Inducible Carboxypeptidase Activity
A Role in Clot Lysis In Vivo
Background An inducible carboxypeptidase activity in human plasma delays tissue-type plasminogen activator (TPA)–induced clot lysis in vitro. We investigated whether carboxypeptidase activity is induced in vivo during thrombosis and thrombolytic therapy in a canine model of myocardial infarction.
Methods and Results By use of synthetic substrate assays, dog plasma was shown to contain an inducible carboxypeptidase activity that is efficiently inhibited by potato carboxypeptidase inhibitor. This inhibitor accelerates TPA-mediated clot lysis in vitro by an average of 27% (n=5, P=.046). Analysis of the inducible carboxypeptidase activity in plasma samples of dogs with electrically induced thrombosis of the circumflex coronary artery treated with TPA revealed that (1) inducible carboxypeptidase activity is increased during thrombosis (8.7±2.0 U/L, P<.013) and thrombolytic therapy (9.9±1.8 U/L, P<.024) compared with baseline (3.2±2.0 U/L); (2) thrombosis is a prerequisite of carboxypeptidase induction during and after TPA infusion, since carboxypeptidase levels were lower in dogs without a coronary thrombus; and (3) a significant positive correlation (r=.6, P<.0069) of carboxypeptidase activity with time to restoration of blood flow was observed.
Conclusions These data indicate that carboxypeptidase activity is induced in vivo and may influence thrombolysis.
The activation of plasminogen to plasmin is subject to intricate regulation; both accelerating and dampening mechanisms are in place to achieve controlled proteolysis of fibrin. Initial proteolysis of fibrin generates new carboxy-terminal lysines, which serve as additional plasminogen binding sites to accelerate its activation and protect plasmin from physiological inhibitors.1 2 Recently, initial evidence has been obtained for a previously unrecognized regulatory mechanism: an inducible plasma carboxypeptidase (Cp) activity capable of removing carboxy-terminal lysine residues may suppress fibrinolysis.3 4
Upon coagulation of blood, Cp activity increases significantly.5 This increment, originally designated Cp U, is different from the constitutively active Cp N of plasma.6 Partial purification and functional analyses suggest that Cp U may be identical to a previously purified and latent Cp, plasma Cp B,7 which can be activated by thrombin and plasmin.8 Therefore, Cp activity may be elicited during thrombosis and/or thrombolysis and exert a controlling influence on fibrinolysis.
Cp activation has yet to be shown to occur in vivo, and a role of Cp in suppressing fibrinolysis is deduced solely from in vitro studies. To address these central issues, we analyzed Cp generation in dogs undergoing coronary thrombosis and thrombolytic therapy with tissue-type plasminogen activator (TPA). Our results demonstrate that Cp activation does, indeed, occur in vivo. Furthermore, this inducible Cp activity may play a significant role in determining the outcome of thrombolytic therapy.
Determination of Cp Activity
Arginine Cp activity was assessed by use of furylacroleyl-alanyl-arginine (Bachem) as a substrate.3 In this assay, 1 U=1 μmol substrate hydrolyzed per minute at 22°C. Inducible Cp activity was determined as the Cp activity sensitive to inhibition by potato carboxypeptidase inhibitor (PCI) (Calbiochem), a plasma Cp B–specific inhibitor.3 Accordingly, inducible Cp is calculated as (Cp without PCI) minus (Cp with PCI) and is expressed in units per liter.
Venous blood samples were drawn into tubes containing anticoagulant (14.3 USP units sodium heparin/mL or 0.38% acid/citrate/dextrose) supplemented with 140 to 200 U aprotinin/mL and 1.4 μmol/L phenylalanyl-phenylalanyl-arginyl-chloromethylketone to inhibit plasmin and thrombin, respectively. Additionally, a serum sample was obtained, with the blood allowed to clot for 1 hour. Preliminary experiments established that the anticoagulants did not affect the inducible Cp activity, that the added protease inhibitors stabilized this activity for up to 6 hours at 22°C, and that storage of samples at −70°C had no effect on the Cp activity. Plasma and serum were prepared by centrifugation at 2000g for 15 minutes and incubated without or with 50 μg/mL PCI for >5 minutes before assay.
