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CARDIOVASCULAR
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama, Japan (K.S., Y.M., K.F., K.K., H.I., E.S., C.K., T.M., E.K., M.N., S.Y.); and Department of Public Health, Showa Pharmaceutical University, Machida, Tokyo, Japan (H.I.)
Received for publication
November 7, 2003
Accepted
January 21, 2004.
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Abstract |
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2-antiplasmin activity to a lesser extent than tPA. In an arteriovenous shunt model, EF6265 (1 mg/kg) enhanced exogenous tPA-mediated thrombolysis under the same conditions that neither EF6265 nor tPA (600 kIU/kg) alone reduced thrombi. EF6265 (1 and 30 mg/kg) did not affect the bleeding time in rats. Moreover, it did not prolong the bleeding time evoked by tPA (600 kIU/kg). These results confirm that circulating procarboxypeptidase B functions as a fibrinolysis inhibitor's zymogen and validates the use of CPB inhibitors as both an enhancer of physiological fibrinolysis in microcirculation and as a novel adjunctive agent to tPA for thromboembolic diseases while maintaining a small effect on primary hemostasis.
). Consequently, these large quantities of generated plasmin can induce thrombolysis and result in hemorrhaging as a side effect (Bloom et al., 1988). Many efforts have therefore been made to identify and develop pharmacologically distinct antithrombotic agents while maintaining a low risk of hemorrhaging.
Plasma procarboxypeptidase B (proCPB; EC 3.4.17.20
[EC]
), also known as thrombin-activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase U, is produced in the liver and circulates in the blood as a zymogen (Bajzar et al., 1995; Bouma and Meijers, 2003). proCPB is proteolytically transformed into the active enzyme, carboxypeptidase B (CPB), by thrombin produced during blood coagulation. Thrombin-mediated activation of plasma proCPB is greatly enhanced in the presence of the cofactor protein thrombomodulin, which is distributed on the plasma membrane of vascular endothelial cells and in circulating plasma in several degraded forms (Ishii, 1994). It has therefore been recognized that activation of proCPB is mainly via thrombin-thrombomodulin complexes on the surface of endothelial cells or in the plasma (Bouma and Meijers, 2003).
Generated plasma CPB down-regulates fibrinolysis by removing carboxy-terminal (C-terminal) lysines from fibrin fibers during blood coagulation (Bajzar et al., 1995; Sakharov et al., 1997). These C-terminal lysine residues, initially exposed through the degradation of fibrin by trace amounts of plasmin in the plasma, bind to the lysine-binding sites of plasminogen and tPA (Fleury and Angles-Cano, 1991; Bajzar et al., 1995). Thus, the binding of tPA and plasminogen is facilitated by the C-terminal lysine residues on the surface of fibrin clots and tPA efficiently activates plasminogen bound to the clot surface without any interference of plasminogen activator inhibitor-1 in plasma (Bajzar, 2000). Plasmin, generated on the fibrin clots, can also escape from the plasmin inhibitor plasma 2-antiplasmin, resulting in efficient fibrinolysis. The C-terminal lysine residues are therefore very important in physiological fibrinolysis. Additionally, the proCPB level in human plasma correlates with the time for clot lysis in healthy individuals (Mosnier et al., 1998) and with an increased risk of deep vein thrombosis (van Tilburg et al., 2000), coronary artery disease (Schroeder et al., 2002; Zorio et al., 2003), and ischemic stroke (Montaner et al., 2003). Based on a novel pharmacological mechanism, the specific inhibitors of the plasma CPB are therefore expected to enhance fibrinolysis by generating more plasmin on the surface of fibrin clots while maintaining a low risk of hemorrhaging.
