Brazilian Journal of Medical and Biological Research (2000) 33: 119-125 tPA clearance and inflammation 119 ISSN 0100-879X

The hepatic clearance of recombinant tissue-type decreases after an inflammatory stimulus

M.R. Nagaoka1,2, 1Laboratório de Hepatologia Experimental, and M. Kouyoumdjian1,2 Departamentos de 2Bioquímica and 3Medicina, and D.R. Borges1,3 Universidade Federal de São Paulo, São Paulo, SP, Brasil

Abstract

Correspondence We have shown that tissue-type plasminogen activator (tPA) and Key words D.R. Borges plasma share a common pathway for liver clearance and that · Liver clearance Laboratório de Hepatologia the hepatic clearance rate of plasma kallikrein increases during the · Inflammation Experimental, UNIFESP · acute-phase (AP) response. We now report the clearance of tPA from Kallikrein Rua Jabuticabeiras, 807 · the circulation and by the isolated, exsanguinated and in situ perfused 05674-011 São Paulo, SP · Tissue-type plasminogen Brasil rat liver during the AP response (48-h ex-turpentine treatment). For activator E-mail: [email protected] the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of Research supported by CNPq 125I-tPA from the circulation of turpentine-treated rats (2.2 ± 0.2 ml/ (No. 520281/96-9) and PRONEX min, N = 7) decreases significantly (P = 0.016) when compared to (No. 41.96.0873.00). Publication normal rats (3.2 ± 0.3 ml/min, N = 6). The AP response does not supported by FAPESP. modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of Received June 7, 1999 turpentine-treated rats (15.5 ± 1.3 µg/min, N = 4) was slower (P = Accepted November 4, 1999 0.003) than the clearance rate by the liver of normal rats (22.5 ± 0.7 µg/ min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 ± 2.4 µg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kal- likrein were not altered during the AP response. These results contri- bute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

Introduction proteolysis of fibrin by . Tissue-type plasminogen activator (tPA) is the physi- Activation of the blood sys- ological initiator of , converting tem under physiological or pathological con- plasminogen into plasmin via specific pro- ditions is responsible for the formation of teolysis. Because of its fibrin-selective ac- intravascular fibrin clots (1,2). Clot dissolu- tion, tPA has been successfully used for tion is a process essential for the mainte- thrombolytic therapy. Since the elimination nance of blood fluidity and is achieved by the of tPA from the circulation is relatively rapid

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(4-6 min in humans), high doses of tPA are Material and Methods required to obtain thrombolysis (3). The liver is the major site for tPA clearance from the Chemicals circulation and the two most important he- patic receptors for tPA clearance are the Recombinant human tPA (Actilyse®) pro- low-density-lipoprotein-receptor-related pro- vided by Boehringer Ingelheim (Mannheim, tein (LRP) on parenchymal cells and the Germany) was used for liver perfusion ex- mannose receptor on liver endothelial and periments; melanoma tPA from Calbiochem Kupffer cells. Other receptors marginally (San Diego, CA, USA) was iodinated and contribute to tPA clearance (4). used for in vivo experiments. Na125I was The liver response to injury occupies a obtained from Amersham (Buckingham, central position in the acute-phase (AP) re- UK). Rat plasma thrombin and porcine pan- sponse. The hepatic modulation of the kal- creatic kallikrein were purchased from Sigma likrein-kinin system is altered during the AP Chemical Co. (St. Louis, MO, USA). Acetyl- response to inflammation, with an increase phenylalanine--pnitroanilide (Ac- in hepatic synthesis of total kininogen (5), T- Phe-Arg-pNA) was synthesized by L. Juliano kininogen (6) and prokallikrein (7). The kal- (Department of Biophysics, UNIFESP, SP, likrein-kinin system is important in the patho- Brazil). The amidolytic substrates S2238 (H- genesis of the inflammatory reaction (8) and D-Phe-Pip-Arg-pNA) for thrombin and is linked to the fibrinolytic system since S2288 (H-D-Ile-Pro-Arg-pNA) for tPA were bradykinin is a potent stimulus of tPA secre- purchased from Chromogenix (Mölndal, tion (9). Sweden). It has been shown that plasma levels of tPA and the complex formed by tPA with Rats plasminogen-activator inhibitor type 1 (PAI- 1) are increased in patients with various liver Adult male Wistar rats weighing 150- diseases (10), perhaps because of impaired 265 g were used according to the Interna- tPA clearance. During the AP response the tional Guiding Principles for Biomedical clearance rate of plasma-kallikrein by the Research Involving Animals (16). perfused rat liver increases (11); in contrast, the liver uptake of an asialoglycoprotein de- Inflammatory stimulus creases during the AP response (12), sug- gesting that the mechanism of endocytosis To study the influence of the AP re- mediated by different lectins is distinctly sponse on the hepatic clearance of tPA, TH modified during the AP response. We have and PoPK, rats received 0.5 ml turpentine oil shown that tPA and plasma kallikrein share a subcutaneously at each of two sites on either common pathway for liver clearance (13). side of the abdomen 48 h before the experi- We now report the clearance of tPA from the ment. The AP response was confirmed by a circulation (in vivo) and by the isolated, the detection of serum 2macroglobulin a exsanguinated and perfused rat liver during ( 2M) by radial immunodiffusion (5). The the AP response. For the sake of compari- specific antibody was donated by A.H. Gor- son, we also studied the hepatic clearance don, National Institute for Medical Research, of porcine pancreatic kallikrein (PoPK), London. which is cleared through the mannose recep- To determine the participation of Kupffer tor (14), and thrombin (TH), which binds cells in the clearance of tPA by the liver of to liver cells but is minimally internalized turpentine-treated rats, GdCl3 dissolved in (15). 0.15 M NaCl was injected through the tail