In Vitro Clot Lysis Assay
Whole blood clots, labeled with tracer 125I-fibrinogen as described,3 were placed in citrated canine plasma (buffered with 1/20 vol/vol of 1 mol/L HEPES, pH 7.5) in the absence or presence of 20 μg/mL PCI. Lysis was induced by the addition of increasing concentrations (30 to 1000 ng/mL) of TPA (Genentech Inc).
Animals and Surgical Procedures
The canine model of electrically induced myocardial infarction has been described previously.9 Briefly, mongrel dogs of either sex were anesthetized with 25 mg/kg sodium pentobarbital and placed on assisted respiration. The heart was exposed by left thoracotomy, and the proximal portion of the circumflex coronary artery was isolated. Coronary blood flow was measured with a Doppler probe (Crystal Biotech). The circumflex coronary artery was stenosed by ≈80% with a vascular occluder, and thrombosis was induced by mechanical denudation of the artery and by electrical injury with 100 μA anodal current until the blood flow was zero. For infusion of TPA and blood sampling, catheters were inserted into both femoral veins and advanced into the inferior vena cava.
Paired t test and linear regressions were calculated by use of Sigma Plot Software (Jandel Scientific). Repeated-measures ANOVA with planned comparisons, using SAS GLM (SAS Inc), was applied to compare baseline levels of Cp with all other comparisons. Results are presented as least-squares mean±SEM. A two-sided value of P<.05 was considered significant.
Constitutive and Inducible Cp Activity in Dog Serum and Plasma
The analysis shown in the Table⇓ indicates that, like human blood, canine blood contains a constitutive and an inducible Cp activity. The activity in serum (163±16 U/L) increased significantly compared with plasma (71±4 U/L, n=8, P<.0005, paired t test), indicative of an inducible Cp activity that is activated during blood coagulation. The increment in Cp activity in serum was fully inhibited by PCI, whereas the constitutive Cp activity in plasma was unaffected. These data indicate that dog and human Cp activities behave in a qualitatively similar manner. For quantitative comparison, although constitutive Cp activities in dog (n=8) and human (n=6) plasma were similar (71±4 and 84±22 U/L, respectively), the inducible Cp activity was higher in canine serum (92±15 and 36±14 U/L).
In Vitro Clot Lysis Studies
125I-Fibrinogen–labeled clots were placed in citrated and buffered dog plasma in the presence or absence of PCI, and TPA was added. The presence of PCI accelerated clot lysis such that the time required for 50% lysis was reduced by an average of 27% (Fig 1⇓). In the absence of TPA, no measurable lysis (≤4%) occurred in 24 hours in the absence or presence of PCI. Thus, as in humans, the canine inducible Cp activity can suppress the rate of fibrinolysis in vitro.
Cp Activation In Vivo During Intracoronary Thrombosis and Thrombolytic Therapy
Electrical injury was used to induce thrombosis of the left circumflex artery in dogs, and blood samples were taken before thrombus formation (baseline), during thrombus formation, and during the thrombolytic therapy with TPA, which was initiated 30 minutes after stable thrombus formation. As shown in Fig 2A⇓, inducible Cp activity (inhibitable by PCI) was absent or very low in plasma before vessel injury, indicating that anesthesia and surgery alone did not induce Cp activity. Injury alone also did not induce Cp activity. However, with occlusive thrombus formation (30 to 120 minutes after injury), inducible Cp activity increased to 8.7±2.0 U/L versus a baseline value of 3.2±2.0 U/L (P<.013, n=7, paired t test) and remained elevated throughout the duration of the experiment (9.9±1.8 U/L; P<.024). In three dogs given TPA in the absence of vascular injury, baseline levels of inducible Cp were maintained (Fig 2B⇓) throughout the experiment (40 minutes). These values were significantly lower than in the thrombosed animals receiving TPA, with 1.5±2.4 U/L at 20 minutes and 1.1±2.4 U/L at 40 minutes after the start of TPA infusion, compared with dogs with a thrombus (7.5±1.5 U/L, P<.025 and 8.3±1.5 U/L, P<.019, respectively). These data suggest that vascular injury resulting in thrombus formation activates latent Cp in vivo.
