis time The standard ECLT and Ca2+-supplemented ECLT (Ca2+-ECLT) were measured using microtiter plates [20]. To determine the effect of aPC on this assay, increasing concentrations of aPC (final concentrations of 0, 1.3, 2.5, 5, 10, 20, 40, and 80 nm) were added prior to clot formation. When required, phospholipids (final concentration 0.4 mm) and protein S (final concentration 100 nm) were added before the addition of human thrombin (final concentration 0.1 U mL−1).

Analyses of thrombin generation and PAI-1 cleavage during euglobulin clot lysis

Thrombin generation from prothrombin in the euglobulin fraction was detected by Western immunoblotting. The fibrin clot was removed by a glass rod at different time intervals after euglobulin clot formation, and the remaining supernatant was mixed with sample buffer for SDS–PAGE. After transblotting onto a nitrocellulose membrane, protein bands were visualized using affinity-purified sheep antihuman prothrombin IgG (Cedarlane, Ontario, Canada), followed by horseradish peroxidase (HRP)-conjugated F(ab)2 fragment of donkey–antisheep IgG (Chemicon Int. Inc., Temecula, CA, USA), and enhanced chemiluminescence Western blotting detector reagents (ECL; Amersham Life Sciences). Prestained broad range molecular weight standards and nitrocellulose membranes were purchased from BioRad Labs (Richmond, CA, USA). Biotin-labeled rpPAI-1-related bands were detected with use of a streptavidin–HRP conjugate (Amersham Life Sciences) after separation of protein bands by SDS–PAGE and transblotting onto nitrocellulose membranes. To assess the molecular weights of the thrombin–PAI-1 and aPC–PAI-1 complexes, human α-thrombin (1 µm) and aPC (0.16 µm) were incubated with biotin-labeled rpPAI-1 in the presence of Vn (0.27 µm) and subjected to SDS–PAGE.

Effect of aPC on t-PA-catalyzed activation of Glu-plasminogen

The initial velocity of Glu-plasminogen activation by t-PA was assayed using a continuous coupled assay. The assays were performed at 37 °C in a buffer containing 50 mm Tris–HCl/100 mm NaCl, pH 7.4. The assay system included Glu-plasminogen (0.5 µm), soluble desA-fibrin (0.12 mg mL−1), t-PA (1.9 nm) and S-2251 (0.4 mm). When required, aPC (10 nm) and protein S (100 nm) were added. The amount of S-2251 hydrolyzed by aPC was estimated using the above assay conducted in the absence of Glu-plasminogen, and this value, though it was almost negligible, was subtracted.

Results

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

Effect of aPC on ECLT

The ECLT (16.7 ± 0.04 h) was significantly shortened by 5 mm Ca2+ (5.3 ± 0.05 h) (Fig. 1), an effect attenuated in the presence of aPC at 80 nm. In contrast, aPC (80 nm) slightly shortened ECLT in the absence of Ca2+ (15.9 ± 0.3 h) (Fig. 1). The concentration of aPC required to quench the effect of Ca2+ on the ECLT was higher than the concentrations required to prolong the APTT. The inclusion of protein S in the assay slightly potentiated the effect of aPC and prolonged the Ca2+-ECLT. The Ca2+-ECLT obtained at 80 nm aPC (16.2 ± 0.06 h) together with protein S was similar to the ECLT without Ca2. A combination of phospholipid and protein S, both of which are essential for the inactivation of FVa by aPC [21], significantly amplified the effect of aPC and attenuated Ca2+-induced shortening of the ECLT (Fig. 2). The longest Ca2+-ECLT (22.9 ± 0.1 h) was obtained at 20 nm aPC. The concentration of aPC required for 50% of its effect was 5–10 nm, which was comparable to that required for a 2-fold prolongation of APTT (2.5 nm). A slight shortening of the ECLT was observed with higher concentrations of aPC (40 and 80 nm) in the presence of protein S, in the absence and presence of Ca2+, respectively.

image

Figure 1. The effect of activated protein C (aPC) on the euglobulin clot lysis time (ECLT) both in the presence and absence of Ca2+. Both the standard ECLT and Ca2+-ECLT were measured in the presence of increasing concentrations of aPC (final concentrations of 0, 1.3, 2.5, 5, 10, 20, 40 and 80 nm). When appropriate, protein S (final concentration 100 nm) was included before the addition of human thrombin (final 0.1 U mL−1). The data are expressed as mean ± standard deviation (n = 3).