Braz J Med Biol Res 33(1) 2000 tPA clearance and inflammation 121 vein at the dose of 10 mg/kg (17) 12 h after administration of 125I-tPA from the carotid turpentine oil injection. The experiments artery catheter into polyethylene tubes con- were conducted 36 h after GdCl3 injection. taining 0.4% sodium citrate. The aliquots were centrifuged at 5000 g for 10 min, and tPA labeling the plasma samples (150 µl) separated and precipitated with 10% trichloroacetic acid. tPA (100 µg) in 0.05 M sodium phos- The radioactivity of the acid precipitable g phate, pH 7.4, was iodinated with 125I (1 protein was determined with a -radiation mCi) using the chloramine T method (18). counter. Unincorporated iodine was removed by gel To analyze the tissue distribution of 125I- filtration on a PD-10 column and eluted with tPA, 60 min after its administration the ab- 50 mM sodium phosphate, pH 7.4. The domen and thorax were opened and liver, amidolytic activity upon S2288 and radioac- heart, lung, kidney and spleen were removed tivity of the aliquots were determined. A and weighed and their radioactivity was meas- preparation of 125I-tPA with a specific radio- ured. activity of 2.5 x 106 cpm/µg protein was used. Liver perfusion in situ

Amidolytic activity The livers were perfused at 37oC as de- scribed (20). Rats were anesthetized with The amidolytic activity of tPA, TH or urethane (1.3 mg/g) and kept alive by artifi- PoPK was assayed by incubating perfusate cial respiration. The livers were perfused aliquots (0.1 ml) at 37oC in a final volume through the portal vein (inflow cannula) and (0.2 ml) of 50 mM Tris-HCl, 12 mM NaCl, thoracic inferior vena cava (outflow can- pH 8.0, for tPA, 15 mM Tris-HCl, 20 mM nula) in an open circuit with 200 ml of EDTA, pH 7.4, for TH, and 50 mM Tris- Krebs-Henseleit bicarbonate solution, pH 7.4. HCl, 1 mM EDTA, pH 9.0, for PoPK, at a The circuit was then closed and the livers final concentration of 0.5 mM S2288, 0.5 were perfused with 30 ml of recirculating mM S2238 and 0.6 mM Ac-Phe-Arg-pNA, Krebs’ solution containing 1 mg/ml bovine respectively (19). The reaction was stopped serum albumin (BSA) for 10 min, at a con- with 0.8 ml of 15% acetic acid and the stant flow of 28 ml/min. The recirculating absorbance of p-nitroaniline was measured Krebs’solution was exchanged with the same at 405 nm. Duplicate assays were conducted volume of Krebs/BSA and a new equilibra- and values varied less than 5%. tion period of 10 min was allowed to elapse. The peristaltic pump was then turned off and Clearance of tPA from the circulation 500 µg tPA, 50 µg TH or 160 µg PoPK was added. After 1 min of mixing and collection Clearance of 125I-tPA from the circula- of aliquot “0”, the peristaltic pump was turned tion was determined in rats anesthetized with on and the perfusion reinitiated. Aliquots (1 urethane (1.3 mg/g). Tracheotomy was per- ml) of the perfusate were collected at 0, 5, 10 formed and catheters were positioned into and 20 min for tPA, 0, 1, 3, 5 and 10 min for the left jugular vein and right carotid artery. TH, and 0, 10, 20 and 30 min for PoPK 125I-tPA (2 x 106 cpm, 0.7 µg) plus 500 µg of experiments. unlabeled tPA was injected intravenously in The percentage (aliquot “0” taken as bolus and flushed with 0.5 ml of 0.15 M 100%) of the residual amidolytic activity in NaCl solution. Blood samples were collected the perfusate aliquots was used to calculate at 2, 5, 10, 20, 30, 40, 50 and 60 min after the half-life of disappearance (t½) and the