Recently, in vivo studies have suggested the involvement of plasma CPB in thrombolysis (Bouma and Meijers, 2003): the induction of plasma CPB activity correlated with the suppression of fibrinolysis in canine coronary thrombosis (Redlitz et al., 1995); CPI [a potato-derived carboxypeptidase inhibitor of carboxypeptidase A (CPA) and CPB], incorporated into a clot created in an isolated segment of the jugular vein, potentiated endogenous thrombolysis (Minnema et al., 1998); and administration of CPI or DL-mercaptomethyl-3-guanidinoethylthiopropanoic acid [MGPA, an inhibitor of CPB and carboxypeptidase N (CPN)] reduced tissue factor-induced microthrombosis in rats (Muto at al., 2003). Additionally, exogenous tPA-induced thrombolysis was potentiated by combined administration of CPI in jugular vein (Nagashima et al., 2000) and abdominal aorta (Klement et al., 1999) thrombosis models in rabbits. A problem in these studies is that CPI and MGPA are neither specific nor potent enough (Nagashima et al., 2000; Muto at al., 2003) for plasma CPB, and as a result physiological analysis of these inhibitors is not conclusive. In spite of these studies, proCPB (TAFI)-deficient mice unexpectedly showed no overt phenotype (Nagashima et al., 2002). The pathophysiological importance of proCPB in thrombosis/thrombolysis is yet to be elucidated (Bouma and Meijers, 2003). Hence, we searched for a specific CPB inhibitor to determine the importance and therapeutic potential of plasma CPB in thrombotic conditions.
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Materials and Methods |
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). CPN was partially purified from human plasma (Skidgel et al., 1988; Bajzar et al., 1995). Enzyme activity of CPN was measured in the rate of hydrosis of hippuryl-lysine (Sigma-Aldrich) in 0.1 M Tris-HCl buffer, pH 7.6 (Hosaka et al., 1998). CPB- and CPN-like activities in rat plasma were evaluated according to a previously reported method (Schatteman et al., 1999). Bovine pancreas CPA (Tanaka et al., 1984) was from Roche Diagnostics (Mannheim, Germany), rabbit lung angiotensin-converting enzyme (ACE) (Cushman and Cheung, 1971) was from Wako Pure Chemicals (Tokyo, Japan). Human plasma plasmin (Kato et al., 1980), human plasma -thrombin (Kawabata et al., 1985), and bovine pancreas trypsin (Kawabata et al., 1988) were from Sigma-Aldrich. Human factor Xa (Morita et al., 1977) was from Hematologic Technologies (Essex Junction, VT). Human factor XIa (Kawabata et al., 1988) was purchased from Enzyme Research Laboratories Inc. tPA (alteplase) (Kyowa Hakko, Tokyo, Japan), tissue factor (Innovine) (Dade International, Miami, FL), and MGPA (Calbiochem-Novabiochem, La Jolla, CA) were used.
Synthesis of EF6265. EF6265 was synthesized in our laboratory with consideration of the previously reported methods (Baylis et al., 1984; Miller et al., 1998; Vassiliou et al., 1999). Detailed procedures and spectroscopic data will be reported on another occasion.
Animals. Male Wistar rats, weighing 200 to 300 g (Charles River Japan, Yokohama, Japan), were used. The experiments were approved by the institutional review board of the Pharmaceutical Research Laboratories of our company and were performed in accordance with the guidelines for animal experiments of the National Institutes of Health.
In Vitro Clot Lysis Assay in Human and Rat Plasma. Blood from healthy donors was collected into 1/10 volume of 3.8% sodium citrate. Platelet-poor plasma was obtained by centrifugation and stored at -80°C. Blood from rats was collected from the abdominal aorta of three rats anesthetized by pentobarbital (50 mg/kg intraperitoneal injection) into a syringe partially filled (1:10 total volume) with 3.8% sodium citrate. Rat plasma was quickly obtained by centrifugation and stored at 4°C. Experimental condition of clot lysis assay in human and rat plasma should be differently set because of the species difference in the reaction of clot formation/degradation (Schatteman et al., 1999). The clot lysis assays using human plasma (Hosaka et al., 1998) and rat plasma (Muto et al., 2003) were performed in the presence of tPA at 30 IU/ml and 60 IU/ml, respectively. A fibrin clot was formed by the addition of CaCl2 (final concentration 10 mM in human plasma and 20 mM in rat plasma) to reach the final volume of the solution 250 µl (human and rat plasma dilutions were 3.75 and 5 times, respectively) in a microtiter plate well, and turbidity was monitored at 600 nm at 37°C for 180 min using a microplate reader (THERMO max; Molecular Devices Corp., Sunnyvale, CA). Effect of EF6265 on the clot lysis assay was evaluated by adding EF6265 at final concentrations of 10-9,10-8,10-7,10-6, and 10-5 M into the assay mixture. In the human plasma assay, the time for clot lysis (clot lysis time) was determined by measuring the time required for absorbance to reach the value halfway between baseline and plateau (Hosaka et al., 1998). Clot lysis time in rat plasma assay could not be measured because the turbidity did not return to the baseline level in the turbidity-time profiles in the clot lysis assay using rat plasma under the condition described above without EF6265. So the total clot remaining in the assay was evaluated by integrating the area under the plasma concentration-time curve (AUC) above the baseline from the turbidity-time profile observed over 180 min, and expressed in "total clot" (Muto et al., 2003).
