THROMBOSIS RESEARCH 63; 299-309,199l 0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
PROTHROMBIN PLASMA CLEARANCE IS NOT MEDIATED BY HEPATIC ASIALOGLYCOPROTEIN RECEPTORS. Jaroslav G. Vostal*and Roy B. McCauley** *Clinical Hematology Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, U.S.A. **Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
(Received
28.2.1991;
accepted
in revised form 2.51991
by Editor C.W. Francis)
ABSTRACT
The hepatic asialoglycoprotein receptor (AGPR) system can glycoefficiently internalize and degrade circulating sialic acids on their which lack terminal proteins Since pro-thrombin is a glycosylated carbohydrate chains. plasma protein, possible involvement of AGPR system in its The half lives clearance from circulation was evaluated. and 1251-asialoprothrombin, of bovine 1251-prothrombin injected intravenously into rats, were 192 and 1.8 minutes, Asialoprothrombin appeared to be cleared by respectively. the hepatic AGPRs since 33% of it accumulated in the liver at 30 minutes and its clearance was competitively blocked of increasing amounts of by simultaneous administration Only 5% of prothrombin accumulated in the asialofetuin. liver at 3 hours and injections asialofetuin in amounts capable of saturating the AGPR for the duration of four asialoprothrombin half lives had no effect on the disappearance of prothrombin. Our observations indicate is readily cleared from that, although asialoprothrombin plasma by the AGPR system, prothrombin is not. Thus these receptors do not appear to be involved in physiological processes that control prothrornbin half life. INTRODUCTION The hepatic asialoglycoprotein receptor (AGPR) mediates plasma clearance of injected glycoproteins which lack terminal sialic acid residues in their carbohydrate chains. Glycoprotein binding AGPR results in receptosome-mediated by the hepatic internalization followed by lysosomal degradation(l,2,3). The efficiency of this uptake mechanism, and the almost ubiquitous presence of terminal sialic acid residues in carbohydrate chains of plasma glycoproteins, has led to the idea that in vivq half life, asialoprothrombin, Key words: prothrombin glycoprotein receptors, glycoprotein turnover, 299
asialo-
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generation of the asialoderivative may be a requirement for clearance of serum glycoproteins(l,3). Clinical support for this theory came from the observation that patients with extensive hepatic disease have increased levels of plasma asialoglycoproteins(4,5). However, other investigators(6,7) have shown that although administration of large amounts of asialoglycoproteins will reduce clearance of other asialoglycoproteins by competition for AGPRs, removal of the corresponding native glycoproteins under such conditions is not affected. Furthermore, no suitable desialating enzyme (neuraminidase), which would be necessary to produce the asialo derivative, has been found in plasma(8). These observations cast a doubt on the AGPR system as a major clearance mechanism of plasma glycoproteins. Prothrombin is a glycosylated plasma clotting protein which contains nine terminal sialic acid residues on three carbohydrate chains (9). Although the mechanism of its plasma clearance is not yet understood, its asialoderivative is cleared from circulation faster than prothrombin(l0). A role for the AGPR system in the degradation of prothrombin was suggested, but not substantiated by showing that blocking the AGPR system prolonged the half life of prothrombin (10). We have compared the clearance of bovine prothrombin and asialoprothrombin in rat circulation under normal conditions and under those which fully block the AGPR with exogenous asialoglycoproteins. When the AGPR system was blocked, only the h?lf life of asialoprothrombin was increased while the half life'of prothrombin remained the same. Our data thus indicate that the AGPR system does not significantly participate in the clearance of native prothrombin.
Prothrombin Isolation Prothrombin was obtained from titrated bovine plasma by a scaled down method of Esnouf et al. (11) and further purified by HPLC gel filtration using tandem I-300 protein columns (Waters Associates, Milford, MA). The resultant protein had a specific coagulant activity of 1200 units/mg protein as measured by a one stage assay(l4) and moved as a single line on SDS-PAGE with an apparent molecular weight of 72 kDa. Purified prothrombin was lyophylized and stored in powder form at -200 C. Preparation of asialoprothrombin Two mg of prothrombin were incubated with 0.3 units of agarose bound neuraminidase (Cl. perfringens, type IV,Sigma, St.Louis. MO) in 15 mM Tris buffer (pH 7.5) and a final volume of 2 ml. The mixture was stirred at 37OC for 60 minutes. Asialoprothrombin was then separated from insoluble neuraminidase by centrifugation and separated from free sialic acid by HPLC gel filtration. Extent of sialic acid removal was assessed by the method of Warren et al. (13) and also by the modified thiobarbituric DMSO method of Skoza and Mohos(l4). Sialic acid standards were treated in an identical manner as the protein unknowns. Usually the neuraminidase removed 85% of the prothrombin sialic acid. Asialofetuin, which was derived from bovine fetuin (Sigma) by using the same methods was 90 % free of sialic acid.