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clearance rate of the proteases (with the aid perfused with China ink (21); histological of the GraphPAD Prism™ software, version examination showed that Kupffer cells from 1.03). The results are reported as mean ± normal rats but not those from GdCl3-treated SEM and significant differences were deter- rats were blackish. mined using the Primer computer software To assure that the labeling process did version 3.02 (McGraw-Hill, Inc., New York, not impair tPA clearance by the liver, we NY, USA). perfused the organ with 125I-tPA (2 x 106 To exclude possible interference with the cpm) plus unlabeled tPA (500 µg) and veri- amidolytic activities of tPA, TH or PoPK by fied that 125I-tPA was cleared (27.4 ± 5.2 µg/ substances liberated from the perfused liver min, N = 5) by the isolated, exsanguinated into the perfusion medium, we incubated the and perfused rat liver at the same rate (P = perfusion medium obtained after 30 min of 0.422) as unlabeled tPA (27.4 ± 5.2 µg/min, perfusion (without any protease added) with N = 5). the . In anhepatic experiments, i.e., a system tPA clearance from the circulation without liver but with the other perfusion procedures maintained, tPA, TH or PoPK The clearance rates and half-lives of 125I- were recirculated for 15-30 min and the tPA were calculated from the plasma elimi- amidolytic activities assayed. nation curves for in bolus injection of the activator in normal or turpentine-treated rats. Results The plasma elimination rate of tPA in tur- pentine-treated rats (2.2 ± 0.2 ml/min, N = 7) Control experiments decreased significantly (P = 0.016) com- pared to normal rats (3.2 ± 0.3 ml/min, N = The in vitro assays showed that the liver 6). The tissue distribution of 125I-tPA is shown perfusate neither inhibited nor increased the in Figure 1. It can be seen that the AP re- activity of the enzymes studied (tPA, TH or sponse did not modify the tissue distribution PoPK). In anhepatic experiments we ob- of the activator and that the liver was the served that tPA, TH or PoPK were not inac- major site for the efficient removal of 125I- tivated after 15-30 min of recirculation. tPA from the circulation in both normal and The AP response was confirmed by the turpentine-treated rats. a detection of 2M in the serum of turpentine- a and/or GdCl3-treated rats; 2M was not de- Hepatic clearance tected in the serum of normal rats. To determine that Kupffer cells were ab- Table 1 shows that the inflammatory lated by the GdCl3 treatment, at the end of stimulus also influenced the clearance of the perfusion experiments the livers were tPA by the isolated liver. The clearance rate Figure 1 - Organ distribution of of tPA by the perfused liver from injured rats 125I-tPA 60 min after administra- 100 Normal (N = 6) (i.e., treated with turpentine or turpentine- tion to normal and turpentine- Turpentine-treated (N = 7) GdCl3) was slower than observed in normal treated rats. 75 animals. 50 On the other hand, the inflammatory stimulus had no effect on the clearance rate Radioactivity (%) 25 of PoPK or TH by the perfused rat liver. The clearance rate of PoPK (160 µg) (Figure 2A) 0 Liver Heart Lung Kidney Spleen by the perfused liver of normal rats (2.4 ± 0.1 µg/min, N = 3) was similar (P = 0.830) to that