Ex Vivo Clot Lysis Assay in Rats. Rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg). EF6265 (at doses of 0.1 or 1 mg/kg) or saline (control) was intravenously administered (n = 5 for each group). Blood samples were collected from the jugular vein into a syringe partially filled (1:10 total volume) with 3.8% sodium citrate. The samples were obtained at 0 (just before EF6265 administration), 5, 15, 30, and 60 min after the intravenous administration of EF6265 or saline. Plasma was quickly obtained by centrifugation and stored at 4°C until clot lysis assay, which was performed as described above.
Plasma Concentration of EF6265 after Intravenous Injection. The blood concentration of EF6265 was measured after intravenous administration to male Wister rats (n = 5). EF6265 was separated by a Symmetry Shield RP18 column (Waters, Milford, MA) using 0.1% formic acid/acetonitrile [75:25 (v/v)] as a mobile phase and quantified using a TSQ7000 tandem mass spectrometry system (Thermo Finnigan, San Jose, CA). Pharmacokinetic parameters were calculated using a noncompartment model by processing with Win-Nonlin professional version 3.1 (Pharsight, Mountain View, CA). The elimination constant (kel) was calculated from the terminal three points. The AUC and the area under the moment curve (AUMC) were calculated using the linear trapezoidal rule. The area under the curve from hour 0 to infinity (AUC0-
), the area under the moment curve from hour 0 to infinity (AUMC0-), the total clearance (CLtot), and the volume of distribution at steady state (Vdss) were calculated as shown below.
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Microthrombosis Model Induced by Tissue Factor. Male Wistar rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg). Microthrombi were induced by the continuous infusion of 0.44 µg/kg tissue factor (volume 5 ml/kg) via the femoral vein of rats over a period of 20 min (Takahashi et al., 1997). EF6265 (at doses of 0.01, 0.1, and 1 mg/kg) or tPA (at doses of 12, 60, and 120 kIU/kg) was intravenously administered (bolus injection) 5 min after the infusion of tissue factor ended (25 min), and the rats were sacrificed at 45 min after the beginning of the tissue-factor infusion (n = 4–7). The kidneys were excised and fixed in 10% neutral buffered formalin. Sections of the kidney were histologically examined after phosphotungstic acid-hematoxylin staining for fibrin thrombi. The percentage of glomerular fibrin deposition (%GFD), a marker for thrombi, was determined as follows: 100 glomeruli were examined, and the number of glomeruli with clear fibrin deposits was expressed as a percentage (Muto et al., 2003). The plasma D-dimer concentration was determined in the latex agglutination test (Aoshima et al., 1998) using the assay kit of LPIA ACE DD dimmer (Dia-Iatron, Tokyo, Japan). The D-dimer concentration, which was determined to be less than the detection limit (0.5 µg/ml), was assumed to be 0.5 µg/ml for the purpose of statistical analysis. Plasma 2-antiplasmin activity was determined using the Enzyme test of APL2 kit (Matsuda et al., 1984) (Daiichi Chemical, Tokyo, Japan).
Thrombus formation/degradation was also evaluated from the radioactivity levels of the blood and organs (Hasegawa et al., 1996) after the injection of 125I-fibrinogen (Amersham Biosciences Inc., Tokyo, Japan) at a dose of approximately 37 kBq/kg 5 min before the infusion of tissue factor started. Radioactivity was measured by a gamma counter (Aloka, Tokyo, Japan). The microthrombi index was defined as a ratio of the radioactivity in the organ to the radioactivity in the reference blood that was obtained at zero time (just before the start of tissue factor infusion) and determined in each rat (Hasegawa et al., 1996).