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Iodination of prothrombin derivatives and albumin Iodination of prothrombin and its asialoderivatives was performed and low concentration of hydrogen using lactoperoxidase peroxide (15). Bovine serum albumin (Sigma, type V) which was used to determine extent of residual blood in excised organs was iodinated by using Iodogen(Pierce) (16). The labelled prothrombin, asialoprothrombin and albumin were separated from free iodine by HPLC gel filtration and the proteins were subjected to SDSPAGE(1-I). Resultant gels were stained with Commassie Blue, dried and autoradiographed. In each case, radioactivity coincided with the stained protein. Iodinated prothrombin and asialoprothrombin had the same specific coagulant activity as their native prothrombin counterpart and both proteins had approximate specific radioactivity of 0.15 uCi/ug. Estimates of circulation clearance of iodinated prothrombin and asialoprothrombin Male Wistar rats (350-45Og) were anesthetized with 50 mg/kg phenobarbital and cannulated with polypropylene catheters through the femoral vein and the opposite femoral artery. Fifteen minutes before the start of the experiment animals were injected with 1000 units of heparin dissolved in saline. Fifty microliters of blood was then drawn from the arterial side and replaced on the venous side by injection of the radioactive protein (20 ug in 50 ul). At each time point, 150 ul of blood was taken from the artery and replaced with saline on the venous side. One hundred microliters of collected blood were added to an equal volume of 10% KI (18) and this mixture was then precipitated with 200 microliters of 10% TCA. Two ml of ice cold 5% TCA were then added to the test tube which was vortexed and centrifuged. The supernate was removed and additional 2 ml of ice cold 5% TCA were added to the pellet which was resuspended and again centrifuged. Finally, the radioactivity in the pellet and the combined supernates was counted. Tissue radioactivity determination At the end of the experiment animals were sacrificed by opening the chest cavity. Tissue samples were removed, rinsed in ice cold saline, blotted dry and their weight determined before radioactivity was estimated on smaller fragments of the organs. Reported tissue values are expressed as percent of the total radioactivity recovered in six organs plus that present in blood (the total recovery of radioactivity ranged in 50-75% of the injected amount). Tissue radioactivity was corrected for residual blood as determined in separate experiments by injecting 1251bovine albumin into circulation and sacrificing the animal 5 minutes later. The organs were excised, sampled for radioactivity as above, and the amount of residual blood in each tissue calculated. The circulation half life of 1251-bovine albumin estimated in separate experiments was 10.5 hours. Data analysis The natural log of the fractional amount of radiolabelled prothrombin initially recovered in blood was graphed versus the time of collection. Plasma survival curves for 1251-asialoprothrombin and 1251-prothrombin were analyzed with computer assisted curve stripping methods by fitting the data to a two exponential term equation(19). The log linear part of the curve was extrapolated to time zero and subtracted from the original curve to obtain the fast phase of the clearance. The half disappearance
301
302
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times(tl/z) were calculated from slopes of lines generated linear regression of the resultant points(tl/z=ln2/slope).
by
EESUJlTS As shown in Figure 1, clearance of 12sI-bovine prothrombin (TCA precipitated radioactivity) from rat circulation was a biphasic process. In the initial phase(solid circles), prothrombin disappeared w‘ith a half life of 39.5 minutes. The second phase(open circles), which remained constant for at least 6 hours, had a half life of 202.9 minutes. As 1251-prothrombin disappeared from blood, its degradation products (TCA soluble radioactivity) in blood(open squares) remained constant after reaching 5% of the original injected radioactivity.
i I -60
-60
tlj2a202.9 minutes
-40
111249.5minutes
d
3b
6b
9-O tlme
li0
150
1eo
(minutes)
Figure 1. Clearance of 1251-bovine prothrombin from rat circulation. The natural log (left axis) of the fraction of remaining TCA percipitable radioactivity (open circles) is graphed versus time. The log linear phase represents blood clearance of prothrombin. When extrapolated to zero time and curve, the half life for subtracted from the original equilibration of prothrombin can be determined (dark TCA soluble radioactivity (right axis, open circles). squares) is expressed as percent of total initially observed radioactivity and represents degradation products generated during clearance of prothrombin. Points are means of seven experiments. S.D. for each point did not exceed ten percent of the mean value. Asialoprothrombin also had a two phase clearance curve (Figure 2). The first phase (solid circles) had a half life of 1.8 minutes was during which approximately 80% of injected radioactivity
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cleared, while the second phase had a half life of 56.8 minutes. the TCA soluble injection, minutes after Ten to twenty radioactivity associated with asialoprothrombin (open squares) in rose sharply, and by 30 minutes, most of the radioactivity blood was TCA soluble. 100
p 'I I .60 .
4
\ \ .60
-
i
t1/2=1.8
t112356.8
minutes
c-
minutes .)
.’
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-20
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0 0
30
10
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(mhEtes)
Figure 2. Clearance of 1251-bovine asialoprothrombin from rat Clearance of asialoprothrombin (closed circles) circulation. was obtained by subtracting the extrapolated log linear phase TCA soluble (open circles). from the original curve open squares) represents (right axis, radioactivity degradation products generated during the clearance of Radioactivity is expressed as percent of asialoprothrombin. total initially observed radioactivity and points are means of five experiments. S.D. for each point did not exceed ten percent of the mean value.
Tissue distribution of the injected prothrombins at the end of the experiments was measured in heart, lung, liver, kidney, spleen and Asialoprothrombin distribution at 30 minutes(Figure 3) diaphragm. was 35% in liver, about 4% in kidneys and spleen and only minimal amounts in other organs. Prothrombin distribution at 3 hours indicated that liver was the major organ of uptake but the uptake was only 5% of the recovered radioactivity since most of the prothrombin remained in circulation(Figure 1). Other organs contained only minimal amounts of radioactivity. Results of injecting increasing amounts of asialofetuin simultaneously with labelled asialoprothrombin are shown in Figure 4A and 4B. Doses of 200, 400 and 600 ug asialofetuin/lOO g body weight caused a progressive increase in the half life for asialoprothrombin from 1.8 minutes in controls to 10, 20 and greater than 30 minutes, respectively. The release of TCA soluble radioactivity (Figure 4B) was correspondingly delayed.
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I
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? ?asialoprothrombin
30
20
10
0
heart
lung
liver
kidney
spleen diaphragm
Figure 3. Tissue distribution of radioactivity from l%prothrombin at 3 hours (dark bars) and 1251-asialoprothrombin at 30 minutes (hatched bars). Bars represent percent of radioactivity found in whole tissues which was corrected for residual blood (Methods) and expressed as percent of total recovered radioactivity. Results are means 2 S.D. of three experiments.