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image

Figure 2. The effect of activated protein C (aPC0, protein S and phospholipid on the euglobulin clot lysis time (ECLT) both in the presence and absence of Ca2+. The standard ECLT and Ca2+-ECLT were measured in the presence of increasing concentrations of aPC (final concentrations of 0, 1.3, 2.5, 5, 10, 20, 40 and 80 nm) together with 100 nm protein S and phospholipid (final concentration 0.4 mm). The data are expressed as mean ± standard deviation (n = 3).

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Effect of aPC on plasminogen activation by t-PA

To determine the mechanism by which aPC inhibits fibrinolysis in this assay system, the possible inhibitory effect of aPC on the t-PA-catalyzed activation of plasminogen was investigated. The increases in absorbance at 405 nm in 10 min of 0.116 ± 0.008 (mean ± SD, n = 3) in controls did not significantly change upon addition of aPC or aPC/protein S to the assays. Thus, t-PA-catalyzed Glu-plasminogen activation in the presence of soluble fibrin was not inhibited by aPC.

Detection of thrombin generation in the euglobulin fraction during ECLT

The activation of prothrombin to α-thrombin in the euglobulin fraction during the ECLT was analyzed by Western blot analysis (Fig. 3). In the absence of both Ca2+ and aPC, three major bands were detected (lane 1). Prothrombin was observed at Mr = 76 000 Da, and its migration was identical to that of purified prothrombin (lane 6). Another major band, the migration of which is identical to that of a fragment generated from prothrombin after digestion by a catalytic amount of thrombin, had an estimated Mr of 62 000 Da. This band does not exist either in plasma or in the normal euglobulin fraction (Fig. 4, lanes 1 and 2) and appears to correspond to prethrombin 1, a cleavage product of prothrombin at Arg155 by thrombin [22]. Treatment of euglobulin by α-thrombin to form the euglobulin clot seemed to have generated prethrombin, as shown in lanes 3 and 4 in Fig. 4. Another band migrating between 116 000 and 200 000 Da corresponds to dimerized prothrombin [23]. In the absence of Ca2+ no detectable α-thrombin was generated up to 3 h (lane 1, Fig. 3). In the presence of Ca2+, however, another major band at Mr = 37 000 Da (lane 3 in Fig. 3, upper panel), the migration of which was identical to that of α-thrombin (lane 5, Fig. 3), appeared 15 min after clot formation. The amounts of α-thrombin did not essentially change up to 3 h (lane 3 in Fig. 3, lower panel). Thrombin generation, in the presence of Ca2+, was attenuated by aPC, and essentially no thrombin was detected at 15 min (lane 4 in Fig. 3, upper panel). Only a faint amount of prothrombin was converted to α-thrombin at 3 h (lane 4 in Fig. 3, lower panel). In the absence of Ca2+, aPC did not modify thrombin generation to a significant degree (lane 2, Fig. 3).

image

Figure 3. Thrombin generation in the euglobulin fraction during the euglobulin clot lysis time (ECLT) assay. Thrombin generation was analyzed by Western blotting employing antiprothrombin polyclonal antibody. Samples were obtained during the ECLT either 15 min (upper panel) or 3 h (lower panel) after clot formation, in either the presence or absence of Ca2+ and activated protein C (aPC) (80 nm). α-thrombin (lane 5) and prothrombin (lane 6) were used as positive controls. The conversion of prothrombin to thrombin is clearly demonstrated only in the presence of Ca2+ (lane 3), which is mostly attenuated by aPC (lane 4). Molecular weight standard indicates 116 000, 97 400, 66 200, 45 000, and 31 000 Da, respectively.

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image

Figure 4. Western blotting of plasma and its euglobulin fraction by antiprothrombin polyclonal antibody. Lane 1, plasma; lane 2, euglobulin fraction; lanes 3 and 4, euglobulin fraction treated by catalytic amounts of α-thrombin either in the absence (lane 3) or presence (lane 4) of Ca2+ for 30 min at 37 °C; lane 5, thrombin; lane 6, prothrombin. Molecular weight standard indicates 200 000, 116 000, 97 400, 66 200, 45 000, and 31 000 Da, respectively.