Arteriovenous Shunt Thrombosis Model. An arteriovenous shunt model in rats (n = 10 for each group) was prepared as reported previously (Hara et al., 1995) with slight modifications. The polyethylene catheter that held a copper wire inside was placed to connect the right carotid artery and the left jugular vein of each rat under pentobarbital anesthesia (50 mg/kg i.p.). The copper wire was removed 20 min after the blood began to circulate through the catheter, and the thrombus weight was measured (Hara et al., 1995). EF6265 at a dose of 1 mg/kg or saline was intravenously administered (bolus injection) 5 min before the circulation of blood. The dose of EF6265 was determined as the dose that showed the maximum enhancement of fibrinolysis in the ex vivo clot lysis assay. tPA at a dose of 600 kIU/kg or saline was administered (bolus injection) 10 min after circulation of the blood in the shunt via the artery just in front of the shunt region. The dose of tPA (600 kIU/kg) was determined because tPA at that dose did not show any reduction of the weight of thrombi in preliminary experiments to evaluate the dose-response effects of tPA.
Bleeding Time. The tails of rats anesthetized with pentobarbital (50 mg/kg i.p.) were incised with disposable surgical blades (Hara et al., 1995). Bleeding of the incised tails was checked by blotting the blood with a filter paper every 30 s for 30 min (n = 5). We measured the control value of bleeding time of each rat before the administration of the drug (prevalue). Thereafter, we measured the bleeding time 1 min after the starting of the bolus administration of the test sample (postvalue). The ratio of the postvalue to the prevalue was calculated as a fold-increase in bleeding time. The test compounds were dissolved in saline and intravenously administered into the femoral vein. The bleeding time longer than 30 min was assumed to be 30 min for the purpose of statistical analysis. In case of evaluation for the effects of combined administration of tPA and EF6265, EF6265 was administered immediately after the tPA administration and a total injection volume for one rat was identical to 2 ml/kg in all groups.
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Results |
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Effect of EF6265 in Clot Lysis in Human and Rat Plasma. Clot lysis assays were performed to examine the effect of EF6265 on clot formation and subsequent clot lysis. A decline in turbidity after clot formation reflects clot lysis, or the fibrinolytic process. EF6265 enhanced tPA-mediated clot lysis in human (Fig. 2, A and B) and rat plasma (Fig. 2, C and D) in a concentration-dependent manner (Fig. 2, B and D). EF6265 maximally enhanced clot lysis at a concentration of about 1 µM in human and rat plasma.
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Enhanced fibrinolysis with the systemic administration of EF6265 into rats was confirmed by an ex vivo assay. tPA-mediated clot lysis was enhanced by EF6265 at a dose of 0.1 mg/kg at 5 (Fig. 3A) and 30 min (Fig. 3B) after intravenous administration, but this enhancement in plasma was reduced in a time-dependent manner (Fig. 3C). In contrast, this enhancement in plasma was maintained until 60 min after intravenous administration of EF6265 at a dose of 1 mg/kg (Fig. 3). These results indicate that systemically administered EF6265 enhanced fibrinolysis in a dose- and time-dependent manner.
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Pharmacokinetic Profiles of EF6265 after Intravenous Administration. The plasma concentration of EF6265 decreased with an elimination half-life of 0.96 ± 0.10 and 0.97 ± 0.06 h at 0.1 and 1 mg/kg, respectively (Fig. 4). The CLtot was 390 ml/h/kg at 0.1 mg/kg and 316 ml/h/kg at 1 mg/kg. The Vdss was 316 ± 32 ml/kg at 0.1 mg/kg and 223 ± 25 ml/kg at 1 mg/kg. The fibrinolysis enhancement by EF6265 in ex vivo clot lysis in rat plasma was explained by the plasma concentration of EF6265. The plasma concentrations at 5 min postdose of 0.1 and 1 mg/kg were 391 ng/ml (0.92 µM) and 6120 ng/ml (14.4 µM), respectively, and those at 60 min postdose of 0.1 and 1 mg/kg were 54.3 ng/ml (0.13 µM) and 589 ng/ml (1.38 µM), respectively. Considering that the greatest enhancement of fibrinolysis occurred with a concentration of 1 µM in the in vitro clot lysis assay (Fig. 2, C and D), it is likely that at a dose of 0.1 mg/kg the EF6265-mediated enhancement was attenuated in a time-dependent manner. In agreement, the greatest enhancement of clot lysis was identified 60 min after the intravenous administration of 1 mg/kg EF6265. Thus, the enhanced ex vivo clot lysis can be explained by the plasma concentrations of EF6265.