4A
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Figure 4. The effects of increasing amounts of injected asialo-fetuin on clearance of 1251-asialoprothrombin. Asialoprothrombin was injected into rat circulation with varied amounts of asialofetuin ( 0 0, L 200, D 400, x 600 ug/lOO kg), with blood collected and TCA precipitated at each (A) TCA precipitable and (B) TCA soluble time point. are expressed as percent of the initial radioactivity Control points are the means of 5 observed radioactivity. animals per curve while experimental points are means of two animals per curve.
To test whether prothrombin clearance was affected by saturating the AGPRs we used two conditions to administer asialofetuin (600 One was a single injection to saturate ug/lOOg) intravenously. the receptors for at least 30 minutes (as judged by experiments in Figure 4) and the other was a series of injections every twenty minutes to assure saturation of the AGPRs for the duration of a 3 In either case, the half life of 1251-prothrombin hour experiment. and the release of TCA soluble radioactivity were unchanged in the presence of asialofetuin (Figure 5).
FIG. 5
0 0
30
60
90
time
120
(minutes)
150
160
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Figure 5. Clearance of 125 I-prothrombin alone compared to its clearance when the AGPR system is saturated. Asialofetuin (600 ug/lOO kg) was given simultaneously as a single dose (a ) or as repeated doses injected every twenty minutes (A). TCA precipitable radioactivity (closed symbols, right axis) TCA soluble radioactivity (open symbols, left axis). Points are means + S.D. of three experiments per curve.
DISCUSSION The AGPR receptor mediated hepatic uptake system has been postulated to be the final degradation step of normal plasma glycoprotein clearance(l,3). However, attempts to directly demonstrate the participation of this receptor in normal clearance of plasma glycoproteins have not been sucessful(5,6). Thus the physiological relevance of this uptake system remains obscure. Since the prothrombin molecule is rich in terminal sialic acid residues and has a relatively fast turnover rate, it is suitable for evaluating the importance of AGPRs in glycoprotein clearance. In coumadin treated rats the half life of prothrombin has been reported to be 5.6 hours (20) to 6 hours (21) based on disappearance of clotting activity. One study (22) of iodinated rat prothrombin clearance in rats gave a half life of 10.6 hours but this value was based on extrapolation of points representing less than 10% of the original injected radioactivity and thus may not represent true prothrombin half life. Examination of the reported blood clearance curves reveals that for approximately 75% of the injected radioactivity the half life was 4-5 hours. These variations in rat prothrombin half life may be due to difference in techniques and our value of 3.2 hours for half life of bovine prothrombin in rat circulation appears to be in the physiologic range. The half life of bovine asialoprothrombin in our experiments was 1.8 minutes and compares favorably with reported half lives of asialoderivatives from other glycoproteins such as fetuin (1.5 minutes)(23) and orosomucoid (1.3 minutes) (7) in rat circulation. The slower second phase of asialoprothrombin clearance(half life of 56.7 minutes), during which the last 20% of the radiolabelled ligand was removed, most likely represents the distribution of partially or fully sialated prothrombin, since about 15% of the sialic acid was not removed by the neuraminidase treatment. An earlier report(lO) showed that clotting activity due to bovine asialoprothrombin injected into prothrombin deficient rats, disappeared faster than clotting activity due to injected bovine prothrombin. However, it was not clear if the AGPR system was responsible for clearing the asialoglycoprotein or if other factors such as changes in susceptibility of this derivative to plasma proteases could have also accounted for its accelerated In our experiments, clearance of decrease in clotting activity. 12sI-asialoprothrombin was competitively blocked by asialofetuin, a with rat AGPRs (24, 25). known to interact glycoprotein progressively delayed the Increasing amounts of asialofetuin clearance of 1251-asialoprothrombin and the highest dose completely
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blocked the fast phase of the clearance for 30 minutes. When removal of 1251-asialoprothrombin by the AGPR system was blocked by asialofetuin, asialoprothrombin remained in circulation undigested. These observations strongly suggest an interaction of asialoprothrombin with the AGPRs and implicate the liver as the site of its uptake and processing. It has been shown that injected bovine or rat asialoorosomucoid are cleared with minimal identical rates from suggesting rat circulation, recognition of asialointerspecies difference in the It is therefore likely that clearance of bovine derivatives(7). asialoprothrombin in our experiments represents the fate of rat asialoprothrombin. In contrast to the effects of asialofetuin on asialoprothrombin, doses of asialofetuin sufficient to saturate the AGPR for four asialoprothrombin half lives did not alter the course of prothrombin clearance. If AGPRs contributed to the disappearance of prothrombin from circulation then a blockade of this system should have prolonged the apparent prothrombin half life. Failure to demonstrate this argues against the necessity of generating an asialoderivative in plasma clearance of prothrombin. Others have the AGPR system with exogenous also found that saturating asialoglycoproteins did not effect the half lives of injected (6,7). Since clearances of native orosomucoid or fetuin prothrombin, orosomucoid and fetuin do not depend on the function of the AGPR system, this mechanism does not appear to be a major factor in the general turnover of plasma glycoproteins. The mechanism and site of prothrombin clearance from circulation While some activated can only be speculated on at present. clotting factors appear to be cleared by initial conversion to an activated state(26), experimental evidence indicates that conversion of prothrombin to thrombin is not a significant factor in prothrombin turnover(27). There may be a receptor mechanism for localizing and digesting prothrombin on surface of hepatocytes since prothrombin fragments generated by an extracellular hepatocyte protease have been shown to influence the synthesis of prothrombin by isolated hepatocytes(28). Similarly, injection of such prothrombin fragments into rabbit circulation has also been associated with increased prothrombin synthesis(29).