Braz J Med Biol Res 33(1) 2000 tPA clearance and inflammation 123 of turpentine-treated rats (2.3 ± 0.3 µg/min, tory stimulus. We assured that labeling of N = 3). Thrombin (50 µg) was efficiently tPA with 125I does not impair its clearance by removed from the circulation by the liver the liver, a phenomenon described for plasma (Figure 2B) of both normal (34.8 ± 2.9 µg/ kallikrein (18). min) and turpentine-treated rats (39.1 ± 2.5 In normal rats, human tPA and rat plasma µg/min) (P = 0.321). kallikrein share some characteristics in their clearance by the liver and a molar excess of Discussion tPA inhibits the hepatic clearance of rat plasma kallikrein (13), suggesting a com- Receptor-mediated endocytosis is a mech- mon pathway inside the organ. After an in- anism that transports macromolecules into the cell following a series of intracellular Table 1 - Clearance of tPA by the isolated and exsanguinated liver of normal, turpen- tine- and/or GdCl -treated rats. transfers through distinct environments. This 3 process is important for the regulation of the ANOVA, P = 0.003; Bonferroni t-test: a>b; a>d; a = c; b = c; b = d; c = d. plasma concentration of many glycoproteins, a b c None Turpentine GdCl3 Turpentine and several types of lectins are involved in d and GdCl3 the initial step (binding) of the internaliza- tion process, which can be modified by patho- AP response - ++ + ++ a logical situations (22). (serum 2M) N10445 When complexed with an inhibitor, Clearance (µg/min) 22.5 ± 0.7 15.5 ± 1.3 17.5 ± 1.4 16.2 ± 2.4 thrombin is cleared from the circulation by hepatocytes via receptor-mediated endocy- tosis; on the contrary, free TH binds to pa- Figure 2 - Clearance of pancreatic renchymal cells but is minimally internal- A 160 kallikrein (A) and thrombin (B) by ized (15). We now report that the uptake of the isolated and exsanguinated liver of normal or turpentine- 145 free TH by the liver of turpentine-treated rats treated rats. is not altered, suggesting that the AP re- 130 sponse does not modify the binding of TH to hepatic cells. The hepatic endocytosis of 115 pancreatic kallikrein is mediated by a specif- kallikrein (µg) Residual pancreatic 100 ic mannose receptor (14) found mainly in 0 5 10 15 20 25 liver endothelial and Kupffer cells. In this Perfusion time (min) study we show that the PoPK clearance by Normal (N = 3) the liver of turpentine-treated rats is not al- Turpentine-treated (N = 3) tered. B It is known that after intravenous infu- 50 sion of tPA three molecular forms may be 40 found in plasma: two active forms (free tPA a and the tPA- 2M complex) and an inactive 30 form (tPA-plasminogen activator inhibitor 1 20 (PAI-1) complex) (23). Our results showed 10 that tPA clearance from plasma decreases in Residual thrombin (µg) turpentine-treated rats and that the AP re- 0 012345 sponse does not modify the in vivo tissue Perfusion time (min) distribution of 125I-tPA, with the liver being Normal (N = 3) 125 responsible for most of the I-tPA cleared Turpentine-treated (N = 3) from the circulation even after an inflamma-

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flammatory stimulus the hepatic clearance ance rate did not change, in agreement with rate of rat plasma kallikrein increased (11), the in vivo experiments reported by Narita et in contrast to the tPA behavior now de- al. (4). scribed. Different behavior during endocy- In the present liver perfusion experiments, tosis of proteins that share a common plasma we used an amount of tPA comparable to the membrane receptor has already been de- dose therapeutically administered to humans scribed: epidermal growth factor and trans- (26). Only when the in vivo administered forming growth factor-a compete for the tPA overcomes the inhibition by PAI-1 in same receptor with different affinities, which plasma will the (free) be therapeuti- could be a consequence of differences in the cally active. It is also known that the throm- mechanisms of intracellular processing (24). bolytic efficacy of tPA is not correlated with The results reported here suggest that the dose administered (26). The result re- receptor-mediated endocytosis is not uni- ported here showing that free tPA clearance formly affected by an AP situation. After an by the liver is compromised during an AP inflammatory stimulus, proteins such as in- situation contributes to the understanding of terleukin-6 and transforming growth factor- this fact. ß synthesized by activated Kupffer cells elicit the AP response in hepatocytes and stellate Acknowledgments cells (25). The clearance rate of tPA by the liver of turpentine-treated rats was mini- We thank Miss Hercilia M. Molina for mally influenced by Kupffer cells since after technical assistance. their ablation by GdCl3 treatment the clear-

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