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Activity of EF6265 in a Microthrombosis Model Induced by a Tissue Factor. The antithrombotic effect of EF6265 was assessed in a thrombosis model induced by tissue factor (Fig. 5). This model is characterized by microthrombi occluding both the capillaries and small vessels of organs. Histological evaluation showed that the infusion of tissue factor formed distinct thrombi in the rat kidney, and the %GFD in the tissue-factor infused control group was 98 ± 1.1%, whereas in saline infused control group %GFD was 0 ± 0.0% (Fig. 5A). At doses ranging from 0.01 to 1 mg/kg, EF6265 attenuated the increase in %GFD in a dose-dependent manner (Fig. 5A) in conjunction with a simultaneous dose-dependent increase in plasma D-dimer levels (Fig. 5B), a marker of fibrin degradation, when the compound was intravenously administered at 25 min (5 min after the end of tissue factor infusion). A similar change in these parameters was observed in rats injected with tPA at doses of 60 kIU/kg or higher (Fig. 5, A and B). These results indicate that EF6265, as well as tPA, ameliorated microthrombosis through the enhancement of fibrinolysis. The effects of EF6265 and tPA in this model were also confirmed by experiments using 125I-labeled fibrinogen. The level of radioactivity was markedly increased in the kidney (Fig. 5C, hatched column) and lung (Fig. 5D, hatched column). Accordingly, the levels of radioactivity in the blood (Fig. 5E, open circle) decreased to about 35% at 25 min and were retained for a further 20 min (Fig. 5E). These results indicate that the labeled fibrinogen in circulating plasma was transformed into fibrin and deposited into the vessels of various tissues such as the kidney and lung. The administration of EF6265 or tPA, at 25 min, significantly inhibited this increase in radioactivity in these organs in a dose-dependent manner (Fig. 5, C and D). The injection of EF6265 slowly reelevated plasma radioactivity levels compared with tPA (Fig. 5E). Additionally, treatment with EF6265 in the thrombosis model did not markedly reduce the plasma levels of free 2-antiplasmin, whereas 60 and 120 IU/kg tPA reduced the free levels to 70 and 40%, respectively, at 35 min (Fig. 5F). These results suggest that EF6265 reduced thrombi in this model and enhanced fibrinolysis, whereas EF6265 produced much less plasmin in circulating plasma than tPA.
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Activity of EF6265 in an Arteriovenous Shunt Thrombosis Model. Although the intraarterial administration of 600 kIU/kg tPA, 10 min after the recirculation of the blood, did not reduce the weight of the thrombus, administration of tPA combined with pretreatment of 1 mg/kg EF6265 significantly reduced thrombus weight to less than 50% of that in the saline control (Fig. 6). This indicates that pretreatment with EF6265 enhanced exogenous tPA-mediated thrombolysis of arterial thrombi formed in the larger vessels.
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Effects of EF6265 on a Bleeding Time. The effect of EF6265 on primary hemostasis was evaluated by determining the bleeding time in rats. tPA produced a dose-dependent increase in the fold-increase in bleeding time, and 600 kIU/kg tPA increased it about 6-fold (Fig. 7A). EF6265 did not prolong the bleeding time at doses of 1 and 30 mg/kg (Fig. 7A). The group that received the combined treatment of tPA and EF6265, at the same doses used in the arteriovenous shunt model (Fig. 6), did not have a significantly prolonged fold-increase in bleeding time compared with the group administered with tPA alone (Fig. 7B). These results show that there is a small effect of EF6265 on primary hemostasis in both condition of the single use and combined use with tPA.