ACNOWLEDGEMENTS The authors would like to thank Dr. N.R. Shulman for helpful discussions and to Dr. B. Marks for continuing encouragement.
1 Ashwell, G & Hartfort, J. Carbohydrate specific receptors in the liver. Ann. Rev. Biochem. 51, 531-34, 1984. 2 Schwartz, A. L. The hepatic asialoglycoprotein Critical Rev. Biochem. 16, 207-33, 1983.
receptor.
CRC
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3 Ashwell, G. & Steer, C. J. Hepatic recognition
and catabolism of serum glycoproteins. J. Am. Med. Assoc. 246, 2358-64,1981.
4 Marshall, J. S., Green, A. M., Pensky, J., Williams, S,., Zinn, A. & Carlson, D. M. Measurement of circulating desialated glycoproteins and correlation with hepatocellular damage. J. Clin. Invest. 54, 555-62, 1974.
5 Sobue, G. & Kosaka, A. Asialoglycoproteins in a case of primary hepatic cancer. Hepato-Gastroenterology27, 200-203, 1980. 6 Wong, K. L., Charlwood, P. 0. A., Hatton, M. W. C & Regoeczi, E. Studies of the metabolism of asialotransferrins: evidence that transferin does not undergo desialization in vivo. Clin. Sci. Mol. Med
46, 763-774,1974.
7 Clarenburg, R. Asialoreceptor is uninvolved in clearing of intact glycoproteins from rat blood. Am.J.Physiol. 224, 6247-6253, 1983. 8 Conzelmann, E. & Sandhoff, K. Glycolipid degradation. Adv. Enzymol. 60:89-216, 1987
and
glycoprotein
9 Mizouchi, T., Yamashita, K., Fujikawa, K., Kisiel, W., & Kobata, A. The carbohydrate of bovine prothrombin J. Biol. Chem. 254, 6419-6425, 1979. 10 Nelsestuen, G. L., & Suttie, J.W. The properties of asialo and aglyco prothrombin. Biochem. Biophys. Res. Comm. 45, 198-203,
1971. 11 Esnouf, P. M., Lloyd, P. H., & Jesty, J. A method for the simultaneous isolation of factor X and prothrombin from bovine plasma. Biochem. J. 131, 781-789, 1973.
12 Hjort, P., Rappaport, S. I. & Owren, P. A. A simple specific one stage prothrombin assay using Rusell's viper venom on cephalin susupension. J. Lab. Clin. Med. 46, 89-97,1955. 13 Warren, L. The thiobarbituric Biol. Chem. 231, 1971-1975, 1959.
acid assay of sialic acids.
J.
14 Skoza, L. & Mohos, S. Stable thiobarbituric acid chromophobe with dimethyl sulfoxide. Biochem. J. 159, 457-462, 1976. A highly stable 15 David, G. S. Solid state lactoperoxidase: of proteins. Biochem. enzyme for simple, gentle iodination Biophys. Res. Comm. 48, 464-71, 1972. 16 Fraker, P. J., & Speck, J. C. Protein and cell ;e;b;a;e chloramide, soluble with sparingly iodinations Ga-diphenyl-glycouril. Biochem. Bi0phyL.I Rk. tetrachloro-3a,
Comm. 80, 849-57, 1978. 17 Laemmli, U. K., Cleavage of structural proteins during assembly of the head of bacteriophage t4. Nature 222, 680-687, 1970.
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18 Chisolm, G. M., Sil, S.A. C Hmiel, S. P. Measurement of the degradation products of radioiodinated proteins. Anal. Chem. 111, 212-219,1981. 19 Mitopoulos, K. A. & Esnouf, M. P. Turnover of factor X and of prothrombin fed on a standard or cholesterol-supplemented diet. Biochem. J. 244, 263-269, 1987. 20 van Oosterom,
A.T. Mattie, H., Hermans, W.T. & Veltkamp, J.J. The influence of the thyroid function on the metabolic rate of prothrombin, factor VII, factor X in the rat. Thromb. Haemostas. 35, 607-619,1976. 21 Bell, R. G. 6 Matschiner, J. T. Synthesis and destruction of prothrombin in the rat. Arch. Biochem. Biophys. 135, 152-59, 1969. 22 Koj, A., Regoeczi, E., Chindemi, P. A. & Gauldie, J. Synthesis and turnover of prothrombin during experimental inflammation in rats. Br. J. Exp. Path. 65, 691-700, 1984. 23 Regoeczi, E., DeBanne, M. T., Hatton, M. W. C. & Koj, A. Elimination of asialofetuin and asialoorosomucoid by the intact rat. Biochim. Biophys. Acta 541, 372-384, 1978. 24 LaBadie, J. H., Chapman, K. & Arowson, N. N. Glycoprotein catabolism in the liver. Biochem. J. 151, 271-279, 1975. 25 Tolleshaug, H., Berg, T. Nilsson, M. & Norm, K. R. Uptake and labeled asialofetuin by isolated rat degradation of I251 hepatocytes. Biochem. Biophys. Acta 499:73-84, 1975. 26 Fuchs, H. E., Trapp, H. G., Griffith, M. J., Roberts, H. R. & Pizzo,S. V. Regulation of factor IX in vitro in the mouse. J. Clin. Invest. 73, 1696-1703, 1984. 28 Takeda, Y. Studies of the effects of heparin, coumadin and vitamin K on prothrombin metabolism and distribution in calves with the use of iodine-125-prothrombin. J. Lab. Clin. Med. 75, 355-377, 1970. 29 Graves, C. B., Munns, T. W., Carlisle, T. L., Grant, G. A.& Strauss, A. W. Induction of prothrombin synthesis by prothrombin fragments. Proc. Natl. Acad. Sci. 78, 4772-4776, 1981.