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Analysis of the modification of supplemented biotin-labeled rpPAI-1 during ECLT

Biotin-labeled rpPAI-1 supplemented in the euglobulin fraction migrates at Mr = 56 300 Da, which is slightly larger in size than non-glycosylated PAI-1 [19], probably due to its biotinylation (Fig. 5). This band remained unchanged in the absence of Ca2+ for at least 3 h, during euglobulin clot lysis (lane 1). When aPC was added, a trace amount of a higher molecular weight signal was detected at approximately 105 100 Da (lane 2), a molecular weight identical with that of the complex between purified aPC and biotin-labeled rpPAI-1 (lane 6). The amount of aPC–PAI-1 complex did not essentially change up to 3 h (lane 2, lower panel). In the presence of Ca2+, large amounts of biotin-labeled rpPAI-1 were detected as a low-molecular-weight form (Mr = 50 500 Da) (lane 3) that represents a cleavage product. A high-molecular-weight band (Mr = 85 200 Da), probably from the biotin-labeled rpPAI-1–thrombin complex, was also detected at 15 min (lane 3, upper panel) and disappeared at 3 h (lane 3, lower panel). The cleaved form therefore appears to be generated by a rapid dissociation of the biotin-labeled rpPAI-1–thrombin complex, as was reported earlier [9]. Interestingly, the biotin-labeled rpPAI-1–thrombin complex disappeared faster in the Ca2+-supplemented euglobulin fraction than in the purified system (lane 5). Proteolytic cleavage of Vn, which accelerates thrombin–PAI-1 complex formation [24] by a template mechanism [25] employing proteases, including plasmin [26], during euglobulin clot lysis may have facilitated the rapid disappearance of the biotin-labeled rpPAI-1–thrombin complex in the euglobulin fraction. After the addition of aPC, the amounts of the low-molecular-weight cleaved form decreased, and the high-molecular-weight band, representing biotin-labeled rpPAI-1–aPC, was detected (lane 4), as in the absence of Ca2+ (lane 2). The attenuation of thrombin generation by aPC in the presence of Ca2+ therefore suppressed both complex formation between thrombin and PAI-1 and cleavage of PAI-1 by thrombin.

image

Figure 5. Modification of biotin-labeled recombinant prokaryotic plasminogen activator inhibitor type 1 (rpPAI-1) in the euglobulin fraction during euglobulin clot lysis time (ECLT). The proteolytic modification of biotin-labeled rpPAI-1 was analyzed during ECLT. The euglobulin fraction was supplemented with biotin-labeled rpPAI-1, and samples were obtained during the ECLT either 15 min (upper panel) or 3 h (lower panel) after clot formation, either in the presence or absence of Ca2+ and activated protein C (aPC) (80 nm). Samples of biotin-labeled rpPAI-1 +α-thrombin (lane 5), and biotin-labeled rpPAI-1 +aPC (lane 6) were employed as positive controls. Biotin-labeled rpPAI-1 formed a high-molecular-weight complex with aPC (lanes 2 and 4), and a high-molecular-weight complex with thrombin only in the presence of Ca2+ (lane 3). A lower molecular weight form of biotin-labeled rpPAI-1 was observed only in the presence of Ca2+ (lane 3). The generation of both the high-molecular-weight complex with thrombin and the lower molecular weight form of biotin-labeled rpPAI-1 was not observed, even in the presence of Ca2+, when aPC was added (lane 5). The molecular weight standards are 116 000, 97 400, 66 200, and 45 000 Da, top-to-bottom, respectively.

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Discussion

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

The shortening of the ECLT by addition of a physiological concentration of Ca2+ appears to be dependent on thrombin generation and the cleavage of PAI-1, both of which were attenuated by aPC. Inactivation of PAI-1 by the thrombin generated plays a role in coagulation-associated enhancement of fibrinolysis. aPC can modulate the over-expression of fibrinolysis by suppressing thrombin generation under physiological conditions.

t-PA is the main plasminogen activator in the vasculature, and it possesses almost full activity in its single-chain form when bound to fibrinogen [27]. The simultaneous presence of active t-PA and PAI-1 in plasma leads to a condition in which the amounts of free t-PA, and thus its specific activity, are determined by the equilibrium between the two proteins. In a purified system, we demonstrated that both Ca2+-bound FXa and thrombin enhanced fibrinolysis by inactivating PAI-1 [28]. In order to understand the physiological relevance of these phenomena, we employed the ECLT assay, which is expressed by the balance between t-PA and PAI-1 [7,20], and is shortened by physiological concentrations of Ca2+, for which FXa is a requisite [17].