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Discussion |
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EF6265 enhanced in vitro tPA-mediated clot lysis. Clot lysis assays of human and rat plasma showed that this enhanced lysis occurred at concentrations greater than 10-8 M EF6265 and that the greatest effects occurred at 10-6 M. These concentrations were almost the same for the inhibition of plasma CPB (Fig. 2, B and D). The ex vivo clot lysis assay showed that the intravenous administration of EF6265 into rats enhanced tPA-dependent fibrinolysis in a dose- and time-dependent manner (Fig. 3). The pharmacokinetic study after intravenous administration of EF6265 (Fig. 4) revealed that the enhanced fibrinolysis (Fig. 3C) could be explained by the blood concentrations of EF6265. Hence, we concluded that to use EF6265 in further pharmacological studies, it was rational to determine both the antithrombotic potency of the CPB inhibitor and the role of plasma CPB in thrombus formation and/or degradation.
EF6265 alone showed clear antithrombotic effects in the tissue factor-induced microthrombosis model (Fig. 5, A–E). The injection of EF6265 as well as tPA attenuated the increases in %GFD (Fig. 5A) and deposition of radiolabeled fibrinogen (fibrin) in the tissues of rats with microthrombosis (Fig. 5, C and D). This inhibition was accompanied by increased levels of D-dimer (Fig. 5B) and plasma radioactivity (Fig. 5E), indicating degradation of fibrin. However, the increases in plasma radioactivity after the administration of EF6265 were slower than after the administration of tPA. These rates of increase in radioactivity could reflect the rate of fibrinolysis. This result suggests that the slow rate of fibrinolysis by EF6265, compared with tPA, which is a direct plasminogen activator (Rijken and Sakharov, 2001; Bouma and Meijers, 2003), could be explained by the enhancement of endogenous fibrinolysis (Speiser et al., 1988), because EF6265 protects the C-terminal lysine residues, which are the binding sites for endogenous tPA and plasminogen, from being cleaved by plasma CPB. Thrombomodulin plays a pivotal role in thrombin-mediated activation of plasma proCPB and thrombin-mediated activation of protein C as a natural anticoagulant (Esmon, 1989). Protein C activation is stimulated by low concentrations of thrombin and higher concentrations of thrombomodulin, whereas proCPB activation is stimulated by high concentrations of thrombin and low concentrations of thrombomodulin (5 nM) and reduced at higher thrombomodulin concentrations (10 nM) (Bouma and Meijers, 2003). Thus, thrombomodulin seems to play a dual role; it dampens the generation of thrombin by enhancing the activation of protein C, and it down-regulates fibrinolysis via the activation of proCPB. Although activation of proCPB and protein C can occur simultaneously, the concentration of thrombomodulin was found to be the factor determining the overall effect. In this context, vessel size might be an important factor, as the effective concentration thrombomodulin increases as blood moves from the aorta to the capillaries (Bouma and Meijers, 2003; Esmon, 1989). Therefore, the effective antithrombotic action of EF6265 in the microthrombosis model might be characterized by the direct enhancement of fibrinolysis through the inhibition of plasma CPB, and the anticoagulant effects of activated protein C generated by high concentrations of endothelial thrombomodulin in the microvessels and capillaries.
EF6265 enhanced fibrinolysis without significantly reducing plasma 2-antiplasmin levels in this model, even though effective doses of tPA did reduce it (Fig. 5F). These results indicate that CPB inhibitors enhance fibrinolysis on the surface of clots (Bouma and Meijers, 2003), whereas effective doses of tPA directly activate plasminogen in circulating blood (Weitz et al., 1993). These results might be related to the low level of bleeding seen with EF6265 in vivo (Fig. 7). Correspondingly, Nagashima et al. (2002) reported that a deficiency in TAFI did not lead to increased bleeding. These results strongly suggest that the effects of CPB inhibitors on primary hemostasis are small.