PROTHROMBIN PLASMA CLEARANCE IS NOT MEDIATED BY HEPATIC ASIALOGLYCOPROTEIN RECEPTORS. Jaroslav G. Vostal*and Roy B. McCauley** *Clinical Hematology Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, U.S.A. **Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
(Received
28.2.1991;
accepted
in revised form 2.51991
by Editor C.W. Francis)
ABSTRACT
The hepatic asialoglycoprotein receptor (AGPR) system can glycoefficiently internalize and degrade circulating sialic acids on their which lack terminal proteins Since pro-thrombin is a glycosylated carbohydrate chains. plasma protein, possible involvement of AGPR system in its The half lives clearance from circulation was evaluated. and 1251-asialoprothrombin, of bovine 1251-prothrombin injected intravenously into rats, were 192 and 1.8 minutes, Asialoprothrombin appeared to be cleared by respectively. the hepatic AGPRs since 33% of it accumulated in the liver at 30 minutes and its clearance was competitively blocked of increasing amounts of by simultaneous administration Only 5% of prothrombin accumulated in the asialofetuin. liver at 3 hours and injections asialofetuin in amounts capable of saturating the AGPR for the duration of four asialoprothrombin half lives had no effect on the disappearance of prothrombin. Our observations indicate is readily cleared from that, although asialoprothrombin plasma by the AGPR system, prothrombin is not. Thus these receptors do not appear to be involved in physiological processes that control prothrornbin half life. INTRODUCTION The hepatic asialoglycoprotein receptor (AGPR) mediates plasma clearance of injected glycoproteins which lack terminal sialic acid residues in their carbohydrate chains. Glycoprotein binding AGPR results in receptosome-mediated by the hepatic internalization followed by lysosomal degradation(l,2,3). The efficiency of this uptake mechanism, and the almost ubiquitous presence of terminal sialic acid residues in carbohydrate chains of plasma glycoproteins, has led to the idea that in vivq half life, asialoprothrombin, Key words: prothrombin glycoprotein receptors, glycoprotein turnover, 299
asialo-
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generation of the asialoderivative may be a requirement for clearance of serum glycoproteins(l,3). Clinical support for this theory came from the observation that patients with extensive hepatic disease have increased levels of plasma asialoglycoproteins(4,5). However, other investigators(6,7) have shown that although administration of large amounts of asialoglycoproteins will reduce clearance of other asialoglycoproteins by competition for AGPRs, removal of the corresponding native glycoproteins under such conditions is not affected. Furthermore, no suitable desialating enzyme (neuraminidase), which would be necessary to produce the asialo derivative, has been found in plasma(8). These observations cast a doubt on the AGPR system as a major clearance mechanism of plasma glycoproteins. Prothrombin is a glycosylated plasma clotting protein which contains nine terminal sialic acid residues on three carbohydrate chains (9). Although the mechanism of its plasma clearance is not yet understood, its asialoderivative is cleared from circulation faster than prothrombin(l0). A role for the AGPR system in the degradation of prothrombin was suggested, but not substantiated by showing that blocking the AGPR system prolonged the half life of prothrombin (10). We have compared the clearance of bovine prothrombin and asialoprothrombin in rat circulation under normal conditions and under those which fully block the AGPR with exogenous asialoglycoproteins. When the AGPR system was blocked, only the h?lf life of asialoprothrombin was increased while the half life'of prothrombin remained the same. Our data thus indicate that the AGPR system does not significantly participate in the clearance of native prothrombin.
Prothrombin Isolation Prothrombin was obtained from titrated bovine plasma by a scaled down method of Esnouf et al. (11) and further purified by HPLC gel filtration using tandem I-300 protein columns (Waters Associates, Milford, MA). The resultant protein had a specific coagulant activity of 1200 units/mg protein as measured by a one stage assay(l4) and moved as a single line on SDS-PAGE with an apparent molecular weight of 72 kDa. Purified prothrombin was lyophylized and stored in powder form at -200 C. Preparation of asialoprothrombin Two mg of prothrombin were incubated with 0.3 units of agarose bound neuraminidase (Cl. perfringens, type IV,Sigma, St.Louis. MO) in 15 mM Tris buffer (pH 7.5) and a final volume of 2 ml. The mixture was stirred at 37OC for 60 minutes. Asialoprothrombin was then separated from insoluble neuraminidase by centrifugation and separated from free sialic acid by HPLC gel filtration. Extent of sialic acid removal was assessed by the method of Warren et al. (13) and also by the modified thiobarbituric DMSO method of Skoza and Mohos(l4). Sialic acid standards were treated in an identical manner as the protein unknowns. Usually the neuraminidase removed 85% of the prothrombin sialic acid. Asialofetuin, which was derived from bovine fetuin (Sigma) by using the same methods was 90 % free of sialic acid.