Correlation Between Cp Activation and Thrombolysis
Because inducible Cp activity delays clot lysis in vitro, we attempted to correlate inducible Cp levels with the success of thrombolytic therapy. In the 19 dogs, the time required for reperfusion (restoration of at least 30% of baseline blood flow) ranged from 10 to 60 minutes, and the inducible Cp levels during thrombolytic therapy (measured 20 minutes after initiation of TPA infusion) ranged from 2 to 44 U/L. When pooled data from 19 dogs were analyzed, a significant (P<.007) positive linear correlation (r=.60) was found between inducible Cp levels and time to reperfusion (Fig 3⇓).
In this study, the role of the inducible Cp system in vascular fibrinolysis was investigated in a canine model of intracoronary thrombosis. Analyses of plasma samples from dogs undergoing thrombolytic therapy after intracoronary thrombosis suggest that Cp activity is induced in vivo and that high levels of this activity are associated with a prolonged time for restoration of blood flow by TPA administration. To the best of our knowledge, this is the first report that delineates the importance of inducible plasma Cp in fibrinolysis in vivo.
Canine blood, like human blood, contains latent Cp activity that is induced during coagulation.5 The inducible Cp activity was efficiently inhibited by PCI, as reported for the purified human enzymes.3 Thus, canine counterparts of the two enzymes are functionally similar. In line with this notion, we found that, as in human plasma, inhibition of inducible Cp by PCI accelerates TPA-induced clot lysis in vitro.3 Even though canine serum contains about three times more inducible Cp than human serum, the effect of PCI on clot lysis was greater in human plasma.3 Thus, the regulatory role of the Cp system in clot lysis may be even more pronounced in humans.
Analyses of dog plasma samples demonstrated a significant increase of inducible Cp over baseline after electrically induced thrombosis and thrombolytic therapy. These findings define conditions that result in Cp activation in vivo. In addition, the level of Cp is positively correlated with the time required for restoration of blood flow, consistent with the proposed role of inducible Cp in modulating fibrinolysis.3 4 Our data are consistent with the hypothesis that thrombin activation and thrombus formation play a significant role in Cp induction. In vitro studies7 suggest that plasmin, as well as thrombin, can activate inducible Cp. Although administration of TPA in the absence of thrombosis failed to trigger detectable Cp activation, lack of reliable assays to quantify plasmin activity in dog plasma did not allow us to exclude plasmin as an activator of Cp. Taken together, these analyses emphasize the potential role of Cp induction in modulating vascular fibrinolysis. Although it is not the exclusive mechanism for suppressing fibrinolysis,10 Cp may play an important physiological role. The possibility of inhibiting the Cp system to improve thrombolytic therapy is a logical extension of these observations.
This work was supported by the NIH (grant HL-17964) and by a grant (Dr Nicolini) and a Postdoctoral Fellowship (Dr Redlitz) from the American Heart Association, Northeast Ohio Affiliate. The authors thank Dr Kristopher L. Arheardt, Biostatistics Department of the Cleveland Clinic Foundation, for help with data analysis.
- Received November 28, 1995.
- Revision received January 31, 1996.
- Accepted January 31, 1996.
- Copyright © 1996 by American Heart Association
Pannell R, Black J, Gurewich V. Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained. J Clin Invest. 1988;81:853-859.
Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest. 1995;96:2534-2538.
Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem. 1995;270:14477-14484.
Wang W, Hendriks DF, Scharpé SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem. 1994;269:15937-15944.
Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem. 1991;266:21833-21838.
Nicolini FA, Lee P, Rios G, Kottke-Marchant K, Topol EJ. Combination of platelet fibrinogen receptor antagonist and direct thrombin inhibitor at low doses markedly improves thrombolysis. Circulation. 1994;89:1802-1809.
Stringer HA, van Swieten P, Heijnen HF, Sixma JJ, Pannekoek H. Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance: studies with thrombi generated in the Chandler loop. Arterioscler Thromb. 1994;14:1452-1458.