We found that prothrombin in the euglobulin fraction was largely converted to α-thrombin within 15 min only in the presence of Ca2+. The conversion was negligible even 3 h after clot formation in the absence of Ca2+. The conversion was attenuated by aPC, and only a fraction of prothrombin was converted to α-thrombin within 3 h. To investigate the possible modification of PAI-1 by either Ca2+-bound FXa or thrombin, we employed biotin-labeled rpPAI-1 for enhanced signal detection. Biotin-labeled rpPAI-1 (5 nm) was cleaved to a smaller fragment only in the presence of Ca2+, whereas it remained essentially intact in the absence of Ca2+. This cleaved form of biotin-labeled rpPAI-1 appears to be generated by a rapid dissociation of the biotin-labeled rpPAI-1–thrombin complex [9], which was observed at 15 min and then disappeared by 3 h. The Ca2+-supplemented euglobulin clot was dissolved more rapidly by an enhanced fibrinolytic activity induced by the thrombin-dependent inactivation of PAI-1. Such enhancement of fibrinolysis as a consequence of PAI-1 inactivation has also been demonstrated using aPC [15], neutrophil elastase [29], contact phase coagulation factors [8], thrombin [9,10], FXa [28], and subtilisin [30].

We then attenuated the activation of FX and prothrombin by aPC and analyzed the effects on the ECLT. aPC successfully diminished thrombin generation and prolonged the Ca2+-dependent ECLT in a dose-dependent manner. Both high-molecular-weight complex formation between thrombin and biotin-labeled rpPAI-1 and the cleavage of biotin-labeled rpPAI-1 were abolished by aPC. These results confirmed that the activation of the coagulation cascade and PAI-1 cleavage by generated thrombin were essential in the shortening of ECLT by Ca2+.

Many lines of evidence have suggested that aPC does not inhibit, but rather enhances fibrinolysis. One of the suggested mechanisms is neutralization of PAI-1 activity as a consequence of high-molecular-weight complex formation with PAI-1 [15,31], especially in the presence of Vn [32]. In the present study, the high-molecular-weight complex formation of aPC with biotin-labeled rpPAI-1 was shown when a high concentration of aPC (80 nm) was added, which appeared to be responsible for the slight shortening of the ECLT in both the presence and absence of Ca2+. However, the magnitude of the shortening of ECLT by aPC was much lower than that of the inhibition of the Ca2+-ECLT induced by the attenuation of thrombin generation. The profibrinolytic activity of aPC might be expressed only under special circumstances, and, as was suggested in previous reports, may not be important under normal conditions [18].

In the present study, we employed the ECLT assay to mimic physiological conditions, and showed that aPC attenuates coagulation-associated enhancement of fibrinolysis. In a situation under which the coagulation cascade is fully activated, and clot lysis is spontaneously induced by intrinsic t-PA in the presence of PAI-1, aPC appeared to inhibit fibrinolysis by attenuating thrombin generation. This agrees with a previous finding that fibrinolytic activity was elevated by infusion of neutralizing antibody to abolish PC activation in a thrombosis model induced by FXa/phospholipid [33]. This mechanism may play an essential role in the development of a severe bleeding tendency, due to disseminated intravascular coagulation (DIC) in animals deficient in control factors of the coagulation cascade such as PC [34], tissue factor pathway inhibitor [35], or antithrombin III [36].

Under physiological conditions, the thrombin-dependent inactivation of PAI-1 appears to play an important role in coagulation-associated enhancement of fibrinolysis. Though its profibrinolytic role has probably been exaggerated, aPC does attenuate coagulation-associated enhancement of fibrinolysis. Thus, we speculate that aPC normalizes highly enhanced fibrinolytic activity after intense activation of the coagulation cascade under pathological conditions, such as DIC. This mechanism seems to be at least partly involved in the beneficial effects of aPC in animal models of thromboembolism [37].

Acknowledgements

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

This work was supported in part by a Grant-in-Aid for Scientific Research (no. 10670040) from the Japan Society for the Promotion of Science (to T.U.) and NIH Grant HL-19982 (to F.J.C.).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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