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Iodination of prothrombin derivatives and albumin Iodination of prothrombin and its asialoderivatives was performed and low concentration of hydrogen using lactoperoxidase peroxide (15). Bovine serum albumin (Sigma, type V) which was used to determine extent of residual blood in excised organs was iodinated by using Iodogen(Pierce) (16). The labelled prothrombin, asialoprothrombin and albumin were separated from free iodine by HPLC gel filtration and the proteins were subjected to SDSPAGE(1-I). Resultant gels were stained with Commassie Blue, dried and autoradiographed. In each case, radioactivity coincided with the stained protein. Iodinated prothrombin and asialoprothrombin had the same specific coagulant activity as their native prothrombin counterpart and both proteins had approximate specific radioactivity of 0.15 uCi/ug. Estimates of circulation clearance of iodinated prothrombin and asialoprothrombin Male Wistar rats (350-45Og) were anesthetized with 50 mg/kg phenobarbital and cannulated with polypropylene catheters through the femoral vein and the opposite femoral artery. Fifteen minutes before the start of the experiment animals were injected with 1000 units of heparin dissolved in saline. Fifty microliters of blood was then drawn from the arterial side and replaced on the venous side by injection of the radioactive protein (20 ug in 50 ul). At each time point, 150 ul of blood was taken from the artery and replaced with saline on the venous side. One hundred microliters of collected blood were added to an equal volume of 10% KI (18) and this mixture was then precipitated with 200 microliters of 10% TCA. Two ml of ice cold 5% TCA were then added to the test tube which was vortexed and centrifuged. The supernate was removed and additional 2 ml of ice cold 5% TCA were added to the pellet which was resuspended and again centrifuged. Finally, the radioactivity in the pellet and the combined supernates was counted. Tissue radioactivity determination At the end of the experiment animals were sacrificed by opening the chest cavity. Tissue samples were removed, rinsed in ice cold saline, blotted dry and their weight determined before radioactivity was estimated on smaller fragments of the organs. Reported tissue values are expressed as percent of the total radioactivity recovered in six organs plus that present in blood (the total recovery of radioactivity ranged in 50-75% of the injected amount). Tissue radioactivity was corrected for residual blood as determined in separate experiments by injecting 1251bovine albumin into circulation and sacrificing the animal 5 minutes later. The organs were excised, sampled for radioactivity as above, and the amount of residual blood in each tissue calculated. The circulation half life of 1251-bovine albumin estimated in separate experiments was 10.5 hours. Data analysis The natural log of the fractional amount of radiolabelled prothrombin initially recovered in blood was graphed versus the time of collection. Plasma survival curves for 1251-asialoprothrombin and 1251-prothrombin were analyzed with computer assisted curve stripping methods by fitting the data to a two exponential term equation(19). The log linear part of the curve was extrapolated to time zero and subtracted from the original curve to obtain the fast phase of the clearance. The half disappearance
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times(tl/z) were calculated from slopes of lines generated linear regression of the resultant points(tl/z=ln2/slope).
by
EESUJlTS As shown in Figure 1, clearance of 12sI-bovine prothrombin (TCA precipitated radioactivity) from rat circulation was a biphasic process. In the initial phase(solid circles), prothrombin disappeared w‘ith a half life of 39.5 minutes. The second phase(open circles), which remained constant for at least 6 hours, had a half life of 202.9 minutes. As 1251-prothrombin disappeared from blood, its degradation products (TCA soluble radioactivity) in blood(open squares) remained constant after reaching 5% of the original injected radioactivity.
i I -60
-60
tlj2a202.9 minutes
-40
111249.5minutes
d
3b
6b
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li0
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(minutes)
Figure 1. Clearance of 1251-bovine prothrombin from rat circulation. The natural log (left axis) of the fraction of remaining TCA percipitable radioactivity (open circles) is graphed versus time. The log linear phase represents blood clearance of prothrombin. When extrapolated to zero time and curve, the half life for subtracted from the original equilibration of prothrombin can be determined (dark TCA soluble radioactivity (right axis, open circles). squares) is expressed as percent of total initially observed radioactivity and represents degradation products generated during clearance of prothrombin. Points are means of seven experiments. S.D. for each point did not exceed ten percent of the mean value. Asialoprothrombin also had a two phase clearance curve (Figure 2). The first phase (solid circles) had a half life of 1.8 minutes was during which approximately 80% of injected radioactivity
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cleared, while the second phase had a half life of 56.8 minutes. the TCA soluble injection, minutes after Ten to twenty radioactivity associated with asialoprothrombin (open squares) in rose sharply, and by 30 minutes, most of the radioactivity blood was TCA soluble. 100
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Figure 2. Clearance of 1251-bovine asialoprothrombin from rat Clearance of asialoprothrombin (closed circles) circulation. was obtained by subtracting the extrapolated log linear phase TCA soluble (open circles). from the original curve open squares) represents (right axis, radioactivity degradation products generated during the clearance of Radioactivity is expressed as percent of asialoprothrombin. total initially observed radioactivity and points are means of five experiments. S.D. for each point did not exceed ten percent of the mean value.
Tissue distribution of the injected prothrombins at the end of the experiments was measured in heart, lung, liver, kidney, spleen and Asialoprothrombin distribution at 30 minutes(Figure 3) diaphragm. was 35% in liver, about 4% in kidneys and spleen and only minimal amounts in other organs. Prothrombin distribution at 3 hours indicated that liver was the major organ of uptake but the uptake was only 5% of the recovered radioactivity since most of the prothrombin remained in circulation(Figure 1). Other organs contained only minimal amounts of radioactivity. Results of injecting increasing amounts of asialofetuin simultaneously with labelled asialoprothrombin are shown in Figure 4A and 4B. Doses of 200, 400 and 600 ug asialofetuin/lOO g body weight caused a progressive increase in the half life for asialoprothrombin from 1.8 minutes in controls to 10, 20 and greater than 30 minutes, respectively. The release of TCA soluble radioactivity (Figure 4B) was correspondingly delayed.
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? ?asialoprothrombin
30
20
10
0
heart
lung
liver
kidney
spleen diaphragm
Figure 3. Tissue distribution of radioactivity from l%prothrombin at 3 hours (dark bars) and 1251-asialoprothrombin at 30 minutes (hatched bars). Bars represent percent of radioactivity found in whole tissues which was corrected for residual blood (Methods) and expressed as percent of total recovered radioactivity. Results are means 2 S.D. of three experiments.