Using the arteriovenous shunt model, we next evaluated the antithrombotic effects of EF6265, as well as combined with tPA, on the larger thrombi formed on a wire in tube (not in blood vessels). EF6265 enhanced lysis induced by exogenously administrated tPA, whereas EF6265 alone did not reduce the thrombus in the arteriovenous thrombosis model (Fig. 6). This effect of the CPB inhibitor with exogenous tPA has been previously reported in venous (Nagashima et al., 2000) and arterial (Klement et al., 1999) thrombosis rabbit models. With the exogenous administration of tPA, plasmin generated in the plasma is important for activation of proCPB as well as the degradation of fibrin (Bouma and Meijers, 2003). This occurs because plasmin can activate plasma proCPB with a low Michaelis constant (Km of 55 nM) without thrombomodulin and exposes the C-terminal lysine residues on fibrin by partial degradation of fibrin clots (Bouma and Meijers, 2003). Therefore, potentiation of EF6265 on tPA-mediated thrombolysis can be explained: plasmin produced by administration of tPA partially degrades fibrin clots and exposes the new C-terminal lysine residues on the clot; the plasmin also activates proCPB, although active CPB is inhibited by EF6265 and the newly exposed C-terminal lysine is protected from CPB; therefore, the binding of both tPA and circulating plasminogen to the newly exposed C-terminal lysine is recruited to the tPA-dependent efficient activation of plasminogen (Bajzar, 2000). In contrast, the lack of antithrombotic effects with a single administration of EF6265 might occur because endogenous tPA was insufficient to dissolve larger thrombi even when plasma CPB was inhibited. When the doses of both compounds were set to levels, such that when combined, both compounds showed antithrombotic effects in the arteriovenous model (Fig. 6), EF6265 did not further lengthen the bleeding time prolonged by tPA (Fig. 7B). Thus, EF6265 may also be expected to be an enhancer of tPA treatment for various thromboses, including myocardial and cerebral infarctions while maintaining a low risk of hemorrhaging.
As described above, EF6265 showed different antithrombotic effects on the two models that were performed in this study. The difference could be explained by the size of thrombi and involvement of the blood vessels around the thrombi. EF6265 was clearly effective on the microthrombosis model in the treatment setting. To further clarify the potential utility of EF6265 in single use for microthrombotic diseases, a model of organ injury induced by microthrombi would be particular interest (such as endotoxemia and thrombotic microangiopathy). On the other hand, pretreatment of EF6265 enhanced the fibrinolysis induced by exogenous tPA in the shunt model. To clarify the potential utility of EF6265 for clinical use in arterial thrombotic diseases with or without tPA, a model of clot formation/degradation on arterial side would be of particular interest (such as FeCl3 injury on an carotid model), because it is in such condition that endogenous tPA is delivered. These further studies are needed to reveal possibilities and limitations of plasma CPB inhibitors in clinical usage.
In conclusion, our results show that plasma CPB regulates pathophysiological fibrinolysis and that targeting plasma CPB is an attractive strategy to uncover novel antithrombotic agents. Using EF6265, a selective inhibitor of plasma CPB, we show the crucial role of plasma CPB in thrombotic conditions, especially in small vessels and capillaries. CPB inhibitors can be novel antithrombotic agents and a fibrinolytic enhancer while maintaining a small effect on primary hemostasis, especially for thrombolysis in the microcirculation and in conjunction with drugs such as tPA for the treatment of various thromboembolic diseases.
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Footnotes |
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ABBREVIATIONS: tPA, tissue-type plasminogen activator; proCPB, procarboxypeptidase B; TAFI, thrombin-activatable fibrinolysis inhibitor; CPB, carboxypeptidase B; CPI, potato-derived carboxypeptidase inhibitor; CPA, carboxypeptidase A; MGPA, DL-mercaptmethyl-3-guanidinoethylthiopropanoic acid; CPN carboxypeptidase N; ACE, angiotensin-converting enzyme; AUC, area under the plasma concentration-time curve; GFD, glomerular fibrin deposition; EF6265, (S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylamino)propyl]hydroxyphosphinoyl]methyl]heptanoic acid.
1 These authors contributed equally to this work. 
Address correspondence to: Dr. Kokichi Suzuki, R&D Planning, Meiji Seika Kaisha Ltd., 580 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan. E-mail: kokichi_suzuki{at}meiji.co.jp
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