4A
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Figure 4. The effects of increasing amounts of injected asialo-fetuin on clearance of 1251-asialoprothrombin. Asialoprothrombin was injected into rat circulation with varied amounts of asialofetuin ( 0 0, L 200, D 400, x 600 ug/lOO kg), with blood collected and TCA precipitated at each (A) TCA precipitable and (B) TCA soluble time point. are expressed as percent of the initial radioactivity Control points are the means of 5 observed radioactivity. animals per curve while experimental points are means of two animals per curve.
To test whether prothrombin clearance was affected by saturating the AGPRs we used two conditions to administer asialofetuin (600 One was a single injection to saturate ug/lOOg) intravenously. the receptors for at least 30 minutes (as judged by experiments in Figure 4) and the other was a series of injections every twenty minutes to assure saturation of the AGPRs for the duration of a 3 In either case, the half life of 1251-prothrombin hour experiment. and the release of TCA soluble radioactivity were unchanged in the presence of asialofetuin (Figure 5).
FIG. 5
0 0
30
60
90
time
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Figure 5. Clearance of 125 I-prothrombin alone compared to its clearance when the AGPR system is saturated. Asialofetuin (600 ug/lOO kg) was given simultaneously as a single dose (a ) or as repeated doses injected every twenty minutes (A). TCA precipitable radioactivity (closed symbols, right axis) TCA soluble radioactivity (open symbols, left axis). Points are means + S.D. of three experiments per curve.
DISCUSSION The AGPR receptor mediated hepatic uptake system has been postulated to be the final degradation step of normal plasma glycoprotein clearance(l,3). However, attempts to directly demonstrate the participation of this receptor in normal clearance of plasma glycoproteins have not been sucessful(5,6). Thus the physiological relevance of this uptake system remains obscure. Since the prothrombin molecule is rich in terminal sialic acid residues and has a relatively fast turnover rate, it is suitable for evaluating the importance of AGPRs in glycoprotein clearance. In coumadin treated rats the half life of prothrombin has been reported to be 5.6 hours (20) to 6 hours (21) based on disappearance of clotting activity. One study (22) of iodinated rat prothrombin clearance in rats gave a half life of 10.6 hours but this value was based on extrapolation of points representing less than 10% of the original injected radioactivity and thus may not represent true prothrombin half life. Examination of the reported blood clearance curves reveals that for approximately 75% of the injected radioactivity the half life was 4-5 hours. These variations in rat prothrombin half life may be due to difference in techniques and our value of 3.2 hours for half life of bovine prothrombin in rat circulation appears to be in the physiologic range. The half life of bovine asialoprothrombin in our experiments was 1.8 minutes and compares favorably with reported half lives of asialoderivatives from other glycoproteins such as fetuin (1.5 minutes)(23) and orosomucoid (1.3 minutes) (7) in rat circulation. The slower second phase of asialoprothrombin clearance(half life of 56.7 minutes), during which the last 20% of the radiolabelled ligand was removed, most likely represents the distribution of partially or fully sialated prothrombin, since about 15% of the sialic acid was not removed by the neuraminidase treatment. An earlier report(lO) showed that clotting activity due to bovine asialoprothrombin injected into prothrombin deficient rats, disappeared faster than clotting activity due to injected bovine prothrombin. However, it was not clear if the AGPR system was responsible for clearing the asialoglycoprotein or if other factors such as changes in susceptibility of this derivative to plasma proteases could have also accounted for its accelerated In our experiments, clearance of decrease in clotting activity. 12sI-asialoprothrombin was competitively blocked by asialofetuin, a with rat AGPRs (24, 25). known to interact glycoprotein progressively delayed the Increasing amounts of asialofetuin clearance of 1251-asialoprothrombin and the highest dose completely
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blocked the fast phase of the clearance for 30 minutes. When removal of 1251-asialoprothrombin by the AGPR system was blocked by asialofetuin, asialoprothrombin remained in circulation undigested. These observations strongly suggest an interaction of asialoprothrombin with the AGPRs and implicate the liver as the site of its uptake and processing. It has been shown that injected bovine or rat asialoorosomucoid are cleared with minimal identical rates from suggesting rat circulation, recognition of asialointerspecies difference in the It is therefore likely that clearance of bovine derivatives(7). asialoprothrombin in our experiments represents the fate of rat asialoprothrombin. In contrast to the effects of asialofetuin on asialoprothrombin, doses of asialofetuin sufficient to saturate the AGPR for four asialoprothrombin half lives did not alter the course of prothrombin clearance. If AGPRs contributed to the disappearance of prothrombin from circulation then a blockade of this system should have prolonged the apparent prothrombin half life. Failure to demonstrate this argues against the necessity of generating an asialoderivative in plasma clearance of prothrombin. Others have the AGPR system with exogenous also found that saturating asialoglycoproteins did not effect the half lives of injected (6,7). Since clearances of native orosomucoid or fetuin prothrombin, orosomucoid and fetuin do not depend on the function of the AGPR system, this mechanism does not appear to be a major factor in the general turnover of plasma glycoproteins. The mechanism and site of prothrombin clearance from circulation While some activated can only be speculated on at present. clotting factors appear to be cleared by initial conversion to an activated state(26), experimental evidence indicates that conversion of prothrombin to thrombin is not a significant factor in prothrombin turnover(27). There may be a receptor mechanism for localizing and digesting prothrombin on surface of hepatocytes since prothrombin fragments generated by an extracellular hepatocyte protease have been shown to influence the synthesis of prothrombin by isolated hepatocytes(28). Similarly, injection of such prothrombin fragments into rabbit circulation has also been associated with increased prothrombin synthesis(29).
ACNOWLEDGEMENTS The authors would like to thank Dr. N.R. Shulman for helpful discussions and to Dr. B. Marks for continuing encouragement.
1 Ashwell, G & Hartfort, J. Carbohydrate specific receptors in the liver. Ann. Rev. Biochem. 51, 531-34, 1984. 2 Schwartz, A. L. The hepatic asialoglycoprotein Critical Rev. Biochem. 16, 207-33, 1983.
receptor.
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3 Ashwell, G. & Steer, C. J. Hepatic recognition
and catabolism of serum glycoproteins. J. Am. Med. Assoc. 246, 2358-64,1981.
4 Marshall, J. S., Green, A. M., Pensky, J., Williams, S,., Zinn, A. & Carlson, D. M. Measurement of circulating desialated glycoproteins and correlation with hepatocellular damage. J. Clin. Invest. 54, 555-62, 1974.
5 Sobue, G. & Kosaka, A. Asialoglycoproteins in a case of primary hepatic cancer. Hepato-Gastroenterology27, 200-203, 1980. 6 Wong, K. L., Charlwood, P. 0. A., Hatton, M. W. C & Regoeczi, E. Studies of the metabolism of asialotransferrins: evidence that transferin does not undergo desialization in vivo. Clin. Sci. Mol. Med
46, 763-774,1974.
7 Clarenburg, R. Asialoreceptor is uninvolved in clearing of intact glycoproteins from rat blood. Am.J.Physiol. 224, 6247-6253, 1983. 8 Conzelmann, E. & Sandhoff, K. Glycolipid degradation. Adv. Enzymol. 60:89-216, 1987
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glycoprotein
9 Mizouchi, T., Yamashita, K., Fujikawa, K., Kisiel, W., & Kobata, A. The carbohydrate of bovine prothrombin J. Biol. Chem. 254, 6419-6425, 1979. 10 Nelsestuen, G. L., & Suttie, J.W. The properties of asialo and aglyco prothrombin. Biochem. Biophys. Res. Comm. 45, 198-203,
1971. 11 Esnouf, P. M., Lloyd, P. H., & Jesty, J. A method for the simultaneous isolation of factor X and prothrombin from bovine plasma. Biochem. J. 131, 781-789, 1973.
12 Hjort, P., Rappaport, S. I. & Owren, P. A. A simple specific one stage prothrombin assay using Rusell's viper venom on cephalin susupension. J. Lab. Clin. Med. 46, 89-97,1955. 13 Warren, L. The thiobarbituric Biol. Chem. 231, 1971-1975, 1959.
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14 Skoza, L. & Mohos, S. Stable thiobarbituric acid chromophobe with dimethyl sulfoxide. Biochem. J. 159, 457-462, 1976. A highly stable 15 David, G. S. Solid state lactoperoxidase: of proteins. Biochem. enzyme for simple, gentle iodination Biophys. Res. Comm. 48, 464-71, 1972. 16 Fraker, P. J., & Speck, J. C. Protein and cell ;e;b;a;e chloramide, soluble with sparingly iodinations Ga-diphenyl-glycouril. Biochem. Bi0phyL.I Rk. tetrachloro-3a,
Comm. 80, 849-57, 1978. 17 Laemmli, U. K., Cleavage of structural proteins during assembly of the head of bacteriophage t4. Nature 222, 680-687, 1970.
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18 Chisolm, G. M., Sil, S.A. C Hmiel, S. P. Measurement of the degradation products of radioiodinated proteins. Anal. Chem. 111, 212-219,1981. 19 Mitopoulos, K. A. & Esnouf, M. P. Turnover of factor X and of prothrombin fed on a standard or cholesterol-supplemented diet. Biochem. J. 244, 263-269, 1987. 20 van Oosterom,
A.T. Mattie, H., Hermans, W.T. & Veltkamp, J.J. The influence of the thyroid function on the metabolic rate of prothrombin, factor VII, factor X in the rat. Thromb. Haemostas. 35, 607-619,1976. 21 Bell, R. G. 6 Matschiner, J. T. Synthesis and destruction of prothrombin in the rat. Arch. Biochem. Biophys. 135, 152-59, 1969. 22 Koj, A., Regoeczi, E., Chindemi, P. A. & Gauldie, J. Synthesis and turnover of prothrombin during experimental inflammation in rats. Br. J. Exp. Path. 65, 691-700, 1984. 23 Regoeczi, E., DeBanne, M. T., Hatton, M. W. C. & Koj, A. Elimination of asialofetuin and asialoorosomucoid by the intact rat. Biochim. Biophys. Acta 541, 372-384, 1978. 24 LaBadie, J. H., Chapman, K. & Arowson, N. N. Glycoprotein catabolism in the liver. Biochem. J. 151, 271-279, 1975. 25 Tolleshaug, H., Berg, T. Nilsson, M. & Norm, K. R. Uptake and labeled asialofetuin by isolated rat degradation of I251 hepatocytes. Biochem. Biophys. Acta 499:73-84, 1975. 26 Fuchs, H. E., Trapp, H. G., Griffith, M. J., Roberts, H. R. & Pizzo,S. V. Regulation of factor IX in vitro in the mouse. J. Clin. Invest. 73, 1696-1703, 1984. 28 Takeda, Y. Studies of the effects of heparin, coumadin and vitamin K on prothrombin metabolism and distribution in calves with the use of iodine-125-prothrombin. J. Lab. Clin. Med. 75, 355-377, 1970. 29 Graves, C. B., Munns, T. W., Carlisle, T. L., Grant, G. A.& Strauss, A. W. Induction of prothrombin synthesis by prothrombin fragments. Proc. Natl. Acad. Sci. 78, 4772-4776, 1981.
